STUDIES OF THE INFECTION OF CULTIVARS OF SHALLOT (ALLIUM 
ASCALONICUM L.) IN GHANA BY ASPERGILLUS NIGER VAN TIEGHEM AND 
THE EFFECT OF THE HOST ON SOME ASPECTS OF THE BIOLOGY OF A. NIGER. 
 
 
A Thesis presented by 
YAHAYA BUKARI B.Sc. (HONS), M. PHIL. 
(10088032) 
In part fulfilment of the requirements for the Ph. D. Degree of the University of Ghana. 
 
 
 
               From: DEPARTMENT OF BOTANY, UNIVERSITY OF GHANA, LEGON. 
 
 
JULY 2014. 
 
 
i 
 
STUDIES OF THE INFECTION OF CULTIVARS OF SHALLOT (ALLIUM 
ASCALONICUM L) IN GHANA BY ASPERGILLUS NIGER VAN TIEGHEM AND THE 
EFFECT OF THE HOST ON SOME ASPECTS OF THE BIOLOGY OF A. NIGER. 
 
 
 
A Thesis presented by 
YAHAYA BUKARI B.Sc. (HONS), M. PHIL. 
(10088032) 
In part fulfilment of the requirements for the Ph. D. Degree of the University of Ghana. 
 
 
 
               From: DEPARTMENT OF BOTANY, UNIVERSITY OF GHANA, LEGON. 
 
 
JULY 2014. 
 
 
ii 
 
DECLARATION 
I, the undersigned, Yahaya Bukari, declare that this thesis is the result of my own research carried 
out in the Department of Botany, University of Ghana, legon, under the supervision of Emeritus 
Prof. G.C. Clerk. 
This work has never been presented either in part or completely, for any degree of this University 
or elsewhere. References cited have been fully acknowledged. 
 
 
 
 
……………………………..                                                        ……………………………………. 
YAHAYA BUKARI                                                            EMERITUS PROF. G.C. CLERK 
(Student) (Supervisor). 
 
 
 
 
 
 
iii 
 
DEDICATION 
This thesis is dedicated to my loving mother, Madam Zenabu Dabonne, to the memory of 
my late father, Mr. Yorda Bukari, and to my sisters and brothers. 
 
 
iv 
 
ACKNOWLEDGEMENT 
My first thanks go to Almighty God who has guarded and guided me throughout this 
programme.  
I wish to express my deepest appreciation to my supervisor Emeritus Prof. G.C. Clerk for his 
guidance and deepest interest throughout the course of this work. I am forever indebted to him 
for all his time and patience for me during this course. May Almighty God continue to bless 
him and his entire family. 
I will like to thank the Head of Department, Prof. G.K. Ameka, for his interest in my work and 
also for making available the department vehicle during my trips to Anloga in the Volta Region. 
I am also grateful to Prof. G.T. Odamtten, Prof.  L. Enu-Kwesi, Prof I.K. Asante, Dr. Ebenezer 
Owusu and all the lecturers of the Department of Botany for their encouragement. May God 
bless them all. 
My sincere thanks go to Mr. Kofi Baako, a chief technologist of the Department for his personal 
interest in my work and who used much of his time to help me in my work and I am grateful 
for all his encouragement during the course of this work. I will like to thank all the technicians 
of the Department as well, especially, Mr. George Akwettey and the former Chief technologist 
Madam Alberta Banson. My thanks also go to the technicians of the Ecological Laboratory of 
the University of Ghana, especially, Mr. Prince Owusu. 
My appreciation also goes to the drivers of the Department Messrs. G. O. Agyeman and Raphael 
Atutra. I am grateful to Messrs Stephen Akpedonu and A. Orizi of Anloga who helped me in 
the collection of soil samples and procurement of shallot bulbs. 
v 
 
Finally my thanks go to all who helped me in the typing of this thesis: Gilbert Boateng, Diana 
Ayegbe, Esther H. Quacoo, Monica Esinam Gasika, Taquiyya Najib and Benedict Quagraine. 
 
There are many others who contributed in diverse ways during this programme who cannot 
individually be mentioned here because of the number. I thank them all. 
 
 
 
vi 
 
TABLE OF CONTENTS 
Page 
DECLARATION .........................................................................................................................ii 
DEDICATION ........................................................................................................................... iii 
ACKNOWLEDGEMENT ......................................................................................................... iv 
LIST OF TABLES .....................................................................................................................xii 
LIST OF FIGURES .................................................................................................................xxii 
LIST OF PLATES ................................................................................................................. xxiii 
ABSTRACT ......................................................................................................................... xxviii 
I INTRODUCTION AND LITERATURE REVIEW ......................................................... 1 
II. MATERIALS AND GENERAL METHODS .................................................................. 22 
        (i)  MATERIALS ............................................................................................................................ 22 
      a.  Shallot bulbs. ............................................................................................................. 22 
      b.  Isolates of Aspergillus niger ..................................................................................... 22 
      c.  Soil Samples. ............................................................................................................. 23 
      d.  Grains of maize, Seeds of Beans, and Tubers of Cassava, Irish Potato and 
           Sweet Potato. ............................................................................................................ 28 
      e.  Chemicals .................................................................................................................. 28 
         (ii)  GENERAL METHODS ........................................................................................................ 29 
      a.  Characteristics of bulbs ............................................................................................. 29 
      b.  Elemental  composition of bulbs .............................................................................. 29 
      c.  Soil analysis .............................................................................................................. 33 
      d.   Preparation of and composition of nutrient media. .................................................. 33 
      e.   Methods of sterilization............................................................................................ 34 
      f   Soil mycofloral studies. ............................................................................................. 35 
vii 
 
Page 
      g.  Colonization of surface of bulbs growing in soil by fungi. ...................................... 36 
      h.  Conidium germination tests ...................................................................................... 37 
      i.  Aspergillus niger growth on shallot bulbs on sale at the vegetable stores ................ 38 
      j.  A. niger culture growth tests. ..................................................................................... 38 
      k.  Growth of A. niger isolates in aqueous extracts of the bulbs. .................................. 39 
      l.  Humidity chambers .................................................................................................... 39 
      m.  Maintenance of constant humidities ........................................................................ 40 
      n.  Study of rot development of wound-inoculated bulb ............................................... 42 
      o.  In Vitro production of Pectic enzymes. .................................................................... 42 
      p.  Maceration test .......................................................................................................... 43 
      q   Determination of productivity of A. niger on ground shallot bulb ........................... 43 
      r   Conidia Longevity test. ............................................................................................. 44 
      s.  Application of Potassium and Urea fertilizers .......................................................... 44 
      t.  Photography ............................................................................................................... 45 
      u.  Statistical Analysis .................................................................................................... 45 
      v.  Experimental precautions. ........................................................................................ 45 
III.  EXPERIMENTAL DETAILS .......................................................................................... 47 
SHALLOT CULTIVARS IN GHANA ...................................................................................... 47 
EXPERIMENT A. Morphology of the two shallot cultivars ............................................... 47 
EXPERIMENT B. Elemental  composition of the bulbs of the two shallot cultivars ......... 47 
        ASPERGILLUS NIGER INFECTION OF THE SHALLOT BULBS. .................................... 48 
EXPERIMENT C. Aspergillus niger growth on bulbs of fresh stocks of the two          
                         shallot cultivars in market stalls. ................................................................. 48 
 
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EXPERIMENT D. Influence of micro-habitats of shallot bulbs on the development           
                         of Aspergillus niger conidiophores ............................................................. 48 
EXPERIMENT E. Culture characteristics of the five test isolates of Aspergillus niger ..... 49 
EXPERIMENT F. Germination of conidia of the five test Aspergillus niger isolates              
and pattern of growth on the swollen leaf bases and the scale                
leaves and fate of the germ tubes. ................................................................ 49 
EXPERIMENT G. Germination of conidia of the five test Aspergillus niger isolates                
in aqueous extracts of bulbs of the two shallot cultivars. ............................ 50 
EXERIMENT H. Shallot bulb inoculation tests using the two shallot cultivars and                
the five test Aspergillus niger isolates ......................................................... 51 
EXPERIMENT I. Rotting of wound-inoculated bulbs at different relative humidities          
using the two shallot cultivars and the five test Aspergillus niger        
isolates. ........................................................................................................ 51 
EXPERIMENT J. In-vitro production of pectic enzymes by the five test Aspergillus           
niger isolates ................................................................................................ 52 
THE SOIL ASPERGILLUS NIGER-SHALLOT BULB COMPLEX. ................................... 53 
EXPERIMENT K. Mycoflora of the bulbs and the rhizosphere of shallot plants raised             
in soil of Anloga farms amended with Urea fertilizer. ................................ 53 
EXPERIMENT L. Response of bulbs of the two shallot cultivars formed in Anloga             
farm soils amended with Urea fertilizer to Aspergillus niger           
inoculation using A. niger Isolates 1 and 2. ................................................. 55 
EXPERIMENT M. Mycoflora of the bulbs and the rhizosphere of shallot plants                 
raised in soil of Anloga farms amended with Potassium fertilizer. ............. 56 
EXPERIMENT N Response of bulbs of the two shallot cultivars formed in Anloga             
farm soils amended with Potassium fertilizer to Aspergillus niger 
inoculation using A. niger Isolates 1 and 2. ................................................. 56 
 
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Page 
EXPERIMENT O.  Mycoflora of the bulbs and rhizosphere of plants of the two                
shallot  cultivars grown in soils of farms of non-alliaceous crops ............... 56 
ROLE OF THE DIFFERENT SHALLOT CULTIVARS IN THE PERSISTENCE              
OF A. NIGER IN THE ECOSYSTEM ....................................................................................... 57 
EXPERIMENT P. Conidiation of Aspergillus niger Isolates 1, 3 and 5 on ground bulb        
tissue of the two shallot cultivars and the germination capacity of the 
conidia... ....................................................................................................... 57 
EXPERIMENT Q. Longevity of conidia formed by Aspergillus niger Isolates 1, 3 and               
5 at different relative humidities. ................................................................. 58 
IV. RESULTS ............................................................................................................................ 60 
SHALLOT CULTIVARS IN GHANA………..……….………………………………….60 
EXPERIMENT A. Morphology of the two shallot cultivars ............................................... 60 
       EXPERIMENT B. Elemental composition of the bulbs of the two shallot  
                                      cultivars……………………………………………………….….....…61  
ASPERGILLUS NIGER INFECTION OF THE SHALLOT BULBS………………...……74  
EXPERIMENT C. Aspergillus niger growth on bulbs of fresh stocks of the two shallot 
cultivars in market stalls. ............................................................................. 74 
EXPERIMENT D. Influence of micro-habitats of shallot bulbs on the development of 
Aspergillus niger conidiophores .................................................................. 77 
EXPERIMENT E. Culture characteristics of the five test isolates of Aspergillus niger ..... 79 
EXPERIMENT F. Germination of conidia of the five test Aspergillus niger isolates and  
pattern of growth on the swollen leaf bases and the scale leaves and         
fate of the germ tubes. .................................................................................. 82 
EXPERIMENT G. Germination of conidia of the five test Aspergillus niger isolates in  
aqueous extracts of bulbs of the two shallot cultivars. .............................. 110 
EXPERIMENT H. Shallot bulb inoculation tests using the two shallot cultivars and the        
five test Aspergillus niger isolates. ............................................................ 117 
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EXPERIMENT I. Infection of wound-inoculated bulbs at different relative humidities        
using the two shallot cultivars and the five test Aspergillus niger       
isolates. ...................................................................................................... 119 
EXPERIMENT J. In-vitro production of pectic enzymes by the five test Aspergillus           
niger Isolates .......................................................................................... …132 
THE SOIL-ASPERGILLUS NIGER-SHALLOT BULB COMPLEX…………………….154  
EXPERIMENT K. Mycoflora of the bulbs and the rhizosphere of shallot plants raised             
in soil of Anloga farms amended with Urea fertilizer ............................... 154 
EXPERIMENT L. Response of bulbs of the two shallot cultivars formed in Anloga             
farm soils amended with Urea fertilizer to Aspergillus niger           
inoculation using A. niger Isolate 1 and 2. ................................................ 171 
EXPERIMENT M. Mycoflora of the bulbs and the rhizosphere of shallot plants raised             
in soil of Anloga farms amended with Potassium fertilizer. ...................... 175 
EXPERIMENT N. Response of bulbs of the two shallot cultivars formed in Anloga             
soils amended with Potassium fertilizer to Aspergillus niger           
inoculation using A. niger Isolates 1 and 2 ................................................ 190 
EXPERIMENT O. Mycoflora of the bulbs and rhizosphere of plants of the two shallot   
cultivars grown in soils of farms of non-alliaceous crops ......................... 196 
ROLE OF THE DIFFERENT SHALLOT CULTIVARS IN THE PERSISTENCE                  
OF A. NIGER IN THE ECOSYSTEM……………………………………………..……..201  
EXPERIMENT P. Conidiation of Aspergillus niger Isolates 1, 3 and 5 on ground bulb       
tissue of the two shallot cultivars and the germination capacity of the 
conidia. ....................................................................................................... 201 
EXPERIMENT Q. Longevity of conidia formed by Aspergillus niger Isolates 1, 3 and              
5 at different relative humidities ................................................................ 208 
 
 
xi 
 
V.   DISCUSSION .................................................................................................................... 213 
VI. SUMMARY ....................................................................................................................... 229 
VII REFERENCES ................................................................................................................. 239 
APPENDICES A-V ................................................................................................................. 263 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
xii 
 
LIST OF TABLES 
Page 
Table 1: Production of shallot (Allium ascalonicum) in Ghana in 2005-2009 (Library         
record: Ministry of Agriculture) ....................................................................................... 9 
Table 2: Sulphuric acid (H2SO4) solutions for maintaining Constant Humidity ......................... 41 
Table 3: Major chemical elements (%) of bulbs of two cultivars of shallot (Allium ascalonicum) 
purchased from Agbogbloshie Market, Accra in October 2012. .................................... 68 
Table 4: Major chemical elements (%) of bulbs of two cultivars of shallot (Allium ascalonicum) 
purchased from Anloga Market in October 2012. .......................................................... 69 
Table 5: Major chemical elements (%) of bulbs of two cultivars of shallot (Allium ascalonicum) 
purchased from Kaneshie Market, Accra in October 2012............................................. 70 
Table 6: Major chemical elements (%) of bulbs of two cultivars of shallot ( Allium ascalonicum) 
purchased from Makola Market, Accra in October 2012. .............................................. 71 
Table 7: Major chemical elements (%) of bulbs of two cultivars of shallot ( Allium ascalonicum) 
purchased from Mallam Atta Market, Accra in October 2012. ...................................... 72 
Table 8: Means of the values of the major chemical elements ( % ) of the bulbs of the two            
cultivars of shallot ( Allium ascalonicum) recorded in Tables 3 to 7. ............................ 73 
Table 9: A. niger growth on the bulbs of the two shallot cultivars at the time of                
purchase from Agbogbloshie Market, Accra ( August 2011) and Anloga               
market, Volta region (January 2014). ............................................................................. 75 
Table 10: Influence of the micro-environment of bulbs of the two shallot cultivars         
purchased from Anloga, Volta region on the development of the conidiophores            
of A. niger. ...................................................................................................................... 78 
Table 11: Characteristics of A. niger Isolates 1, 2, 3, 4 and 5 as test fungi in EXPERIMENTS        
F-J, L, N, P and Q. .......................................................................................................... 80 
Table 12: Germination of conidia of A. niger Isolate 1 in light of 76 lux in 12 hours                   
in Potato Dextrose Broth (PDB) suspension drops on different component                
parts of bulbs of shallot (Allium ascalonicum) ............................................................... 85 
Table 13: Germination of conidia of A. niger Isolate 2 in light of 76 lux in 12 hours in        
Potato Dextrose Broth (PDB) suspension drops on different component parts               
of bulbs of shallot (Allium ascalonicum). ....................................................................... 87 
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Table 14: Germination of conidia of A. niger Isolate 3 in light of 76 lux in 12 hours in        
Potato Dextrose Broth (PDB) suspension drops on different component parts               
of bulbs of shallot (Allium ascalonicum). ....................................................................... 89 
Table 15: Germination of conidia of A. niger Isolate 4 in light of 76 lux in 12 hours in        
Potato Dextrose Broth (PDB) suspension drops on different component parts               
of bulbs of shallot (Allium ascalonicum). ....................................................................... 91 
Table 16: Germination of conidia of A. niger Isolate 5 in light of 76 lux in 12 hours in        
Potato Dextrose Broth (PDB) suspension drops on different component parts               
of bulbs of shallot (Allium ascalonicum). ....................................................................... 93 
Table 17: Germination of conidia of A. niger Isolate 1 at 32±2oC in light of 76 lux in                
12 hours in suspension drops of Glucose of different concentrations on parts                 
of bulbs of the two cultivars of shallot (Allium ascalonicum). ....................................... 95 
Table 18: Germination of conidia of A. niger Isolate 2 at 32±2oC in light of 76 lux in               
12 hours in suspension drops of Glucose of different concentrations on parts                
of bulbs of the two cultivars of shallot (Allium ascalonicum). ....................................... 96 
Table 19: Germination of conidia of A. niger Isolate 3 at 32±2oC in light of 76 lux in                
12 hours in suspension drops of Glucose of different concentrations on parts                
of bulbs of the two cultivars of shallot (Allium ascalonicum). ....................................... 97 
Table 20: Germination of conidia of A. niger Isolate 4 at 32±2oC in light of 76 lux in               
12 hours in suspension drops of Glucose of different concentrations on parts                 
of bulbs of the two cultivars of shallot (Allium ascalonicum) ........................................ 98 
Table 21: Germination of conidia of A. niger Isolate 5 at 32±2oC in light of 76 lux in               
12 hours in suspension drops of Glucose of different concentrations on parts                 
of bulbs of the two cultivars of shallot ( Allium ascalonicum). ...................................... 99 
Table 22: Germination of conidia of A. niger Isolate 1 at 32±2oC in light of 76 lux in               
12 hours in suspension drops of Galactose of different concentrations on parts             
of bulbs of the two cultivars of  shallot ( Allium ascalonicum). ................................... 100 
 
 
 
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Page 
Table 23: Germination of conidia of A. niger Isolate 2 at 32±2oC in light of 76 lux in                
12 hours in suspension drops of Galactose of different concentrations on parts              
of bulbs of the two cultivars of  shallot ( Allium ascalonicum). ................................... 101 
Table 24: Germination of conidia of A. niger Isolate 3 at 32±2oC in light of 76 lux in                
12 hours in suspension drops of Galactose of different concentrations on parts              
of bulbs of two cultivars of  shallot  ( Allium ascalonicum). ........................................ 102 
Table 25: Germination of conidia of A. niger Isolate 4 at 32±2oC in light of 76 lux in               
12 hours in suspension drops of Galactose of different concentrations on parts             
of bulbs of the two cultivars of  shallot ( Allium ascalonicum). ................................... 103 
Table 26: Germination of conidia of A. niger Isolate 5 at 32±2oC in light of 76 lux in                
12 hours in suspension drops of Galactose of different concentrations on parts              
of bulbs of the two cultivars of shallot ( Allium ascalonicum). .................................... 104 
Table 27: Germination of conidia of A. niger Isolate 1 at 32±2oC in light of 76 lux in                
12 hours in suspension drops of sucrose of different concentrations on parts                 
of bulbs of the two cultivars of  shallot (Allium ascalonicum). .................................... 105 
Table 28: Germination of conidia of A. niger Isolate 2 at 32±2oC in light of 76 lux in               
12 hours in suspension drops of Sucrose of different concentrations on parts                 
of bulbs of the two cultivars of  shallot ( Allium ascalonicum). ................................... 106 
Table 29: Germination of conidia of A. niger Isolate 3 at 32±2oC in light of 76 lux in                
12 hours in suspension drops of Sucrose of different concentrations on parts                  
of bulbs of the two cultivars of  shallot ( Allium ascalonicum). ................................... 107 
Table 30: Germination of conidia of A. niger Isolate 4 at 32±2oC in light of 76 lux in                
12 hours in suspension drops of Sucrose of different concentrations on parts                  
of bulbs of the two cultivars of  shallot ( Allium ascalonicum). ................................... 108 
Table 31: Germination of conidia of A. niger Isolate 5 at 32±2oC in light of 76 lux in               
12 hours in suspension drops of Sucrose of different concentrations on parts                  
of bulbs of the two cultivars of  shallot ( Allium ascalonicum). ................................... 109 
Table 32: Germination of conidia of A. niger Isolate 1 in fluid of bulbs of the two               
cultivars of shallot ( Allium ascalonicum) for 12 hours at 32±2oC............................... 111 
 
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Table 33: Germination of conidia of A. niger Isolate 2 in fluid of bulbs of the two            
cultivars of shallot (Allium ascalonicum) for 12 hours at 32±2oC................................ 112 
Table 34: Germination of conidia of A. niger Isolate 3 in fluid of bulbs of the two            
cultivars of shallot (Allium ascalonicum) for 12 hours at 32±2oC................................ 113 
Table 35: Germination of conidia of A. niger Isolate 4 in fluid of bulbs of the two            
cultivars of shallot (Allium ascalonicum) for 12 hours at 32±2oC................................ 114 
Table 36: Germination of conidia of A. niger Isolate 5 in fluid of bulbs of the two            
cultivars of shallot (Allium ascalonicum) for 12 hours at 32±2oC................................ 115 
Table 37: Extent of rot of bulbs of shallot (Allium ascalonicum) either surface-             
inoculated or wound-inoculated with mycelium discs of different A. niger            
isolates and store at 32±2oC in humid atmosphere (100% R.H.) for 3 days................. 118 
Table 38: Infection of bulbs of shallot (Allium ascalonicum) wound-inoculated with      
mycelium of A. niger Isolate 1 and incubated at 50-100% relative humidity                  
at 32±2oC. ..................................................................................................................... 121 
Table 39: Infection of bulbs of shallot (Allium ascalonicum) wound-inoculated with     
mycelium of A. niger Isolate 2 and incubated at 50-100% relative humidity                  
at 32±2oC ...................................................................................................................... 122 
Table 40: Infection of bulbs of shallot (Allium ascalonicum) wound-inoculated with       
mycelium of A. niger Isolate 3 and incubated at 50-100% relative humidity                  
at 32±2oC ...................................................................................................................... 123 
Table 41: Infection of bulbs of shallot (Allium ascalonicum) wound-inoculated with      
mycelium of A. niger Isolate 4 and incubated at 50-100% relative humidity                  
at 32±2oC. ..................................................................................................................... 124 
Table 42: Infection of bulbs of shallot (Allium ascalonicum) wound-inoculated with       
mycelium of A. niger Isolate 3 and incubated at 50-100% relative humidity                  
at 32±2oC ...................................................................................................................... 125 
Table 43: Effect of Relative Humidity on sporulation of A. niger Isolate 1 on bulbs of          
shallot (Allium ascalonicum) at 32±2oC under 12 hour day- night cycle in 10      
days ............................................................................................................................... 126 
 
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Table 44: Effect of Relative Humidity on sporulation of A. niger isolate 2 on bulbs                   
of shallot (Allium ascalonicum) at 32±2oC under 12 hour day- night cycle                     
in 10 days ...................................................................................................................... 127 
Table 45: Effect of Relative Humidity on sporulation of A. niger Isolate 3 on bulbs                    
of shallot (Allium ascalonicum) at 32±2oC under 12 hour day night cycle                      
in 10 days. ..................................................................................................................... 128 
Table 46: Effect of Relative Humidity on sporulation of A. niger Isolate 4 on bulbs                   
of shallot (Allium ascalonicum) at 32±2oC under 12 hour day-night cycle                      
in 10 days ...................................................................................................................... 129 
Table 47: Effect of Relative Humidity on sporulation of A. niger Isolate 5 on bulbs                    
of shallot (Allium ascalonicum) at 32±2oC under 12 hour day-night cycle                      
in 10 days ...................................................................................................................... 130 
Table 48a: Growth of A. niger Isolate 1 grown in Czapek-Dox broth medium (pH 7.0)               
at 32±2oC under a 12-hour day-night cycle in 12 days. ................................................ 133 
Table 48b: Growth of A. niger Isolate 2 grown in Czapek-Dox broth medium (pH 7.0)              
at 32±2oC under a 12-hour day-night cycle in 12 days. ................................................ 134 
Table 48c: Growth of A. niger Isolate 3 grown in Czapek-Dox broth medium (pH 7.0)              
at 32±2oC under a 12-hour day-night cycle in 12 days. ................................................ 135 
:Table 48d: Growth of A. niger Isolate 4 grown in Czapek-Dox broth medium (pH 7.0)             
at 32±2oC under a 12-hour day-night cycle in 12 days. ................................................ 136 
Table 48e: Growth of A. niger Isolate 5 grown in Czapek-Dox broth medium (pH 7.0)              
at 32±2oC under a 12-hour day-night cycle in 12 days. ................................................ 137 
Table 49a: Mean maceration time of Potato tuber discs by Aspergillus niger Isolate 1         
filtrate and calculated enzyme activity of the culture filtrate. ...................................... 138 
Table 49b: Mean maceration time of Potato tuber discs by Aspergillus niger Isolate 2        
filtrate and calculated enzyme activity of the culture filtrate. ...................................... 139 
Table 49c: Mean maceration time of Potato tuber discs by Aspergillus niger Isolate 3        
filtrate and calculated enzyme activity of the culture filtrate. ...................................... 140 
Table 49d: Mean maceration time of Potato tuber discs by Aspergillus niger Isolate 4        
filtrate and calculated enzyme activity of the culture filtrate. ...................................... 141 
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Table 49e: Mean maceration time of Potato tuber discs by Aspergillus niger Isolate 5        
filtrate and calculated enzyme activity of the culture filtrate. ...................................... 142 
Table 50: Growth of Aspergillus niger isolates in Bean Meal Extract at 32±2oC under                a 
12-hour day-night cycle in 8 days.  ............................................................................... 144 
Table 51: Growth of Aspergillus niger isolates in Cassava Dextrose Broth at 32±2oC          under 
a 12-hour day-night cycle in 8 days.  ............................................................................ 145 
Table 52: Growth of Aspergillus niger isolates in Oat Meal Extract at 32±2oC under                   
a 12-hour day-night cycle in 8 days  ............................................................................. 146 
Table 53: Growth of Aspergillus niger isolates in Potato Dextrose Broth at 32±2oC             
under a 12-hour day-night cycle in 8 days  ................................................................... 147 
Table 54a: Growth of Aspergillus niger Isolate 1 in aqueous extract of bulbs of shallot            
(Allium ascalonicum) at 32±2oC under a 12-hour day-night cycle in 8 days.  ............. 148 
Table 54b: Growth of Aspergillus niger Isolate 2 in aqueous extract of bulbs of shallot                       
( Allium ascalonicum) at 32±2oC under a 12-hour day-night cycle in 8 days.  ............ 149 
Table 54c: Growth of Aspergillus niger Isolate 3 in aqueous extract of bulbs of shallot              
(Allium ascalonicum) at 32±2oC under a 12-hour day-night cycle in 8 days  .............. 150 
Table 54d: Growth of Aspergillus niger Isolate 4 in aqueous extract of bulbs of shallot            
(Allium ascalonicum) at 32±2oC under a 12-hour day-night cycle in 8 days  .............. 151 
Table 54e: Growth of Aspergillus niger Isolate 5 in aqueous extract of bulbs of shallot         
(Allium ascalonicum) at 32±2oC under a 12-hour day-night cycle in 8 days. .............. 152 
Table 55: Growth of Aspergillus niger isolates in Sweet Potato Dextrose Broth                 
medium at 32±2oC under a 12-hour day-night cycle in 8 days. ................................... 153 
Table 56: Fungal species isolated with Potato Dextrose Agar from freshly prepared              
plots at Anloga for cultivation of shallots ( Allium ascalonicum) immediately         
before planting. ............................................................................................................. 155 
.Table 57: Fungal species isolated with Sabouraud Agar from freshly prepared plots                  
at Anloga for cultivation of shallot ( Allium ascalonicum) immediately before 
planting. ........................................................................................................................ 156 
Table 58: Mineral and chemical composition of soil used for cultivation of shallot                
from Anloga. ................................................................................................................. 157 
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Table 59: Morphology of bulblets of the two shallot cultivars developing in the           
greenhouse under a 12- hour day-night cycle in Anloga shallot farm soil          
containing different quantities of Urea fertilizer. ......................................................... 160 
Table 60: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR)                 
and rhizosphere (RH) soils, and from soil particles adhering to bulbs (BS)                    
of the two shallot cultivars grown in unamended Anloga shallot farm soil                     
for 60 days. ................................................................................................................... 163 
Table 61: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR)                
and rhizosphere (RH) soils, and from soil particles adhering to bulbs (BS)                    
of the two shallot cultivars grown in Anloga shallot farm soil treated with               
1.94g of Urea for 60 days. ............................................................................................ 165 
Table 62: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR)               
and rhizosphere (RH) soils, and from soil particles adhering to bulbs (BS)                     
of the two shallot cultivars grown in Anloga shallot farm soil treated with               
3.88g of Urea for 60 days.  ........................................................................................... 167 
Table 63a: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in                  
unamended soil, wound-inoculated with mycelium of A. niger Isolates 1                     
and 2 and incubated  at 50-100% relative humidity at 32±2oC for 3 days.  ................. 172 
Table 63b: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in soil               
amended with, 1.94 g of Urea fertilizer, wound-inoculated with mycelium of               
A. niger Isolates 1 and 2 and incubated at 50-100% relative humidity at                
32±2oC for 3 days. ........................................................................................................ 173 
Table 63c: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in soil                   
amended with 3.88 of Urea fertilizer wound-inoculated with mycelium of                    
A. niger Isolates 1 and 2 and incubated at 50-100% relative humidity at               
32±2oC for 3 days. ........................................................................................................ 174 
Table 64: Morphology of bulblets of the two shallot cultivars developing in the           
greenhouse under a 12- hour day-night cycle in Anloga shallot farm soil          
containing different quantities of Potassium fertilizer. ................................................. 177 
 
xix 
 
Page 
Table 65: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR)               
and rhizosphere (RH) soils, and from soil particles adhering to bulbs (BS)                    
of the two shallot cultivars grown in unamended Anloga shallot farm soil                    
for 60 days. ................................................................................................................... 180 
Table 66: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR)                
and rhizosphere (RH) soils, and from soil particles adhering to bulbs (BS)                    
of the two shallot cultivars grown in Anloga shallot farm soil treated with                
3.33g of Potassium for 60 days. .................................................................................... 182 
Table 67: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR)                
and rhizosphere (RH) soils, and from soil particles adhering to bulbs (BS)                    
of the two shallot cultivars grown in Anloga shallot farm soil treated with              
6.67g/l of Potassium for 60 days. ................................................................................. 184 
Table 68: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR)                
and rhizosphere (RH) soils, and from soil particles adhering to bulbs (BS)                     
of the two shallot cultivars grown in Anloga shallot farm soil treated with                
10.00 g/l of Potassium for 60 days. .............................................................................. 186 
Table 69: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR)               
and rhizosphere (RH) soils, and from soil particles adhering to bulbs (BS)                    
of the two shallot cultivars grown in Anloga shallot farm soil treated with               
13.33 g/l of Potassium for 60 days.  ............................................................................. 188 
Table 70a: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in unamended           
soil, wound-inoculated with mycelium of A. niger Isolates 1  and 2 and             
incubated at 50-100% relative humidity at 32±2oC for 3 days. .................................... 191 
Table 70b: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in soil               
amended with, 3.33 g/l of Potassium fertilizer wound-inoculated with               
mycelium of A. niger Isolates 1 and 2 and incubated at 50-100% relative            
humidity at 32±2oC for 3 days. ..................................................................................... 192 
 
 
 
xx 
 
Page 
Table 70c: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in soil                
amended with 6.67 g/l of Potassium fertilizer wound-inoculated with                 
mycelium of A. niger Isolates 1and 2 and incubated at 50-100% relative              
humidity at 32±2oC for 3 days. ..................................................................................... 193 
Table 70d: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in soil               
amended with, 10.00 g/l of Potassium fertilizer wound-inoculated with             
mycelium of A. niger Isolates 1 and 2 and incubated at 50-100% relative            
humidity at 32±2oC for 3 days. ..................................................................................... 194 
Table 70e: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in soil               
amended with 13.33 g/l of Potassium fertilizer wound-inoculated with              
mycelium of A. niger Isolates 1 and 2 and incubated at 50-100% relative           
humidity at 32±2oC for 3 days. ..................................................................................... 195 
Table 71: Some morphological and colour characteristics of 20-day old bulblets of the two 
shallot cultivars developing in soils of indicated non-alliaceous crop farms in the 
greenhouse under 12-hour day-night cycle. .................................................................. 197 
Table 72: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR)               
and rhizosphere (RH) soils, and from soil particles adhering to bulbs (BS)                    
of the two Shallot cultivars grown in cassava ( Manihot esculenta) farm soils              
for 20 days .................................................................................................................... 198 
Table 73: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR)               
and rhizosphere (RH) soils, and from soil particles adhering to bulbs (BS)                    
of the two shallot cultivars grown in groundnut ( Arachis hypogea) farm soils             
for 20 days.  .................................................................................................................. 199 
Table 74: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR)                
and rhizosphere (RH) soils, and from soil particles adhering to bulbs (BS) of the       
two shallot cultivars grown in maize ( Zea mays) farm soils for 20 days. ................... 200 
Table 75: Growth of Aspergillus niger Isolate 1 on blended bulb of the two cultivars of       
shallot (Allium ascalonicum) under a 12-hour day-night cycle at 32±2oC. .................. 202 
 
 
xxi 
 
Page 
Table 76 Growth of Aspergillus niger Isolate 3 on ground bulb of the two cultivars                   
of shallot (Allium ascalonicum) under a 12-hour day-night cycle at 32±2oC. .............. 203 
Table 77: Growth of Aspergillus niger Isolate 5 on blended bulbs of the two cultivars                  
of shallot (Allium ascalonicum) under a 12-hour day-night cycle at 32±2oC. .............. 204 
Table 78: Degree of sporulation of Aspergillus niger Isolates 1, 3 and 5 grown on             
blended tissue of bulbs of the two shallot cultivars under a 12-hour day-night            
cycle at 32±2oC for 7 days. ........................................................................................... 205 
Table 79: Germination of conidia produced by 7-day old Aspergillus niger Isolate 1     
incubated in liquid organic nutrients at 32±2oC for12 hours.  ...................................... 206 
Table 80: Germination of conidia produced by 7-day old Aspergillus niger Isolate 3      
incubated in liquid organic nutrients at 32±2oC for12 hours. ....................................... 207 
Table 81: Germination of conidia produced by 7-day old Aspergillus niger Isolate 5      
incubated in liquid organic nutrients at 32±oC for12 hours. ......................................... 208 
 
 
 
 
xxii 
 
LIST OF FIGURES 
Page 
Figure 1: Length, width and weight of both Pale-brown and Pink cultivars of shallot        (Allium 
ascalonicum) purchased from the Anloga market. ...................................................................65 
Figure 2: Class-lengths and class-widths of the mature leaves of the Pale-brown                  
and Pink shallot (Allium ascalonicum) cultivars .......................................................................66 
Figure 3: Germination of conidia of the five test Aspergillus niger isolates in fluids    
expressed from ground bulbs of the Pale-brown (  ) and Pink (   ) shallot cultivars 116 
Figure 4a: Viability of conidia of three Isolates of Aspergillus niger formed on               
ground Pale-brown (  ) and Pink (  ) shallot bulbs and stored at different         
Relative Humidities at 32±2oC under 12-hour Day-Night cycle ............................................210 
Figure 4b: Viability of conidia of three Isolates of Aspergillus niger formed on              
ground Pale-brown (  ) and Pink (  ) shallot bulbs and stored at different         
Relative Humidities at 32±2oC under 12-hour Day-Night cycle. ...........................................211 
Figure 4c: Viability of conidia of three Isolates of Aspergillus niger formed on               
ground Pale-brown (  ) and Pink (  ) shallot bulbs and stored at different         
Relative Humidities at 32±2oC under 12-hour Day-Night cycle. ...........................................212 
 
 
xxiii 
 
LIST OF PLATES 
Page 
Plate 1. Photograph showing first stage of preparation of raised shallot plots at Anloga……..24 
Plate 2. Photograph of a prepared plot at Anloga showing manure heaps                                   
to be incorporated into the sandy soil…….…………………………………….……25 
Plate 3. Photograph showing pure stands of 30-day old (TOP) and 60-day old  
             (BOTTOM) plants of the Pink cultivar of shallot (Allium ascalonicum)...................26. 
 Plate 4. Photograph showing 60-day Pink cultivar of shallot plants (Allium             
              ascalonicum )inter-planted with pepper plants (Capsicum annum) at Anloga……..27 
Plate 5. Photographs of bulbs of Pale- brown (TOP) and Pink ( BOTTOM) cultivars                
             of shallot ( Allium ascalonicum)………………………………………………….….62 
Plate 6. Photographs of 30-day old plants of the Pale-brown (TOP) and Pink            
            (BOTTOM) cultivars of shallot (Allium ascalonicum)……………………………….63 
Plate 7. Photographs of plots of 60-day old plants of shallot ( Allium ascalonicum)  
             distinguishing between the Pale-brown cultivar (TOP) and the Pink cultivar  
             (BOTTOM)……………………………………………………………………….….64 
Plate 8. Photograph showing colonies of Aspergillus niger on the inner and outer scale      
             leaves of  naturally infected shallot bulb (Allium ascalonicum) of Pale-brown  
             cultivar on sale at the Agbogbloshie market….…………………………...………....76 
Plate 9:  Photographs of cultures of Aspergillus niger Isolates 1, 2, 3, 4 and 5……………....81 
 
 
 
xxiv 
 
Page 
Plate 10. Photographs showing wound-inoculated bulbs of the Pale-brown (TOP) and  
               Pink (BOTTOM) cultivars of shallot ( Allium ascalonicum) and incubated at 
               different relative Humidities ( From left: 100, 90. 80, 70, 60 and 50 %     
               R.H.). ……………………………………………………………………………..131 
Plate 11. Photograph showing fungi isolated from freshly prepared soil of the shallot 
                 farm at  Anloga using Sabouraud  Agar………..………………………………...158 
 Plate 12. Photograph showing fungi isolated from the bulb surface of Pale-brown  
                cultivar of shallot ( Allium  ascalonicum) using Sabouraud Agar ( BOTTOM)  
                and Potato Dextrose Agar(TOP)……………………………………..…………..169. 
Plate 13. Photograph showing fungi isolated from the bulb surface of Pink cultivar of  
               shallot   ( Allium  ascalonicum) using Sabouraud Agar ( BOTTOM) and 
               Potato Dextrose Agar (TOP)…………………………………….………………...170 
 
 
xxv 
 
LIST OF APPENDICES 
                                                                                                                                               Page 
 
APPENDIX A: Lengths, diameter and weights of bulbs of shallot ( Allium ascalonicum)....... 208 
APPENDIX B: Percentage of conidia of Aspergillus niger Isolate 1 formed on macerated               
bulbs of the two cultivars of Allium ascalonicum viable after storage for 10              
days at different relative humidities at 32±2oC under 12-hour day-night                  
cycle. (Percentage germination based on 400-500 observed conidia). ......................... 263 
APPENDIX C: Percentage of conidia of Aspergillus niger Isolate 1 formed on macerated             
bulbs of the two cultivars of Allium ascalonicum viable after storage for 20              
days at different relative humidities at 32±2oC under 12-hour day-night                  
cycle. (Percentage germination based on 400-500 observed conidia). ......................... 265 
APPENDIX D: Percentage of conidia of Aspergillus niger Isolate 1 formed on macerated              
bulbs of the two cultivars of Allium ascalonicum viable after storage for 40              
days at different relative humidities at 32±2oC under 12-hour day-night 
cycle.(Percentage germination based on 400-500 observed conidia). .......................... 266 
APPENDIX E: Percentage of conidia of Aspergillus niger Isolate 1 formed on macerated              
bulbs of the two cultivars of Allium ascalonicum viable after storage for 80              
days at different relative humidities at 32±2oC under 12-hour day-night 
cycle.(Percentage germination based on 400-500 observed conidia). .......................... 267 
APPENDIX F: Percentage of conidia of Aspergillus niger Isolate 1 formed on macerated             
bulbs of the two cultivars of Allium ascalonicum viable after storage for 120            
days at different relative humidities at 32±2oC under 12-hour day-night 
cycle.(Percentage germination based on 400-500 observed conidia). .......................... 268 
APPENDIX G: Percentage of conidia of Aspergillus niger Isolate 1 formed on macerated             
bulbs of the two cultivars of Allium ascalonicum viable after storage for 160            
days at different relative humidities at 32±2oC under 12-hour day-night                   
cycle . (Percentage germination based on 400-500 observed conidia). ........................ 269 
APPENDIX H: Percentage of conidia of Aspergillus niger Isolate 1 formed on macerated             
bulbs of the two cultivars of Allium ascalonicum viable after storage for 200            
xxvi 
 
days at different relative humidities at 32±2oC under 12-hour day-night                  
cycle. (Percentage germination based on 400-500 observed conidia). ......................... 270 
APPENDIX I: Percentage of conidia of Aspergillus niger Isolate 3 formed on macerated              
bulbs of the two cultivars of Allium ascalonicum viable after storage for 10              
days at different relative humidities at 32±2oC under 12-hour day-night                  
cycle. (Percentage germination bas based on 400-500 observed conidia).................... 271 
 APPENDIX J: Percentage of conidia of Aspergillus niger Isolate 3 formed on macerated             
bulbs of the two cultivars of Allium ascalonicum viable after storage for 20              
days at different relative humidities at 32±2oC under 12-hour day-night                  
cycle. (Percentage germination based on 400-500 observed conidia). ......................... 272 
APPENDIX K: Percentage of conidia of Aspergillus niger Isolate 3 formed on macerated             
bulbs of the two cultivars of Allium ascalonicum viable after storage for 40              
days at different relative humidities at  32±2oC under 12-hour day-night                  
cycle (Percentage germination bas based on 400-500 observed conidia)..................... 273 
APPENDIX L: Percentage of conidia of Aspergillus niger Isolate 3 formed on macerated             
bulbs of the two cultivars of Allium ascalonicum viable after storage for 80              
days at different relative humidities at 32±2oC under 12-hour day-night                  
cycle. ( Percentage germination based on 400-500 observed conidia). ........................ 274 
APPENDIX M: Percentage of conidia of Aspergillus niger Isolate 3 formed on macerated             
bulbs of the two cultivars of Allium ascalonicum viable after storage for 120            
days at different relative humidities at 32±2oC under 12-hour day-night                  
cycle. (Percentage germination based on 400-500 observed conidia). ......................... 275 
APPENDIX N: Percentage of conidia of Aspergillus niger isolate 3 formed on macerated             
bulbs of the two cultivars of Allium ascalonicum viable after storage for 160            
days at different relative humidities at 32±2oC under 12-hour day-night                   
cycle .( Percentage germination based on 400-500 observed conidia). ........................ 276 
APPENDIX O: Percentage of conidia of Aspergillus niger Isolate 3 formed on macerated             
bulbs of two cultivars of Allium ascalonicum viable after storage for 200                  
days at different relative humidities at 32±2oC under 12-hour day-night                   
cycle (Percentage germination based on 400-500 observed conidia). .......................... 277 
xxvii 
 
APPENDIX P: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated             
bulbs of the two cultivars of Allium ascalonicum viable after storage for 10              
days at different relative humidities at 32±2oC under 12-hour day-night                  
cycle. (Percentage germination based on 400-500 observed conidia). ......................... 278 
APPENDIX Q: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated             
bulbs of the two cultivars of Allium ascalonicum viable after storage for 20              
days at different relative humidities at 32±2oC under 12-hour day-night                   
cycle. (Percentage germination based on 400-500 observed conidia). ......................... 279 
APPENDIX R: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated              
bulbs of the two cultivars of Allium ascalonicum viable after storage for 40               
days at different relative humidities at 32±2oC under 12-hour day-night                  
cycle. (Percentage germination based on 400-500 observed conidia). ......................... 280 
APPENDIX S: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated              
bulbs of the two cultivars of Allium ascalonicum viable after storage for 80              
days at different relative humidities at 32±2oC under 12-hour day-night                   
cycle. (Percentage germination based on 400-500 observed conidia). ......................... 281 
APPENDIX T: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated            
bulbs of the two cultivars of Allium ascalonicum viable after storage for 120            
days at different relative humidities at 32±2oC under 12-hour day-night                  
cycle. (Percentage germination based on 400-500 observed conidia). ....................... 2822 
APPENDIX U: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated           
bulbs of the two cultivars of Allium ascalonicum viable after storage for 160            
days at different relative humidities at 32±2oC under 12-hour day-night                  
cycle. (Percentage germination based on 400-500 observed conidia). ......................... 283 
APPENDIX V: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated           
bulbs of the two cultivars of Allium ascalonicum viable after storage for 200            
days at different relative humidities at 32±2oC under 12-hour day-night                  
cycle. (Percentage germination based on 400-500 observed conidia). ......................... 284 
 
xxviii 
 
ABSTRACT 
 The relationship between five randomly selected isolates of Aspergillus niger from naturally 
infected bulbs of shallot and two shallot cultivars (Allium ascalonicum L.) was investigated. 
The two cultivars were “Pale-brown” and “Pink”- named for the colours of their scale leaves. 
A. niger causes bulb rot of shallots which have important uses in food and traditional medicine 
in Ghana The relationship showed by the cultivars were similar in some aspects and different 
in many other aspects, indicating physiological differences between the cultivars. 
In the field, the Pale-brown plants were bigger (mean length of 3.62±0.06 cm and mean width 
of 2.71±0.05 cm) with greener and more lush leaves. Chemical analysis of the typically larger 
bulbs of Pale-brown cultivar showed higher mean concentrations of calcium (0.164%), 
magnesium (0.054%), nitrogen (1.456%), phosphorus (0.406%), potassium (1.142%) and 
sodium (0.179%). In comparison, the respective percentage values for the Pink cultivar were 
0.145, 0.046, 1.192, 0.367, 0.979 and 0.175. The mean length and the mean width of the Pink 
bulbs were also 3.26±0.08 cm and 2.08±0.08 cm respectively. 
Any nutrients secreted by the scale leaves and swollen leaf bases were not able to stimulate 
germination of conidia of A. niger in distilled water droplets placed on them. The conidia did 
not also germinate in very dilute (1:40) droplets of Potato Dextrose Broth. Higher 
concentrations of Potato Dextrose Broth, galactose, glucose and sucrose induced appreciable 
levels of conidium germination. When that happened, the different types of leaves of the bulbs 
showed differences in their influence on the conidia. Conidium germination was better on the 
scale leaves than on the swollen leaf bases while percentage germination on the corresponding 
organs was better and germ tubes were longer on the Pale-brown cultivar than on the Pink 
cultivar. 
The mycelium of A. niger, in addition, grew better in extract of bulbs of the Pale-brown cultivar 
and rotted bulbs of the wound-inoculated Pale-brown cultivar faster than the Pink bulbs. 
Conidiophores produced in various tests were larger on Pale-brown bulbs than on Pink bulbs. 
Conidia formed by A. niger growing on ground bulbs of the two cultivars showed some 
similarities and differences when they were stored in atmospheres of different relative 
humidities, from zero to 100% R.H., provided and maintained with appropriate concentrations 
of sulphuric acid. The two sets of conidia lived longest at 60 to 100% R.H. and died quickest at 
zero to 50% R.H. At the best survival humidity of 100% R.H., 52.1, 51.2 and 35.8% of  conidia 
of A. niger Isolates 1, 3 and 5 respectively  formed on the Pale-brown cultivar medium were 
still alive on the 200th day. The corresponding values for conidia formed on Pink cultivar 
medium were 43.8, 42.3 and 32.3 per cent, respectively. At each percentage viability 
assessment, conidia formed on the Pale-brown cultivar medium showed higher survival 
potential. There was positive correlation between spore longevity and the length of the germ-
tubes. 
The two shallot cultivars clearly stimulated fungi in the neighbourhood of their bulbs and roots 
over 60 days. Studies using the soil dilution plate method identified Aspergillus as the dominant 
genus in the phyllosphere, rhizosphere and non-rhizosphere soils. The remaining genera were 
Fusarium, Penicillium and Rhizopus. The Aspergilli were A. flavus, A. niger, and A. terreus. A. 
niger was the most abundant, constituting more than half the fungus population. Application of 
xxix 
 
Urea and Potassium fertilizers separately to the soil did not alter the pattern of the influence of 
the shallot cultivars on the fungal populations. The pattern was also the same in different farm 
soils under continuous cultivation with different crops. 
Because of the restraining effects of various sorts of the Pink cultivar on Aspergillus niger, it 
was considered to be the better cultivar for expanded shallot cultivation in Ghana and suitable 
for breeding improved stocks to reduce the threat of A. niger to the shallot industry. 
 
1 
 
I INTRODUCTION AND LITERATURE REVIEW 
Shallots (Allium ascalonicum) and onions (Allium cepa) are among the most important vegetables 
in Ghana. Shallots were introduced   in Ghana in 1800 from Anecho in neighbouring Republic of 
Togo and cultivated in the country for more than a century before the introduction of onion in 
1930 (Adomako, 1959). Shallots, anyway, are preferred to the onions because of their shorter 
growth cycle, better tolerance to disease and drought stresses, longer shelf life and because of 
their distinct flavour (Brewster, 1990; Currah and Proctor, 1990; Grubben, 1994; Pathak, 1994; 
Sumiati, 1994; Abbey et al., 1998). Furthermore, shallot appears to contain more flavanoids and 
phenols than other alliaceous plants (Yang et al., 1955). Finally shallot plants produce clusters of 
several bulb splits that number from two to twelve pieces, with an ideal marketable size compared 
to the single bulb produced by onion plants. 
 
Shallots and onions are used in many households and almost daily. They are grown primarily 
for the bulbs although the green leaves may also be consumed. They can be used raw in salads 
and more importantly serve as essential ingredients in various dishes. In certain regions of West 
Africa, the green leaves are ground, moulded into balls and used for seasoning dishes. The bulbs 
of shallots and onions have antimicrobial properties which can reduce contamination by 
bacteria, protozoa or helminthes in salads. 
 
In traditional medicine shallot is used to treat boils, wounds, coughs, bronchitis, asthma, gastro-
intestinal disorders and headache. The juice of the bulbs is rubbed on the body of a person with 
fever. Also, in some parts of Ghana, they are used as antidote for snake bite. It is known that 
2 
 
the scent of onions and shallots repels snakes and they are planted near homes to achieve that 
purpose  (Sinnadurai and Abu, 1977). 
 
Both shallot and onion bulbs contain sulphosides, the most important being cystein sulphoside. 
Upon bruising the bulb, these are degraded by allinase to release pyruvic acid and alkyl-
thiosulphinates which turn into sulphides and disulphides. The volatile allinase product is the 
well known lachrymatory factor in onions and shallots. (Nabos, 1971). The degradation of 
sulphoxides can be influenced by external conditions such as boiling and frying. The combined 
taste of sulphides and caramelized sugars give flavours to fried onions.  
 
The shallot bulb, as reported by Soitout (1969) contains 81.8% moisture. A 100g dry weight of 
the bulb contains 67cal. total energy, 9g total protein; 0.3g fat; 15.4g total carbohydrates; 0.7g 
fibre; 0.6g ash, 36mg calcium; 45mg phosphorus; 0.8mg iron; 12mg sodium; 334mg sulphur; 
5I.U. vitamin A; 0.04mg thiamine; 0.02mg riboflavin; 0.3mg niacin and 2mg ascorbic acid. The 
corresponding data given by Soitout (1969) for onion bulbs were 88.0% water and 31cal. 
energy;   1. 5g total protein; 0.6g fat; 7.2 g total sugar; 0.g carbohydrates; 0.4 mg thiamine; 
0.02mg riboflavin; 0.1mg niacin; 7mg vitamin C; 30mg calcium; 0.5g iron; 16.5mg 
magnesium; 35mg phosphorus and 7mg sodium in a 100g of edible portion of the bulb. 
 
The high water content of the bulbs certainly affects the shelf life of onions and shallots as well 
as other vegetable crops as growth of bacteria and fungi is greatly encouraged. Bulb rot in 
3 
 
storage is largely caused by Erwinia sp (Norman, 1992) and Pseudomonas sp. (Clerk, 2012. 
Personal communication) and the fungi Aspergillus niger and Fusarium solani (Clerk, 1974). 
 
Stomata are a significant target for plant pathogenic bacteria. The motile Pseudomonas and 
Erwinia cells enter shallot bulbs by the stomata and sub-stomatal chambers are sites of initial 
proliferation. There is evidence that bacteria generally also cause infection by penetrating 
wounds of even minute proportions. Indeed, Crosse et al. (1972) demonstrated that exposure of 
the vascular system of shoots of apple (Malus silvestris Mill.) apex insured xylem penetration 
by Erwinia amylovora and subsequent systemic infection. Entry of bacteria through leaf traces 
provide the rationale for reports of dormant bud infections (e.g. Baldwin and Goodman, 1963; 
Dowler and Peterson, 1967; Dueck and Quame, 1973).  
 
Upon entry into vascular plants, extensive multiplication by bacteria occurs either 
intercellularly or in the xylem. Fox et al. (1971) and Fox (1972) recorded penetration of wounds 
and subsequent spread through electron micrographs of potato tuber tissues infected by Erwinia 
carotova var atroseptica. Spread was primarily between storage parenchyma cells and restricted 
infection to xylem tissues. Wallis et al. (1973) also observed the same phenomenon in their 
ultra-structural histopathology of cabbage ( Brassica olereceae var capitata) leaf infected with 
Xanthomonas campestris. Generally bacteria do not multiply in phloem tissue, probably 
according to Goodman    (1976), due to high soluble concentration of the phloem which is 
inhibitory to bacterial replication. 
4 
 
The dry rot of Erwinia infection of shallots is visualized as the result of limited degradation of 
the tissues and desiccation of the unmacerated dead tissues. The genus Pseudomonas, on the 
other hand, contains species with remarkably diverse biochemical capacities that ensures more 
intense degradation. The ability to carry out some of these activities depends on plasmids 
(Nester et al., 1975). 
The symptoms of the respective shallot rots caused by Fusarium solani and Aspergillus niger 
in storage are very distinctive. Shallot bulbs infected by F. solani are covered by a fluffy dirty 
white mycelium of the fungus whereas A. niger produces flat mycelium bearing masses of black 
conidia, for which it is given the name, ‘black mould’. Black mould is by far the more common 
infection. In both, colonization of the bulbs proceeds rapidly and infection spreads quickly 
among bulbs in storage even though the conidia in the two fungi which constitute the major 
dispersal agents possess different physiological characteristics. Whereas the macro-conidia and 
microconidia of F.solani germinate very well, 98.4 and 90.5 per cent, respectively, in distilled 
water in six hours at the optimum temperature (28 oC) (Halm, 1971), A. niger conidia do not 
germinate in distilled water (Kesse, 1995). In spite of this, A. niger is cosmopolitan flourishing 
on all sorts of substrates. It is common in soil ( Khan et al., 2001; Thom and Raper, 1945), 
parasitic on plants of 37 genera ( Farr et al, 1989), contaminant of harvested fruits and 
vegetables ( Sneh et al., 1991) and all sorts of plant products ( Pitt and Hopking, 1997; Peronne 
et al., 2001; Perfect et al., 2009) , probably because  conidial germination is stimulated by very 
low concentrations of sugars and amino acids ( Kesse, 1995 ). 
 
A. niger is equally notable in industry as a very useful microorganism in industry because of its 
ability to carry out various metabolic processes. It is the source of many enzymes, for example, 
5 
 
amylase, amyloglucosidase, cellulases, glucoamylase, invertase, lactase pectinases, proteases 
etc. (Bennett, 1985; Ward, 1989). Besides the production of useful enzymes certain strains of 
A. niger are used in industry in the preparation of citric acid and gluconic acid used in the food 
industry. The most important organic acid produced by fungi is citric acid. Both have been 
assessed as acceptable for daily intake by the World Health Organization (WHO) and Food and 
Drug Administration (FDA) of the United States of America (USA) (Schuster et al, 2002). The 
annual production of citric acid by fermentation of A. niger is presently 350,000 tonnes (Ward, 
1989). About 70% of it is used in the food industry while 10% is used to produce cosmetics and 
pharmaceutical products (Bigellis, 1991).  Another value of A. niger in the biotechnology 
industry is the production of magnetic isotope-containing variants of biological macro-
molecules for Nuclear Magnetic Resonance (NMR) analysis (Staiano et al., 2005). 
The rotting of bulbs by A. niger is accompanied by infiltration of the tissues by the mycotoxins, 
fuminisin B2 and ochratoxins, synthesized by the fungus (Abarca et al., 1994; Noonimabc et 
al., 2009; Edwin et al., 2010). Such contaminated bulbs are unsuitable for consumption (May 
and Adams, 1997; Schuster et al., 2002; Noonimabe et al., 2009; Al-Abdalall, 2009). 
Fuminisins and ochratoxins may cause immunotoxicity, carcinogenicity and hepatoxicity 
(Louthrenoo et al., 1990). 
Oxalic acids, kojic acids and cyclic pentapeptides are another set of products of A. niger of 
moderate to high acute toxicity. Oxalic acid causes pulmonary oxalosis ( Nakagawa et al., 
1999). 
 
6 
 
The third category of A. niger-related hazard to health of humankind is the result of direct 
contact with the human body. There are two major cases of great concern to persons who, by 
vocation, are continually associated with shallots. 
Superficial contact allergy to fungal spores depends on prior sensitivation to specific antigen. 
Later exposure to larger quantities of the same spores may give rise to skin irritation of the type 
observed in a farmer who had smut, Ustilago maydis (Preininger, 1937-1938) allergy. Similarly, 
one of the characteristics lesions seen in the ‘ maladie des Cannes de Provence” occurs on the 
shoulders of the workmen who carry bundles of the reeds affected by fungi especially Papularia 
sphaerosperma (Mandoul et al., 1954) on their shoulders. Also, Alternaria tenuis is thought to 
cause allergic eczema in dogs and horses ( Allen, 1945). Louthrenoo et al. (1990) provided 
evidence of such relationship between Aspergillus  niger and humankind. 
 
All types of fungal spores and the associated structures, such as conidiophores, which achieve 
the air-borne state can theoretically be inhaled. A. niger is naturally included. The range of air-
borne spore concentration is vast. The concentration depends on the fungus species, time of the 
day and season of the year. Even the concentration of outside and air spora of enclosed area 
differ considerably. Aspergillus spores, for instance, occur in much smaller numbers in the 
outside air and probably rarely exceed 500 per m3 but within a farm building following the 
shaking of the mouldy hay, Lacey and Lacey (1964) found up to 21 million per m3 
 
No comparative data are yet available for conidia of A. niger in the atmosphere outside and 
inside market stalls in Ghana handling shallots and onions. A significantly high concentration 
7 
 
of spores in the air of the buildings will consequently increase intake by those worker there, 
even if exposure times are shorter than outside. Definitive  estimation  of the actual numbers in 
future studies is essential considering the fact that A. niger conidia are encountered  among the 
important fungal spores involved in respiratory allergy in humankind ( Austwick, 1966; 
Edwards and Al Zubaidy, 1977). 
Whatever the circumstance, persons persistently handling A. niger-infected shallots bulbs will 
be exposed to another medical hazard: otomycosis. It is a subacute or chronic superficial fungal 
infection of the external auditory canal and auricle. A. niger is the most common cause, with 
occasional cause by Aspergillus flavus and Aspergillus fumigatus (Berjak and Arya 1970; Than 
et al., 1980; Mugliston et al., 1985; Poulose et al., 1989; Lucente, 1993; Yehia et al., 1990). 
The disease is worldwide in distribution. Otomycosis is more prevalent in warm, humid 
climates, particularly in the rainy season as opposed to dry season. The most common form of 
the disease is termed ‘ otitis externa’, first described by Chandler ( 1968). By the estimation of 
Osguthorpe and Nielson ( 2006) chronic otitis externa affects 3.0-5.0 per cent of the population. 
 
Cases of otitis externa described in the reports in the preceding paragraph mention a number of 
symptoms including redness and swelling of the external ear canal, ear pain always 
accompanied with a sensation of irritation and itching, a feeling of pressure and fullness inside 
the ear, a thin and watery discharge from the ear, swollen and sore lymph nodes in the throat 
and above all the presence of growth of projecting Aspergillus coniodiophore from the walls of 
the ear canal. 
 
8 
 
Above all, an important consequence of rotting of shallot bulbs in storage is the loss of planting 
material. For, unlike onions which flower and produce viable seeds for propagation, shallot 
genotypes rarely flower in tropical regions. Many shallot genotypes are, therefore, only clonally 
propagated (Jones and Mann, 1963; Currah and Proctor, 1990; Messiaen et al., 1994). 
 
There is no information on the magnitude of post-harvest losses of shallot bulbs in Ghana. 
Hayden (1989) estimated a 30 per cent post-harvest loss of onions (Allium cepa) in Sudan, and 
Fedaku (1989) recorded the same level of loss (25-35 per cent) in Ethiopia, but for all 
horticultural crops in general. It is probable that the average yearly level shallot production of 
34,262.8 tonnes, calculated from the productions data of the Ministry of Agriculture of the 
Government of Ghana reproduced in Table 1, might suffer approximately the same level of 
post-harvest loss. 
 
 
 
 
 
 
 
 
 
 
 
9 
 
 
 
 
 
 
 
Table 1: Production of shallot (Allium ascalonicum) in Ghana in 2005-2009 (Library record: 
Ministry of Agriculture) 
 
                         Year           Area of Cultivation (Hectare)          Yield (Metric Tonnes) 
 
 
2002005                   4,000        30,000 
2002006        4,300        30,014 
2002007        4,800        34,000 
2002008        4,900        39,000 
2022009        4,950        40,000 
 
 
 
 
 
 
10 
 
There are two sets of microflora of stored plant products: field microflora and storage 
microflora. The field species appear on the products during development, maturation and before 
harvest. The storage species are the post-harvest invaders together with the viable field species 
transferred from the field into storage. 
 
Field fungi of Red Creole cultivar of onion (Allium cepa) included species of Aspergillus, 
Cladosporium, Fusarium and Rhizopus with Aspergillus niger constituting 65 per cent of fungal 
isolates of the dry season crop (January – March, 1989) and 74 per cent of the rainy season crop 
(June – August, 1989) (Teye, 1994). Most of the species survived many weeks of storage, 
thereby transformed into storage fungi as well. Penicillium cyclopium was the only new 
colonizer during storage of the bulbs worthy of note. In this instance, the pre-harvest and post-
harvest bulbs provided physiologically similar substrates which supported practically the same 
fungal communities. 
 
In circumstances where the substrates and the ambient environmental conditions differ, clearly 
distinct fungal communities with different component species would develop. For example, the 
field fungi of maize (Zea mays) grains include species of Alternaria, Cladosporium, Fusarium  
and Helminthosporium which invade the maturing grains when the moisture content is high and 
they are metabolically active (Christensen, 1957). These fungi require moisture level in 
equilibrium with a relative humidity of 90% or more. The storage fungi arrive after harvest and 
mostly xerophilic as the grains, by then, contain very little water. The Aspergillus species are 
the most important storage fungi of maize grains (Mislivec and Tuite, 1970). They are 
xerophilic and they grow at low water activity (0.70 – 0.75 aw) (Magan and Lacey, 1987). 
11 
 
It has long been known that numerous diffusible compounds of low molecular weight leak from 
actively growing plants and their release is not metabolically mediated. Leakage is the loss of 
compounds along electro-chemical potential gradients by simple diffusion. On the other hand, 
by an active metabolic process, poorly diffusible, high molecular weight compounds are also 
transported across membranes. These compounds are energy-carrying metabolites which act as 
an ecologically favourable matrix for the evolution of complex biological communities. 
 
In soil, roots and other subterranean structures become covered by a slime or mucilage. Root 
mucilages can be classified into two types. The first is secreted by outer root cap cells as 
droplets containing sloughed cells which cover the root tips of axenically grown roots (Miki et 
al., 1980; Rougier, 1981). The second is a firm, mucilagenous layer overlying the epidermal 
cells and coating the root hairs (Oaddes, 1978). 
 
Miki et al. (1980) recommended the use of the terms root cap mucilage’ and ‘epidermal 
mucilage’ to designate the two types of mucilage on axenically grown roots. The term 
‘mucigel’, first introduced by Jenny and Grossenabcher (1963) to describe the mucilagenous 
material at the surface of roots grown on non-sterile soils, which could be applied to both types 
of mucilage detected on axenic roots, and thereby to the mucilagenous material of soil-grown 
developing shallot bulbs. The mucilages contain in addition, slime of bacterial cells on the plant 
surfaces as well as products of decomposing root hairs and exfoliated epidermal cells. 
 
The mucigel occupies a key position at the plant structure-soil interface and may well be of 
significance to plants, soils and the soil microflora. The following are the most important 
12 
 
contributions to root growth proposed: (a) root hairs and their associated mucigel may help 
anchoring of the young seedling in the soil (b) the adsorption of fine clay particles to mucigel 
constituents in the first in the formation of stable micro aggregates around plants roots 
(Turchenek and Oades, 1976): (c) reduction of friction between the growing roots tip and the 
soil (Oades, 1978: Rougier, 1981): (d) protection of root from desiccation (Greenland, 1979), 
toxic ions (Horst et al., 1982) or metal injury (Barlow, 1975; Drew, 1979); (e) improvement of 
the root-soil contact facilitates water and nutrient movement of the dry soil or water-stressed 
plants, there is an increased production of mucigel by the roots which results in a cylinder of 
soil tightly adhering to the roots maintaining a hydraulic conductivity between root and soil 
(Mambiar, 1976; Martin, 1977; Drew, 1979; Foster, 1982); (f) mucilage adsorps ions and exerts 
some selectivity on the uptate of ions (Rovira  et al., 1983): and (g) acid phosphatase and 
esterase in mucigel (Felipe et al., 1979) hydrolyze organic phosphate esters in the soil into 
inorganic phosphate which could then be assimilated by plants. 
 
The acid phosphatase and esterases and the vast range of organic compounds including amino 
acids, auxins, carbohydrates, enzymes, flavones, growth factors, nucleic acid derivatives, 
organic acids, phenol derivatives and vitamins and the various microorganisms they influence 
have been extensively reviewed (e.g. Oades, 1978; Foster, 1982; Rougier and Chaboud 1985). 
The composition of mucigel is generally specific for a plant species and even cultivars of a 
species. Consequently, host species or cultivars have recognizable characteristic microflora. 
 
Whether or not it is utilized as a food source by microorganisms, mucigel is a favourable 
ecological microhabitat which promotes their rapid growth and special activities. For example, 
13 
 
mucigel compounds play a role in the recognition of maize roots by Phytophthora cinnamon, 
zoospores (Hinch and Clarke, 1980; Irving and Grant, 1984). Umali-garcia et al. (1980) 
considered that mucilage may be helpful for the nitrogen- fixing bacterium Azospirillum in 
regulating transport of oxygen originating from the soil; the process of formation of nodules by 
legume roots is triggered off by vitamin B, in the roots exudate which stimulates the growth of 
the Rhizobium population close to the root; the interaction can only proceed when the 
Rhizobium cells covert tryptophan in the root exudate to Indole Acetic Acid (IAA), and the 
bacterial slime polysaccharide stimulates the production of the enzymes polygalacturonase 
(PG) by the legume root cells which then finally catalyze the breaking of the linkages in the 
cellulose microfobrils at the apex of the root hair leading to the development of the infection 
thread. Mucigel may represent a favourable site for the establishment and development of 
mycorrhizae (Reid and Bowen,1979), and the mucigel may suppress or eliminate activities of 
soil pathogens, first by being inhibitory to the pathogens or secondly by either parasitism, 
competition or antibiosis by beneficial mucigel species. 
An exhaustive search of the pertinent literature failed to reveal any information on pathogenesis 
of A. niger on shallot bulbs in storage. Teye ( 1994), however, identified A. niger in her studies 
as a formidable storage fungus of bulbs of the Red Creole and Texas Grano cultivars of onion 
( Allium cepa) in Ghana. She reported that bulbs of the onions still retained viable A. niger 
colonies after they had been cured for 30 days, then irradiated with 0.5 Gy gamma rays and 
then stored at room temperature for 90 days. 
 
Woldestadik (2003) noted marked differences in the extent of rotting of three cultivars of 
shallot, DZ- Sht-78, DZ-Sht-91 and Fedis in Ethiopia. The rotting affected 44 percent of Fedis 
14 
 
bulbs and about 26 percent of DZ-Sht-91 bulbs. That of DZ-Sht-78 lay about median between 
these two extremes. The report did not, however, specify the cause of rotting. 
Resistance or susceptibility of onion and shallot bulbs to post-harvest fungal contaminants may 
be determined by chemical antimicrobial defense mechanisms. For example, Smalley and 
Hansen (1962) observed that growth of Penicillium corynbiferum, which attacks garlic (Allium 
sativum L.) was less inhibited by garlic extracts than was the growth of 17 species of Penicillium 
from other hosts. 
 
As has been mentioned earlier at the beginning of this chapter, the compound alliin ( S-allyl-L-
cysteine-sulfoxide) is converted  by the enzyme allin-lyase to allicin ( Diallyl-disulfide-oxide). 
Eventually, allicin is converted to diallyl- disulphide in plants of the genus Allium. The role of 
allicin and related compounds in antimicrobial defence mechanism in shallot remains to be 
determined. 
 
The classical case of pre-formed inhibitory compounds emanating from the leaves of high 
plants and preventing the germination of potential parasites is that of the onion metabolites 
protocatechuic acid and catechol which confer resistance to smudge caused by Colletotrichum 
circinans in yellow-scaled onions by virtue of the sporostatic activity of these compounds ( 
Walker and Stahmann, 1965). In this case not only germination is inhibited but the substance 
caused lysis of the spores, particularly if any of them do germinate. Shallots have not been 
studied for these compounds. 
15 
 
Aspergillus niger has been considered, on the one hand, as a strict saprophyte (Commonwealth 
Institute, 1966; Farr et al., 1989) and, on the other, a serious facultative parasite . Indeed, 
Lorbeer et al. (2000) gave a detailed account of their findings on the pathogenesis of A. niger 
attacking onion plants (Allium cepa). They inoculated the seeds of onion with A. niger conidia, 
sowed them in sterile soil and followed the course of infection of the various organs of the 
plants. The hyphae invaded the roots, the basal disc and the leaves. In a subsequent experiment, 
flowers of different ages were inoculated with A. niger conidia. The hyphae were eventually 
detected in all parts of the flowers including the ovules and developing seeds. 
Saprophytes and facultative parasites employ the same enzyme systems to dismantle the various 
plant cell constituents.  Obligate parasites (Van Sumere et al., 1957) and mycorrhizas 
(Williamson and Hadley, 1970) also have the ability to produce cell wall degrading enzymes. 
The production of their enzymes, however, must be under adequate control lest the invaded 
tissues become macerated and the host cells killed. 
 
Based on the concept of primary cell wall structure, pectic components of unaltered cell walls 
will be attacked first rendering other wall polymers susceptible to enzymatic hydrolysis. The 
pectic enzymes have been grouped according to the following criteria: (a) the mechanism by 
which the α-1, 4 glycosidic bond is split, (that is, hydrolytic or lytic) (b) enzyme specificity for 
a substrate, that is, pectin or pectic acid, and (c) position in the pectic chain at which cleavage 
occurs, that is, random or terminal point of attack (Bateman and Millar, 1966; Rombouts and 
Pilnik, 1972). The enzymes specific for pectin are referred to as pectinmethylgalacturonases. In 
contrast, the polygalacturonases are specific to pectic acid. If the enzyme attacks the terminals 
of the pectic chains and releases only monomeric products, it is termed an 
16 
 
exopolygalacturonase. If the attack is random and oligomers are released, the enzyme would be 
called an endopolygalacturonase. Different activities of the enzymes release different reaction 
products. 
 
The multi- enzyme hypothesis of enzymatic decomposition of cellulose recognizes a C1, Cx and 
β-glucosidase (Reese, 1963). Cellulose is an insoluble crystalline substance in its native form. 
Microorganisms capable of utilizing native cellulose are believed to produce an enzyme 
designated C1 which acts on native cellulose by destroying its crystalline structure and exposing 
the glucose chains to β-1, 4 endoglucanases, termed Cx enzymes. The Cx enzymes degrade the 
glucan chains to cellobiose. Conversion of cellulose to glucose also requires a cellobiase or β- 
glucosidase (King and Vessal, 1969). 
Similarly, there are specific enzymes which break down other cell components in herbaceous 
plants like shallots. Glucanase, oxylanase, mannonase, mannosidase, glucosidase which 
hydrolyze the hemicellulose fraction of the plant cell wall. Phospholipases hydrolyze the 
acylester and phosphate ester bonds of phospholipids of cell membranes ( Gatt and Barenholz, 
1973). While proteolytic enzymes hydrolyze peptides in proteins. Primary classification of 
proteolytic enzymes is based on the catalytic mechanism of the active site (Smith, 1960; 
Matsubara and Feder, 1970). 
 
 Invading pathogenic fungi of aerial parts of plants first come in contact with the cuticle 
covering of the epidermal cells and penetrate by enzymatic dissolution. Cuticle also covers the 
17 
 
cells in the substomatal cavity. In addition, cuticular layer is present in the palisade and spongy 
mesophyll cells ( Sitholey, 1971). 
Cuticle has a framework of cutin, a polymer of crosswise esterified mono-, di-, and, trihydroxy 
fatty acids linked by peroxide bridges ( carboxycutin). Other components are waxes, cellulose 
and pectin ( Van den Ende and Linskens, 1974). 
 
Cutin is attacked by a number of cutinolytic enzymes. The principal enzyme is cutin esterase 
which by hydrolytic cleavage of ester links delivers substrates for oxidizing enzymes. For 
instance, the product oleic acid is acted on by oleic oxidase, which in turn, produces substrates 
for linoleic oxidase. Accompanying the oxidizing enzymes is the peroxidase, carboxycutin 
peroxide, which catalyzes the release of oxygen from carboxycuttin by loosening the peroxide 
bridges (Van den Ende and Linskens, 1974). 
 
These enzymes make it possible for microorganisms like Spilocaea oleagina which live sub or 
intracuticularly to use the cuticle as a food source ( Ruinen, 1966) and decay of cutin and 
cuticular components by microorganisms in their environment ( de Vries et al., 1967). 
 
Aspergillus niger may damage the host plant by other means. It produces the phytotoxic 
compounds called malformins. The toxin cause curvatures and grotesque plant malformations 
( Curtis, 1958; Postlethwait and Curtis, 1959). Malformin is a polypeptide. There are two types 
of malformin- ‘old’ and ‘new’ malformins. ‘Old' malformin is a neutral cyclic pentapeptide ( 
18 
 
D-leucyl-L-iso-leucyl-D-cysteinyl-L-Valyl-d-cysteine anhydride) and the ‘new’ malformin 
contains an allo-isoleucine ( Narumo and Curtis, 1961; Takahashid and Curtis, 1961). Many 
plant species are not susceptible to malformin ( Curtis, 1961). 
Shallot plants take averagely ten weeks from the time of sprouting to reach maturity. By that 
time the green leaves have turned pale green or brown and lost much of their turgidity. The 
plants are pulled and laid out on the ground for some days to dry the leaves thoroughly. Because 
of the small size of the bulbs, they are separated into many lots of manageable sizes and the 
leaves of each lot plaited together into a “pigtail” by which the bunch is handled. Spread of A. 
niger among the bulbs in the bunch and, indeed, the store houses happens in two ways. One is 
by mycelial growth whereby the hyphae from an infected bulb reaches healthy contact bulbs. 
The second and faster process involves air transport of the essential dispersive conidia. 
 
The conidia are passively liberated by blow-off, conidia blown off their conidiophores by wind, 
and by shake-off, when the conidiophores are agitated. An important attribute of a spore from 
the point of dispersal is its retention of the power to germinate. The fact that the spore in the air 
can remain in suspension for a long time is of no significance if at the end of its journey it is 
incapable of germination. 
 
Very great differences have been found in the survival potential of different kinds of fungal 
spores.  The viability of all spores decrease with time and the rate of loss of vigour is dependent 
on the inherent characteristics (genome) of the spore, and upon environmental conditions, 
19 
 
especially, with light, temperature and humidity (Gottlieb, 1950; Hawker, 1950; Cochrane, 
1958). 
Generally, longevity increases with decreasing light intensity, and blue light appears to be the 
most active portion of the visible spectrum. Rotem and Aust (1991) found that exposure to 
sunlight affected the survival of conidia of Alternaria macrospora and Botrytis cinerea in the 
same way as ultraviolet light. One protection mechanism against injurious rays is offered by 
the pigments of some spores, and many correlations between colour of spores and resistance to 
light have been made. For example, Dillon-West and Halman (1932) showed among early 
mycological physiological studies that white and orange spores of Puccinia graminis tritici are 
more easily killed by ultraviolet light than are grey and red spores. 
 
Kesse (1995) studied the longevity of conidia of four Aspergillus species isolated from mouldy 
maize grains in atmospheres of zero to 100% R.H. Interestingly, she recorded four different % 
R.H viability relationships. Incidentally, the conidia of all the four species did not germinate at 
100% R.H. and the experimental conidia were stored at zero, 20, 40, 60, 80 and 100% R.H in 
light for 56 days. 
 
A. clavatus conidia survived longest at 0.0 and 100% R.H. and lost viability quickest at 20 – 
80% R.H. A. flavus conidia survived longest at 0.0 – 40% and 100% R.H. and lost viability 
quickest at 80% R.H. A. niger conidia survived longest at 100% R.H and viability decreased 
with decreasing percentage relative humidity, and, A. tamarii conidia survived longest at 0.0% 
R.H. and viability decreased with increasing percentage relative humidity. 
20 
 
The work of Kesse (1995) did not compare longevity in light and dark. The period of study of 
56 days was also rather short. For, many investigations have shown that the physiology of some 
fungal species dramatically changes beyond a certain age. Hahne (1925), for instance, indicated 
that spores of Tilletia levis and Tilletia tritici less than two years old germinated in dark or light 
whereas spores more than two years old could not germinate in dark. Later, Gassner and 
Niemann (1955) also reported the importance of age in the response of smut spores to light. 
Teyegaga and Clerk (1972), on the other hand, showed that in dark, Cercospora canescence 
conidia lost viability sooner at high relative humidities than at low, whereas conidia exposed to 
light survived longer at medium humidities close to 40 % R.H. than at high and low humidities. 
Survival of A.niger conidia should be re-examined. 
There are commonly cultivars of many crop plant species. In a particular species, the cultivars 
differ from each other in many ways, including form, function, and response to the environment 
and reaction to diseases. A good understanding of A. niger decay of shallot is needed in order 
to develop management strategies that would increase returns to producers. In an environment 
where A. niger is ubiquitous, any shallot cultivar that would exhibit an appreciable level of 
resistance to the fungus could be preferred to ensure enhanced productivity. It is also assumed 
that a resistant cultivar would not support the usual prolific sporulation associated with A. niger 
and would be a less capable source of the bulb infection units – the conidia. 
 
The thesis contains results of investigations carried out mainly on the: 
(a) growth, vegetative morphology and chemical composition of the bulbs of the two shallot 
cultivars, Pale brown and Pink, cultivated in Ghana; 
21 
 
(b) pathogenesis of five A. niger isolates, isolated from infected shallot bulbs; 
(c) relationship between the soil fungi and the shallot bulbs during development; 
(d) effects of Potassium and Urea fertilizers on the phyllosphere  and rhizosphere mycoflora 
of the shallot plants and the response of bulbs of 60 day old plants formed in the treated 
soils to inoculation with A. niger. 
(e) Influence of shallot cultivars on longevity of A. niger conidia. 
 
22 
 
II. MATERIALS AND GENERAL METHODS 
(i)  MATERIALS 
      a.  Shallot bulbs. 
            Pale-brown and Pink cultivars of shallot (Allium ascalonicum) cultivated in Ghana were 
used in this investigation. In Anloga district where the bulbs were periodically obtained, 
there are three shallot growing seasons in a year: a major season from April to August 
and two minor seasons, January to March and September to December. New harvested 
bulbs were purchased in bulk at the end of March, August and December in order to 
standardize the ages of the experimental bulbs. The bulbs were stored in a refrigerator 
at 4oC and used within 30 days of purchase.  
      b.  Isolates of Aspergillus niger 
            Five isolates of Aspergillus niger obtained from naturally infected shallot bulbs were 
used in this investigation. Bulbs of each cultivar showing signs of A. niger infection 
purchased  from the Agbogbloshie were placed in open Petri Dishes standing in 
desiccators with internal atmosphere of 100% R.H. provided by distilled water. The 
bulbs were examined after 5 days incubation at 32oC, and sub-cultures were made of as 
many A. niger colonies on the bulbs as possible on Potato Dextrose Agar. The isolates 
were sub-cultured on two further occasions to obtain pure cultures. 
           By numerous cultural tests, five isolates with clearly distinguishable characteristics as 
shown in EXPERIMENT E of the Results were selected for the various pathogenicity 
and conidial longevity tests. Stock cultures of the five isolates were maintained on PDA 
23 
 
in McCartney tubes at 4oC in the refrigerator. The stock cultures were sub-cultured 
fortnightly. 
 
      c.  Soil Sample. 
            Sandy loam soil samples with similar history were obtained from the shallot farms at 
Anloga were used as planting material where necessary. Shallots are grown on raised 
beds (Plate 1). Because of the annual flooding by the sea, the farmers neutralize the salty 
silt left by the floods by adding river sand and organic manure consisting mainly of 
poultry manure and cow dung (Plate 2). At an advance stage of growth, the shallot plants 
are inter-cropped with egg plants, pepper and tomato (Plates 3 and 4). Because of the 
dense cropping, the soil samples sometimes needed in large quantities, could be 
conveniently collected from freshly prepared plots before planting. 
 
 
 
 
 
 
 
 
24 
 
 
 
 
 
PLATE 1. Photograph showing first stage of preparation of raised shallot plots at Anloga 
 
 
25 
 
 
 
 
Plate 2. Photograph of a prepared plot at Anloga showing manure heaps to be incorporated 
into the sandy soil. 
26 
 
 
 
   x  1/10 
PLATE 3. Photograph showing pure stands of 30-day old (TOP) and 60-day old (BOTTOM)  
plants of the Pink cultivar of shallot ( Allium ascalonicum). 
 
27 
 
 
 
 
                                                                                                                                             X 2/5 
Plate 4. Photograph showing 60-day Pink cultivar of shallot plants (Allium ascalonicum) inter-
planted with pepper plants (Capsicum annum) at Anloga. 
 
 
28 
 
      d.  Grains of Maize, Seeds of Cowpea and Tubers of Cassava, Irish Potato and Sweet   
            Potato. 
            Sound grains of maize ( Zea mays L.), seeds of cowpea ( Vigna unguiculata L. (Walp.)  
and tubers of cassava (Manihot esculenta L.), Irish Potato ( Solanum tuberosum) and 
Sweet Potato ( Ipomea batatas L.) used in the preparation  of culture media were 
purchased from the Madina  Market and stored at 4oC in the refrigerator until needed. 
      e.  Chemicals 
           Chemicals were purchased from Oxoid Ltd. London and Accra Chemists Ltd, Accra. 
 
 
 
 
 
 
 
 
 
 
 
29 
 
(ii)  GENERAL METHODS 
       a.  Characteristics of growing plants. 
             Bulbs of both cultivars of shallot, Pale-brown and Pink, were kept in a refrigerator of a 
temperature of 4oC for 60 days before planting. This was done to break the dormancy 
of the bulbs. Bulbs of each cultivar approximately the same size, weighing between 
6.55g and 7.14g were selected for planting. Each bulb was planted in a 20cm by 12 cm 
black polythene bag filled with sandy-loam soil of the same weight from Anloga. The 
bulbs were each watered with 300 ml of water  every day, and the plants they produced 
after sprouting were kept in the open for 60 days after which the lengths of leaves were 
measured with a meter rule while the width at the broadest point were measured by 
means of a Vernier caliper. 
 
     b.  Elemental composition of bulbs 
           To determine the biochemical composition of the bulbs, they were cut into pieces and 
air dried for two days. After that the samples were milled and used for the analysis.  
   
(i) Estimation of total Nitrogen  
            The Micro-Kjeldahl method of AOAC (1984) was used for the nitrogen determination. 
The procedure involved digestion, distillation and titration. 
 
 
 
30 
 
             Digestion   
            An amount of 0.5g of the sample was weighed into each of three 250ml digestion tubes. 
A small scoop of selenium reaction mixture and 8ml of concentrated sulphuric acid were 
added to each and whirled. The tubes were then placed on a pre-heated digestion block. 
The samples were heated continuously until they were clear. They were then removed 
from the block digester and allowed to cool. 
            During digestion, organic and inorganic nitrogen were converted to ammonium salt by 
wet oxidation in the presence of concentrated sulphuric acid with a suitable catalyst that 
promoted the oxidation of organic matter.  
            Sample + Conc. H2SO4                           (NH4)2 SO4 + CO2 
 
 
            Distillation and Titration 
           This step involved the separation of ammonium salt from the concentrated H2SO4.  In 
strong alkaline solution, gaseous ammonium is distilled and trapped in standard acid 
solution. Distilled water was first used in the distillation apparatus to clean it for a few 
minutes. The Kjeldahl extract was diluted with about 30ml of distilled water. Exactly 
10ml of 0.1 N HCl was put in a conical flask containing two drops of Tashiro indicator 
and placed under a condenser. The tip of the condenser was dipped into the receiving 
acid in order to avoid any loss of ammonia. The digestion tube was fixed to the distilling 
end and about 15ml of 40% NaOH added automatically. This was distilled for about 
four minutes within which about 40ml of the distillate was collected in a conical flask. 
The flask was lowered and the tip of the condenser washed with distilled water. The 
31 
 
flask containing the distillate was removed and titrated with 0.1N NaOH to obtain a 
green end point. Percentage nitrogen was calculated using the formula: 
 
 
% N in soil sample=  
( a−b) x 0.01 X 14 X v X 100
1000 𝑋 𝑤 𝑋 𝑎𝑙
  
 
where v  = final volume of digestion= 100ml 
                                                  w = weight of sample taken in grams 
                al = aliquot of solution taken for analysis 
                                                0.01 = Molarity of acid 
14 = Molar weight of Nitrogen 
 
 
(ii)  Estimation of Calcium, Iron, Magnesium and Potassium levels  
            The wet oxidation method ( IITA, 1981) was used in the determination of calcium, iron, 
magnesium and potassium levels. Half a gram of sample was weighed into each of three 
Erlenmeyer flasks. Five millilitres of concentrated sulphuric acid were added to each 
flask and the flask placed on pre- heated sand bath in a fume chamber. Thirty percent 
Hydrogen peroxide ( H2O2) was added drop by drop until the solution turned colourless. 
The flasks were then removed from the sand bath and the contents allowed to cool. Each 
was diluted with distilled water and filtered into 100ml volumetric flask and made up to 
the volume mark with distilled water. Potassium levels were determined using the flame 
photometer while calcium, iron and magnesium levels were determined from readings 
32 
 
on the Atomic Adsorption Spectrometer ( Perkin Elmer Atomic Absorption 
Spectrometer, Model PinAAcle 900T) 
 
(iii) Total Phosphorus determination( Bray 1 Method) 
 
            The Bray method (Bray and Kutz, 1945) was used for phosphorus determination. This 
process involved in the digestion of the sample, titration with ammonium in the presence 
of P- nitrophenol to give a yellow colour and spectrophotometer used to read the optical 
density by which the amount of phosphorus was calculated. 
 
            Digestion 
            Half gram of the sample was weighed into a 125ml Erlenmeyer flask, 5ml of the 
digestion mixture (1.5 parts of HCIO3) added and placed on a pre-heated sand bath.  The 
sample was heated until it fumed and H2O2 was added drop by drop until the solution 
turned colourless. The sample was taken off to cool and diluted with distilled water. It 
was filtered into 100 ml volumetric flask and made up to the mark with distilled water. 
            Titration and reading on the spectrophotometer. 
            Aliquots of 2ml of the digested samples were dispensed into 50ml volumetric flasks. A 
volume of 30ml of distilled water was added to each flask and a drop of P- nitrophenol 
added. A few drops of ammonium solution were added until the colour turned yellow. 
Eight millilitres of the ascorbic acid mixture were added and the volume made to the 
mark with distilled water. The optical densities of the samples were read using the 
33 
 
spectrophotometer. The reading was used in the calculation below to obtain the 
phosphorus concentration in the sample. 
 
P (ppm)=  Spectrophotometer reading x Volume of sample 
sample weight x volume of aliquot 
 
      c.   Soil analysis 
            The levels of sodium, potassium, calcium, magnesium were determined using the wet 
oxidation method, phosphorus using the Bray 1 method and that of nitrogen using the 
Micro-Kjeldahl method. These methods are described in Experiment b. 
 
      d.   Preparation of and composition of nutrient media. 
            Potato Dextrose Agar (PDA) was prepared with 200g of tuber of Irish potato, 10g 
dextrose, 12g Agar and 1000ml distilled water. The Peeled tubers were washed and cut 
into smaller pieces. An amount of 200g was weighed and boiled in 500ml distilled water 
in an aluminium pan until they started to break up. The fluid was collected in a 500ml 
beaker by using a muslin sieve, left to stand and cool and then poured into a 1L 
measuring cylinder and topped up to the 1000ml mark. An amount of 10g of Dextrose 
and 12g of Agar were added and the mixture was transferred into a 2L Erlenmeyer flask 
and heated in a water-bath to melt the agar. The medium was subsequently dispensed 
into medicinal flats and autoclaved at 121oC for 30 minutes. 
34 
 
             Potato Dextrose Broth (PDB) was also prepared with 200g of tuber of Irish potato, 
dextrose, 10g; Agar, and 1000ml distilled water. This provided a liquid medium (broth) 
as was done in the case of PDA without the Agar.  
            Sabouraud Dextrose Agar was prepared with 10g peptone, 20g Dextrose and 1.5g 
Agar. They were dissolved in 1L of distilled water in a 2L Erlenmeyer flask and heated 
in a water-bath to dissolve the agar completely. The medium was then dispensed into 
medicinal flats and autoclaved. 
            Czapek Dox Broth medium was prepared by dissolving Sucrose, 30.0g; Sodium 
nitrate, 3.0g; Dipotassium phosphate, 1.0g; Potassium chloride, 0.5g; Magnesium 
sulphate, 0.5g; and Ferrous sulphate, 0.01g in 1L distilled water in a 2L Erlenmeyer 
flask. Aliquots of 250ml each were dispensed into four medicinal flats and autoclaved. 
            Ground shallot bulb medium was prepared by grinding five peeled shallot bulbs in a 
mortar into a paste. The paste was spread evenly in a Petri Dish and autoclaved. 
             Shallot bulb Extract. Healthy shallot bulbs without the covering of scale leaves, were 
ground in a mortar and the fluid strained with a muslin cloth. The extract represented 
undiluted standard concentration which was diluted to different strengths as desired for 
some conidium germination tests. 
 
     e.   Methods of sterilization. 
           i. Erlenmeyer flasks, pipettes and Petri dishes were immersed in potassium dichromate 
solution (K2Cr2O7), 100g; conc. H2SO4;distilled water, 1000ml); for 48 hours. They 
were next washed under running tap and rinsed twice with distilled water. They were 
35 
 
air-dried and then sterilized in an electric oven for six hours at 160˚C. Pipettes were 
wrapped in brown paper. 
          ii. Glass slides and solid watch glasses were washed with detergent, rinsed under running 
tap and stored in 70% ethanol. The slides were flamed-sterilized just before use. 
        iii. Media and distilled water were autoclaved for 30 minutes at a temperature of 121˚C at 
a pressure of 1.1kg per square centimeter (6.9x103 Pa). Cotton wool plugs were 
temporary covered with aluminum foil caps to prevent the penetration of any condensed 
water during autoclaving. 
      iv. The inoculation room and lamina flow chamber were sprayed with 10% aqueous Dettol 
solution, 20 minutes prior to plating of the agar media and inoculation. Inoculation of 
all other media was carried out also in the pre-treated inoculation room. 
      v. Forceps, inoculation needles and loops, and scalpels were flamed to red-heat and air-
cooled before use. 
 
      f   Soil mycofloral studies. 
           Soil from freshly collected farm plots at Anloga were used in this experiment. A small 
quantity of soil weighing 1.0g was put in 10 ml of sterile distilled water in a McCartney 
tube. The tube was shaken vigorously to ensure a thorough dispersion and the mixture 
used to prepare dilution series down to 1:1000 using sterile distilled water. One millilitre 
of this final dilution was mixed with 20 ml of either sterile cooled molten PDA or 
Sabouraud Agar containing a few drops of 1.0% streptomycin solution in each of the 
three replicates sterile Petri dishes. The mixture was mixed by a gentle circular motion 
36 
 
and allowed to set. The plates were incubated at 30oC. Colonies of fungi which 
developed were counted after 5 days and the fungal isolates identified.  
 
      g   Colonization of surface of bulbs  and roots of shallot growing in soil by fungi. 
           Shallots plants of the two cultivars were raised in Anloga farm soil in potting plastic 
bags in the greenhouse of the Botany Department for this investigation. At sampling 
time, non-rhizosphere soil was collected by removing 2 cm-long core soil with a sterile 
No. 6 cork borer (1cm in diameter) from the root-free region of the potting bag. Plugs 
of soil samples from the bags of each treatment were pooled, pulverised and thoroughly 
mixed. Phyllosphere and Rhizosphere soils were collected by loosening up the soil and 
gently pulling off the potting bag. The plants were carefully lifted to preserve as much 
of the root system as possible. Excess soil was then removed from the roots by gentle 
shaking of the plants. The closely adhering soil remaining on the bulbs and roots 
constituted the phyllosphere and rhizosphere soils, respectively. The bulbs were 
separated from the root systems using a sterile scalpel and the two placed in separate 
transparent polythene bags. The bags were then vigorously shaken to dislodge the 
respective adhering soils. 
            Soil dilution plate method was used to isolate the fungi in the soils. Serial dilutions were 
prepared for each of the soils by adding one gram of soil to 100ml sterile distilled water 
contained in 250ml Erlenmeyer flask. The soil suspension was vigorously shaken by a 
mechanical shaker( Whirli Mixer Tm, Wm/250/SCP/2) for 30 minutes. Immediately 
following dispersion, a suspension of 1:1,000 was made. One millilitre of this dilution 
was transferred into a sterile 9cm-diameter Petri dish and 20 ml of cooled (40oC) molten 
37 
 
Sabouraud Agar, supplemented with Rose bengal ( 0.05 g/l) to slow down growth of 
fast growing species, and a few drops of streptomycin ( 0.002g/l) to prevent bacterial 
growth on the plates. The inoculum was evenly dispersed in the agar medium by 
oscillating the dish gently by a circular movement after addition of the cool agar medium 
before it set. The Petri dishes were incubated in an inverted position at 32±2oC for 5 
days. The fungal species which developed were identified and the number of colonies 
of each species recorded. Counts of fungal population were used to calculate the 
quantity of fungi in unit soil.  
 
    h.   Conidium germination tests 
            Slide method 
            Conidia were carefully gathered with sterile inoculating loop and transferred into the 
germination liquid medium in McCartney tubes. The spore suspensions were shaken 
after adding a drop of Tween 80 by hand for 10 minutes to give uniform dispersion. The 
number of spores in suspension for every germination test was strictly standardised to 
400,000-500,000 per millilitre of medium with the aid of a haemocytometer. Sterile 
Petri dishes, each containing a sterile glass slide lying on moist filter paper formed the 
incubation chamber. Using a sterile dropping pipette, three individual drops of the spore 
suspension (about 0.01ml in volume) were placed on each slide and the Petri dish closed 
and incubated at 32±2oC for 24 hours. At the end of the incubation period, the spores 
were stained with cotton blue (0.01%) in lactophenol. If observation could not be made 
immediately, drops of N/40 formaldehyde were added, in addition to the stain to make 
sure the development of the spore was arrested. Percentage germination for any 
38 
 
treatment was based on 400-500 observed conidia was recorded. A conidium with 
discerniable germ-tube was considered to have germinated. The lengths of 20 randomly 
selected germ-tubes were also measured with an eye-piece graticule. Using the same 
method, conidial suspension drops were placed on small pieces (1.0 x 1.0cm) of either 
scale leaves or swollen leaf bases lying on the slides. 
 
i. Aspergillus niger growth on shallot bulbs on sale at the vegetable stores 
            Commercial shallot bulbs sold on the markets showed varying degrees of A. niger 
contamination. Bulbs of both Pink and Pale-brown cultivars were kept in desiccators for 
three days. The bottom of the desiccators was filled with water to provide 100% R.H. 
The percentage of bulbs showing external A. niger growth in each case was recorded. 
The coloured papery scale leaves were carefully detached, examining for further growth 
on them and beyond.  
 
     j.   A. niger culture growth tests. 
           Some experiments involved growth of A. niger on solid agar media in Petri dishes and 
in liquid media in 250 ml Erlenmeyer flasks. A Petri dish contained 20 ml of the medium 
and a 250ml Erlenmeyer flask contained 30 ml of the medium. The diameter of the 
growing colony on the agar medium was measured periodically along two diameters at 
right angles to each other drawn at the bottom of the Petri dish. Mycelium produced in 
the liquid medium in the Erlenmeyer flask was harvested after the desired period of 
incubation on a pre-weighed filter paper, dried at 80oC for 12 hours and weighed. The 
dry weight of the mycelium was then determined. 
39 
 
      k.   Growth of A. niger isolate in aqueous extracts of the bulbs. 
            An amount of 40g of the fleshy leaf base of the bulb was blended in 20ml of distilled 
water and the mixture was strained with muslin cloth to obtain clean extract free from 
residues. Thirty millilitres of the extract of each was put into a 250ml Erlenmeyer flask 
and the initial pH of was recorded.  The medium was autoclaved and inoculated with a 
3 mm diameter A. niger culture disk taken from the growing edge of the culture. The 
flask was incubated at 32±2oC for 5 days. The pH of the medium was measured again. 
The mycelium was harvested on a pre-dried filter paper, dried at 80oC for 24 hours and 
weighed. The dry weight of the mycelium was calculated. 
 
      l.   Humidity chambers 
            Wide-mouthed glass jars with screw-cap lids. 
            Solid watch glasses, each measuring 3.7x 3.7 cm with a well 1.6 cm in diameter and 1.0 
cm deep, served as Van Tieghem cells for conidium longevity test. Dry conidia of each 
of the different A. niger isolates were placed separately in wells of the watch glasses 
which were carefully placed in wide-mouthed glass bottles containing different 
concentrations of Sulphuric acid. 
            Petri dishes 
            Petri dishes were used for some of the experiments which required 100 % R.H. A 
shallow pool of distilled water was put in a clean Petri dish to provide an atmosphere of 
100% R.H.  
 
40 
 
            Desiccators 
            Desiccators were also used as humidity chambers to incubate shallot bulbs .The edge of 
the lid of the desiccator was smeared with petroleum jelly to make it air-tight when 
placed in position. 
 
     m.   Maintenance of constant humidities 
            Constant relative humidities ranging from 10 to 90% were maintained with solutions of 
sulphuric acid (H2SO4) of different concentrations according to the data of Solomon 
(1952) and as shown in Table 2. Nominal zero per cent R.H. was maintained with 
anhydrous calcium chloride. Water provided 100% R.H. 
 
 
 
 
 
 
 
 
 
41 
 
Table 2: Sulphuric acid (H2SO4) solutions for maintaining Constant Humidity 
(Data of Solomon, 1952) 
 
                                       Weight of                       Volume of distilled 
                              % R.H.                   Sulphuric acid (g)                   water (ml) 
                 100                    0.00                                    100.00 
                             90                                17.91         82.09 
                              80                               26.79         73.21 
                             70                                33.09         66.91 
                             60                                38.35         61.65 
                             50                                43.10         56.90 
                             40                                47.71         52.29 
                             30                                52.45         47.55 
20                               57.76         42.24 
10                                64.45                 35.55 
 
 
 
 
 
 
42 
 
      n.   Study of rot development of wound-inoculated bulbs 
           The outer brown scales of the bulb were removed and the ‘naked’ bulbs, surface-
sterilized by immersion in 5% sodium hypochlorite solution for 10 minutes. The bulbs 
were rinsed twice in two changes of sterile distilled water.  
           A shallow wound was made at the equator of the bulb with a sterile No 1 cork borer into 
which a 3 mm A. niger culture disc was placed face down. The wound was covered with 
the “lid” and sealed with petroleum jelly. The bulbs were placed in watch glasses and 
incubated at a relative humidity of 100% at 32±2oC.  The diameter of rot that developed 
was measured daily along two planes at right angles to each other and the mean 
calculated. 
 
     o.   In Vitro production of Pectic enzymes. 
            The procedure adopted by Tortoe (1997) was followed. Czapek-Dox medium 
containing 1% Pectin was prepared.  For each exercise, fourteen 250ml Erlenmeyer 
flasks were each filled with 35ml of the Czapek-Dox medium and autoclaved.  Two of 
the flasks were taken after autoclaving, their contents pooled together and the initial pH 
determined. Each of the remaining 12 flasks were inoculated with a 3mm culture disc 
of the isolate of A. niger. The flasks were incubated at 32±2oC. Three flasks were 
withdrawn after 3, 6, 9 and 12 days, respectively, and the mycelium harvested, using 
filter paper. The harvested mycelia were dried at 80oC for 18 hours and weighed to 
obtain the dry weight. 
 
 
43 
 
 
     p.   Maceration test 
           The pooled culture filtrates of Section ‘o’ above were divided into two lots. With the No. 
8 cork borer, plugs of Irish potato tuber were removed and placed in distilled water in a 
clean Petri dish. The plugs were carefully cut into 0.5 mm thin discs with a surgical 
scalpel.The discs were washed in sterile distilled water and drained on filter paper. Two 
millilitres of culture filtrate were put in a mini- Petri dish and the pH adjusted to pH 5.0 
by adding 0.5ml of 0.1M Citrate Buffer of pH 5.0. Six of the discs were placed in the 
preparation. At 5-minute intervals, a disc was picked and gently pulled from two 
opposite ends with a pair of blunt forceps. The disc was placed back in the medium if it 
was still firm and did not pull apart. It came to a time that each of the six disc were 
pulled apart. Each of the discs were pulled apart at a particular time. The time was noted 
and the disc discarded. 
            The enzyme activity was calculated by the formula: 
(1/ Mean time of maceration of 6 discs) x 100. 
 
       q   Determination of productivity of A. niger on ground shallot bulb 
            Ground shallot paste described in Section ‘d’ was inoculated with a 3mm culture disc 
of A. niger and incubated at 32±2oC. The degree of sporulation was determined after 10 
days. Four discs were removed from the plate with a No. 7 cork borer from four 
symmetrical positions equidistant from the centre of the culture. Each disc was placed 
in a McCartney tube holding 10ml of distilled water, containing Tween 20, and shaken 
44 
 
vigorously to disperse the spores. The number of spores per ml. of suspension was 
determined with a haemocytometer. 
      r   Conidial Longetivity test. 
           Conidia of A. niger were removed in the dry state into clean watch glasses and spread 
into thin layer in the watch glasses. The watch glasses with their load of conidia were 
placed in a series of shallow pools of sulphuric acid of different concentrations in wide 
screw glass jars (8.5cm high and 10.0 cm in diameter). The sulphuric acid solution 
provided respective relative humidities of 10, 20, 30, 40, 50, 60, 70, 80, and  90% R.H 
as indicated in Table 2, while other jars containing anhydrous calcium chloride provided 
a nominal 0% R.H. and distilled water maintained 100% R.H. The jars were incubated 
on the laboratory bench under normal day-night light regime. The problem of irregular 
electric light delivery did not allow the setting of tests under continuous light and 
continuous dark over a protracted period. 
           Viability of test conidia was determined after desired intervals, by germinating samples 
of conidia transferred from the jars into PDB at 32±2oC for a period of 24 hours, after 
which the percentage germination was determined and germ-tubes lengths measured. 
 
      s.   Application of Potassium and Urea fertilizer 
            Various amounts of 50, 100, 150 and 200g of Potassium fertilizer (Multi-K) were used 
to prepare different concentrations of the fertilizer. Each was thoroughly dissolved in 
15 L of water and stored in plastic gallons. An aliquot of 300ml of each preparation was 
applied weekly to the respective plants. In the case of Urea fertilizer, two levels of 
45 
 
concentrations were used. Each of the potting bags in one set was treated with 1.98g 
and in the other set 3.88g of Urea per bag. 
           Samples of shallot bulbs of the different treatments were uprooted at intervals of 20, 40 
and 60 days for various assessments.  
 
      t.   Photography 
            The photographs in this thesis were taken with a Sony Cyber-Shot camera, model No. 
DSC-W330. 
 
       u.   Statistical Analysis 
           Experimental results where necessary, were analysed statistically with  SCHEFFE’S 
CALCULATED CONFIDENCE LIMITS AT 95% PROBABILITY. (Kershaw, 1973). 
 
     v.   Experimental precautions. 
i. Glassware were kept scrupulously clean. Particularly, glassware already in 
circulation in the laboratory were first scrubbed with detergent to remove all traces 
of chemicals and grease. 
ii. In the assessment of conidia percentage germination, conidia were counted in all 
suspension drops of each treatment. 
iii. When measuring the length of germ-tubes, those at the edge of the suspension drops 
were avoided as these were under more aerated condition than the submerged germ-
tubes. 
46 
 
iv. Similarly, conidia in clumps which might have been influenced by chemical 
substances of adjacent conidia, were not included in the assessment of percentage 
germination and measurement of lengths of germ-tubes. 
v. In any of the A. niger growth experiments, in the event of the incidence of bacterium 
or fungus contamination on any of the replicate cultures, the entire set of replicates 
was discarded and the exercise repeated. 
vi. In order to avoid cross contamination, experiment with only one A. niger Isolate was 
set up on any particular day. 
vii. Metal screw-caps of glass containers (McCartney tubes, medicinal flats) were kept 
loose during autoclaving and then tightened after autoclaving. 
 
 
 
 
 
 
 
 
 
 
 
 
 
47 
 
III.  EXPERIMENTAL DETAILS 
SHALLOT CULTIVARS IN GHANA 
EXPERIMENT A. Morphology of the two shallot cultivars 
There are two distinctive cultivars of shallots (Allium ascalonicum) cultivated in Ghana. They 
have been designated “Pale-brown” and “Pink” cultivars in this thesis. Their distinct 
characteristics were studied in Experiment A and the observations made are supported by 
histograms (Figs 1 and 2) and photographs (Plates 5, 6 and 7). 
 
EXPERIMENT B.  Elemental composition of the bulbs of the two shallot cultivars 
The morphological differences between the two cultivars were identified in EXPERIMENT A. 
Differences in chemical composition of the two cultivars may also exist. The levels of the 
components may influence the growth of Aspergillus niger in the bulbs and affect pathogenesis. 
Five batches of bulbs of Pale-brown cultivar and Five batches of Pink cultivar were purchased 
in the same month, October 2012, and analyzed. They were purchased from the markets of 
Anloga in the Volta region and of Agbogbloshie, Kaneshie, Makola and Mallam Atta in Accra. 
Bulbs of approximately the same size were selected for the analysis. For any test five bulbs 
were taken, sliced and pooled. The chemical components identified and   their concentrations 
are shown in Tables 3 to 8. 
 
 
 
48 
 
ASPERGILLUS NIGER INFECTION OF THE SHALLOT BULBS. 
EXPERIMENT C. Aspergillus niger growth on bulbs of fresh stocks of the two shallot 
cultivars in market stalls.  
It was noticed that bulbs of both cultivars purchased on any occasion included samples bearing 
the spreading dark colonies of A. niger. In order to quantify these natural infections, shallot 
bulbs were purchased from Agbogbloshie market in Accra and Anloga market in the Volta 
region in August 2011 and January 2014, respectively. One hundred bulbs each of the two 
cultivars, Pale-brown and Pink, were selected from the produce obtained from Agbogboshie 
market. The same was done for the produce purchased from Anloga market. Each was 
meticulously scrutinized and A. niger growth detected at various locations of each bulb was 
recorded. The percentages of these sides bearing A. niger growth for each cultivar are shown in 
Table 9 and example of colonization of the scale leaves is depicted in Plate 8. 
 
EXERCISE D. Influence of micro-habitats of shallot bulbs on the development of 
Aspergillus niger conidiophores.  
As a sequence, the morphology of A. niger at the various sites was studied. The various sites 
constituted different micro-habitats which might affect A. niger differently. That supposition 
was investigated in this Experiment. Samples of the fungus taken from the different sites were 
mounted on glass slides in Lactophenol cotton blue and studied under high power of the 
microscope. Quantitative data obtained are tabulated in Table 10 
 
49 
 
EXPERIMENT E. Culture characteristics of the five test isolates of Aspergillus niger. 
Variation is a common phenomenon among microbial species. This produces variance with 
distinctive characteristics. Any investigation that incorporates variants yields a better 
information on the subject matter. This research therefore, selected five of the numerous A. 
niger isolates obtained from naturally infected bulbs of the two shallot cultivars and used them 
as the test fungi for this experiment. The growth rate, colour of the culture and habit of growth 
of the mycelium were used to distinguish these five Isolates, designated 1,2,3,4 and 5. 
Table 11 contains the details of the characteristics of the five Isolates and Plate 9 shows the 
cultures, growing on Potato Dextrose Agar in Petri dishes. 
 
EXPERIMENT F. Germination of conidia of the five test Aspergillus niger Isolates and 
pattern of growth on the swollen leaf bases and the scale leaves and fate of the germ tubes. 
The conidia of the five Isolates of A. niger were taken from five day old respective cultures and 
suspended in Potato Dextrose Broth (PDB) of different concentrations to find their germination 
capacity in the different media and to study the growth habit of the germ-tubes. The germination   
media used for each isolate were Potato Dextrose Broth of four dilutions viz,1:10, 1:20, 1:30 
and 1:40 v/v (PDB: sterile distilled water). Distilled water constituted control medium. Conidia 
in these suspensions were incubated for 12 hours in light intensity of 76 lux. The suspension 
drops were incubated on surfaces of detached parts as follows: (a) outer surface of the external 
leaf scale (b) inner surface of the external leaf scale (c) outer surface of the internal leaf scale 
(d) inner surface of the internal leaf scale (e). outer surface of the outermost swollen leaf base. 
 
50 
 
The bulb parts were separately placed in 5% sodium hypochlorite solution in sterile Mc-Cartney 
tubes for five  minutes to surface sterilize them  and then rinsed in sterile distilled water and air 
dried on sterile filter paper. Six square pieces, each measuring 5x 5 mm, were cut from each 
specimen and inoculated. For each, the desired test surface was inoculated with a drop of spore 
suspension. Three pieces for each treatment were withdrawn after 12 hours, stained with 
lactophenol cotton blue and used in the germination assessment. The results obtained are 
presented in Tables 12 to 16. The remaining three pieces were withdrawn after 24 hours, stained 
and the pattern of growth of the germ-tubes studied.  Since there was good germination 
especially at the higher concentrations of PDB, a subsequent germination test was carried out 
to find out the level of germination which could be supported by individual selected sugars, 
galactose, glucose and sucrose solutions of concentrations of 0.125, 0.25, 0.5 and 1.0 percent. 
In this experiment the outer surfaces of the external and internal leaf scales and the outermost 
swollen leaf base were used. The spore suspensions were incubated for 12 hours and the results 
are tabulated in Tables 17 to 31. In both experiments the lengths of germ tubes of 20 germinated 
conidia in each case were measured and the mean calculated. 
 
EXPERIMENT G. Germination of conidia of the five test Aspergillus niger isolates in 
aqueous extracts of the two shallot cultivars. 
 In the event of bruising of the bulb, the conidia of A. niger will come in contact with exudates 
from these wounds. The next germination test was, therefore, carried out to find whether these 
exudates are rich in nutrients that may support good germination of the conidia and growth of 
the germ-tubes as in the case of Potato Dextrose Broth. The conidia were germinated in the 
51 
 
fluids expressed from ground bulb, which constituted standard concentrations and dilutions of 
½, 1/4, 1/8 and 1/16 of the fluid. 
Distilled water was the control medium. Spore suspension drops on sterile slides were incubated 
in humid Petri dishes at 32±2oC under light illumination of 76 lux. There were three replicate 
slides for each treatment. The results, both percentage germination and mean length of germ-
tubes, are shown in Tables 32 to 36 and Fig 3. 
 
EXPERIMENT H. Shallot bulb inoculation tests using the two shallot cultivars and the 
five test Aspergillus niger Isolates.  
Good germination of the conidia and healthy growth of the germ-tubes occurred at the higher 
concentrations   of the expressed fluid of the bulbs in the preceding experiment. This suggested 
the probable absence of toxic compounds in the fluid. The germ-tubes will also probably grow 
well in the tissues of the bulbs of both cultivars. This suggestion was examined in this 
experiment. Bulbs of approximately the same size were surface sterilized by immersing in   10% 
sodium hypochlorite solution for five minutes and rinsed in sterile distilled water after the 
covering pigmented scale leaves had been removed. Bulbs of each cultivar were divided into 
two sets. One set was surface inoculated by placing 3 mm diameter culture discs, face down on 
the bulbs. The second set was wound-inoculated as described in “ Materials and General 
Methods” with 3mm diameter culture discs of the A. niger isolates. Both sets were stored in 
humidity chambers with internal atmosphere of 100% R.H. The diameters and depths of rots 
which developed after 3 days were measured and recorded. There were four replicates in each 
treatment. The mean values of the measurements appear in Table 37.  
52 
 
EXPERIMENT I. Rotting of wound-inoculated bulbs at different relative humidities 
using the two shallot cultivars and the five test Aspergillus niger isolates. 
Humidities prevailing in shallot stalls rarely attain 100% R.H. The rate of rotting occurring 
under those conditions may, therefore, be slower than those recorded in Experiment H. The 
relation between the percentage relative humidity of the environment and the rate of rotting was 
investigated in this experiment. The same procedure was followed but, on this   occasion 
incubating different batches of the inoculated bulbs at 50, 60, 70, 80, 90 and 100% R.H for 10 
days and the effect of the different relative humidities on the extent of rotting of the bulbs, on 
the   development of the conidia apparatus and the degree of sporulation of the A. niger isolates 
are reported in Tables 38-47. Plate 10 is a photograph of one set of the inoculated bulbs at the 
end of the incubation period. 
 
EXPERIMENT J. In-vitro production of pectic enzymes by the five test Aspergillus niger 
Isolates 
 Rotting by A. niger is the symptom of the degradation activities of the pectic enzymes it 
produces. The differences in the rate of rotting by the different Isolates of A. niger observed in 
the previous experiment possibly reflects differences in the ability to produce these enzymes. 
This was verified by examining the positive correlation between growth rates and 
corresponding enzyme activity of the filtrates of A. niger isolates . In this experiment, tests were 
carried out to: 
(a) establish growth rate of the A. niger strains in Czapeck Dox medium 
(b) establish enzyme activities of the Czapeck Dox medium filtrate of the A. niger isolates. 
53 
 
(c) find out whether the order of growth performance in the Czapeck Dox medium of the 
A. niger isolates will be altered by change in substrate, by growing the Isolates in Bean Extract, 
Cassava Dextrose Broth, Oat Extract, Potato Dextrose Broth and the Sweet Potato Broth. 
There were four replicate 250ml Erlenmeyer flasks in every test, each containing 30 ml of the 
medium. The inoculated flasks were incubated for 8 days under 12- hour day-night cycle, at 
32±2oC. 
The results of the numerous tests are tabulated in Tables 48a-e, 49a-e, 50, 51, 52.53, 54a-e and 
55. Because the volume of the culture filtrate diminished very drastically during growth of the 
cultures, the replicate filtrates were pooled on the 12th day to provide adequate volume of fluid 
for the determination of a single final pH for any particular treatment. 
 
 
THE SOIL ASPERGILLUS NIGER-SHALLOT BULB COMPLEX. 
 
EXPERIMENT K. Mycoflora of the bulbs and the rhizosphere of shallot plants raised in 
soil of Anloga farms amended with Urea fertilizer. 
Knowledge of the response of A. niger in soil to the presence of the shallot plants is imperative 
for complete understanding of A. niger-shallot plant association. If organs of shallot plants   
secrete stimulatory compounds, growth of A. niger in the vicinity will be much stimulated. It is 
not uncommon for certain fungal species in soil to be associated with specific living roots. 
54 
 
Pertinent experiments were designed to study the sort of relation between A. niger and the two 
shallot cultivars in soil. 
Anloga shallot farmers routinely add manure to the freshly prepared shallot plots prior to 
planting. Manure adds nitrogen compounds to soils. The effects of known quantities of nitrogen 
was studied by adding to the soil different concentrations of Urea. The bulbs of each of the two 
shallot cultivars were planted in several black polythene bags (20 x 12 cm). Each bag contained 
1200g of Anloga farm soil. For each shallot cultivar, the bags were divided into three batches 
of 15 bags each and the bags were planted with a single bulb each. Urea powder weighing 1.94g 
and 3.88g respectively were added to the bags of two batches. Bags of the third batch received 
no urea. The potted bulbs were placed in the green house under a 12-hour day/night cycle. 
The densities of fungi on the surface of the developing bulblets, in the rhizosphere and of those 
in the non rhizosphere soil were determined at 20, 40 and 60 days after planting. By the 20th 
day, the parent bulb had disintegrated and the bulblets had become independent and self-
sustaining. At sampling time, three bags of each treatment were withdrawn for the estimation 
of fungal populations. Non rhizosphere soil was collected at a distance of 2.0 cm from the 
developing bulbs to a depth of 2.0 cm with a sterile cork-borer. Samples of the replicate bags 
were pooled, pulverized and thoroughly mixed. One gram was then taken and used to estimate 
the fungal population by the soil dilution plate method. Rhizosphere soil estimation of the 
fungal population, also by the soil dilution plate method, was collected from gently uprooted 
plants as described under General Methods. The same method was used to determine the density 
of fungal population of soil particles adhering to the surfaces of the bulbs which in essence was 
phyllosphere population. There were three replicate Petri plates for each determination. The 
results, presented as number of Colony Forming Unit (CFU’s) x 103 per g of soil are recorded 
55 
 
in Tables 60 to 62. The results also include data on the stage of development of the bulbs at the 
estimation time. Plates 11, 12 and 13 are examples of photographs of colonies of fungal species 
of Anloga soil and of rhizosphere soils of 40 day old growing bulbs. 
 
EXPERIMENT L. Response of bulbs of the two shallot cultivars formed in Anloga farm 
soils amended with Urea fertilizer to Aspergillus niger inoculation using A. niger Isolates 
1 and 2. 
It was expected that an additional supply of nitrogen to the shallot plants would result in an 
enhanced metabolism and growth. A more robust plant that would be less susceptible would be 
one of the important consequences. Experiment L studied the relative susceptibility of bulbs 
formed in soils amended with the different concentrations of Urea used in the preceding 
Exercise. 
Following the procedure used in Experiment H, bulbs harvested on the 60th day after planting 
were wound inoculated with mycelium of A. niger Isolates 1 and 2. Samples were stored at 50, 
60, 70, 80, 90 and 100% relative humidity for three days after which the extent of infection was 
assessed. There were three replicate bulbs for each urea concentration. 
Tables 63a-c contain measurements of diameters of the rotted areas recorded. 
 
 
 
56 
 
EXPERIMENT M:  Mycoflora of the bulbs and the rhizosphere of shallot plants raised 
in soil of Anloga farms amended with Potassium fertilizer. 
 Experiment K was repeated in this investigation but with different objective. Potassium as a 
fertilizer is well documented. It also has a unique characteristic of increasing the permeability 
of plant cell membranes. Higher concentrations will in the first place cause an increased leakage 
from the bulbs and roots into the soil encouraging greater fungal colonization of the surfaces of 
the plants. Secondly, the increase in cell membrane permeability on the application of 
potassium may cause faster   penetration of cell degrading enzymes of invading fungal parasites 
into host tissues. 
The effect of Potassium fertilizer applied at concentrations of 0.00, 3.33, 6.67, 10.00 and 13.33 
g/l on the density of fungi colonizing the shallot plant surfaces was the subject of Experiment 
M. 
The effect of the Potassium fertilizer at these concentrations on the susceptibility of 60 day old  
bulbs to A. niger infection constituted EXPERIMENT N: ‘Response of bulbs of the two 
shallot cultivars formed in Anloga  soils amended with Potassium fertilizer to Aspergillus 
niger inoculation using A. niger Isolates1 and 2’ following the procedure of Experiment L. 
The results are tabulated in Tables 64 to 70. 
 
EXPERIMENT O.  Mycoflora of the bulbs and rhizosphere of plants of the two shallot 
cultivars grown in soils of farms of non-alliaceous crops. 
The results of Experiment K and M revealed overwhelming preponderance of A. niger growth 
around the shallot plants. It was considered necessary to verify whether this pattern of 
57 
 
colonization would be replicated in farm soils which had supported non-alliaceous crops, 
namely, cassava (Manihot esculenta), groundnut (Arachis hypogea) and maize (Zea mays). 
Bulbs of the two shallot cultivars were used according to the procedure described in 
Experiments K and M but without fertilizers. The results are recorded in Tables 72 to 74. 
 
ROLE OF THE DIFFERENT SHALLOT CULTIVARS IN THE PERSISTENCE OF 
A. NIGER IN THE ECOSYSTEM 
EXPERIMENT P. Conidiation of Aspergillus niger Isolates 1, 3 and 5 on ground bulb 
tissue of the two shallot cultivars and the germination capacity of the conidia. 
Growth of A. niger on the subterranean organs of the shallot plants was overwhelming. The 
bulbs and the roots will contribute largely to persistence of A. niger in the soil. Innumerable 
quantities of A niger conidia, are produced by hyphae growing on the post-harvest bulbs as 
shown in Plate 8. These, as dispersal units, are likely to contribute, to a far greater measure, to 
the persistence of the fungus in the ecosystem, provided, they live long. For any shallot cultivar 
to play this role well, the bulb should be a suitable medium substrate for growth and congenial 
for sporulation. The comparative suitability of the bulbs of the two cultivars was investigated 
in the following experiments using A. niger Isolates 1, 3 and 5. 
(a) Vegetative growth on ground bulb tissues of the two shallot cultivars. 
(b) Degree of sporulation on ground bulb tissue. 
(c) Germination capacity of the conidia in nutrient solutions. 
58 
 
Rate of vegetative growth was provided by daily measurements of diameters of the cultures. 
There were four replicates Petri dishes for   each test. The measurements are recorded in Tables 
75 to 77. 
Discs of identical diameters of the cultures were shaken in similar identical quantities of 
distilled water at the end of incubation period of 7 days. The density in the suspensions were 
determined using a Haemocytometer. The calculated densities are shown in Table 78. 
 
Vigour of the conidia at harvest, 7 days after inoculation, was tested by suspending them in 
Bean Meal Extract, Potato Dextrose Broth and Sweet Potato Extract and incubating them on 
glass slides at 32±2oC. The percentage germination and mean length of germ-tubes were 
recorded after 12 hours. Tables 79 to 81 contain the results of this test. 
 
EXPERIMENT Q. Longetivity of conidia formed by Aspergillus niger Isolates 1, 3 and 5 
at different relative humidities. 
Finally, the longetivity of the conidia of A. niger Isolates 1, 3 and 5 was investigated. Dry 
conidia were removed from seven day old cultures and the samples kept in solid watch glasses 
standing in bottles with screw caps. The bottles contained 0.5 cm deep solutions of Sulphuric 
acid of different concentrations which provided and maintained 10, 20, 30, 40, 50, 60, 70, 80,  
and 90% Relative humidity. Zero and 100% Relative humidities were provided by and 
maintained by dry anhydrous Calcium chloride and distilled water, respectively. The 
preparations were kept at 32±2oC under 12- hour day night cycle. Some of the conidia of each 
treatment were suspended in Potato Dextrose Broth at 32±2oC after specific intervals. The 
59 
 
percentage of the conidia which germinated after 12 hours represented the percentage viability 
of the sample. Germ-tube of the germinated conidia were also measured as an indicator of the 
vigour of the germinated conidia. When a conidia sample registered zero percentage 
germination, samples for that particular watch glass were used in the next two consecutive 
germination tests to confirm the zero results before tests were discontinued. The experiment 
was run over a total period of 200 days from January 2012 to July 2012. The results are 
presented in Appendices B to V and Fig 4. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
60 
 
IV. RESULTS 
EXPERIMENT A. Morphology of the shallot cultivars 
The two shallot cultivars, Pale-brown and Pink, have many similarities. There are also many 
distinct differences by which they can be identified. 
  Features common to the bulbs of the two cultivars observed were: 
1. Conical shape of the bulbs as shown in Plate 5, 
2. Two inner and outer, coloured scale leaves; 
3. White-coloured swollen leaf bases; 
4. Swollen leaf bases of same thickness; 
5. Small cylindrical discs. 
The differences between the bulbs of the two cultivars were as follows: 
1. Pale-brown and Pink pigmentation of the scale leaves of the Pale-brown and Pink 
cultivars respectively 
2. Larger bulbs of the Pale-brown cultivar. 
i. The respective mean lengths of the bulbs of the Pale-brown and Pink cultivars 
were 3.62±0.06 and 3.26±0.08cm which by the calculated Scheffe’s 
Confidence limit were significantly different at the 5% level of probability, and 
the corresponding ranges were 3.1 to 4.18 and 2.66 to 3.83cm as shown by the 
histograms in Fig. 1. 
ii. The respective mean widths of the bulbs were 2.71±0.05 and 2.08±0.08cm, 
which by the calculated Scheffe’s confidence limit, were significantly different 
61 
 
at 5% level of probability, and the corresponding ranges were 2.50-3.08 and 
1.65-2.51cm as observed in the histograms in Fig. 1 
3. As a consequence of the difference in sizes, the respective mean weights of the bulbs 
of the Pale-brown and Pink cultivars were 8.79±0.27 and 5.98±0.29g, which by the 
calculated Scheffe’s Confidence Limit were significantly different at the 5% level of 
probability, and the corresponding ranges were 6.48 to 10.95 and 4.43 to 7.82g 
according to the data in Appendix A. 
4. The growing plants produced by these bulbs could be easily differentiated. 
i. Pale-brown cultivar plants were larger than Pink cultivar plants as observable 
in Plate 6 of 30 day old potted plants. The histograms in Fig.2 contain details 
of the lengths and widths of mature leaves of 60-day old plants 
ii. The lengths of the mature leaves of the Pale-brown and Pink cultivars ranged 
from 34.3 to 49.9 and 33.9 to 46.2cm, respectively, with corresponding means 
of 40.96±4.5 cm and 38.67± 3.1cm respectively. 
iii. The width of the mature leaves of the Pale-brown and Pink cultivars ranged 
from 4.33 to 6.10 and 3.54 to 5.98mm, respectively, with corresponding means 
of 5.38±0.9mm and 4.84±0.7mm. 
iv. In the field the Pale-brown cultivar crop looked greener and more lush than the 
Pink cultivar of the same age crop as shown in Plate 7. 
 
 
62 
 
 
 
 X 1 1 4⁄  
 Plate 5. Photographs of bulbs of Pale- brown (TOP) and Pink ( BOTTOM) 
 cultivars of shallot ( Allium ascalonicum) 
63 
 
 
          
                                                                                                                            X 1/8 
PLATE 6. Photograph of 30-day old plants of the Pale-brown (TOP) and Pink (BOTTOM)  
cultivars of shallot (Allium ascalonicum). 
64 
 
 
 
 
Plate 7. Photographs of plots of 60-day old plants of shallot ( Allium ascalonicum) distinguishing 
between the plants of the Pale-brown cultivar (TOP) and the Pink cultivar (BOTTOM).
65 
 
0
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Lengths of bulbs Width of bulbs 
bulbs 
Weight of bulbs 
Key: 1: 1.61-1.90 cm      4: 2.51-2.80 cm 
         2: 1.91-2.20 cm      5: 2.81-3.10 cm 
         3: 2.21-2.50 cm      6: 3.10-3.40 cm 
Key: 1: 4.01-5.00 g      4: 7.01-8.00 g   7: 10.01-11.00g 
         2: 5.01-6.00 g      5: 8.01-9.00 g 
         3: 6.01-7.00 g      6: 9.01-10.00 g 
Key: 1: 2.51-2.80 cm      4: 3.41-3.70 cm 
         2: 2.81-3.10 cm      5: 3.71-4.00 cm 
         3: 3.11-3.40 cm      6: 4.01-4.30 cm 
Pink 
cultivar 
Pale-brown 
cultivar 
Figure 1: Length, width and weight of both Pale-brown and Pink cultivars of Shallot (Allium ascalonicum) purchased from the Anloga market. 
66 
 
0
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15
20
25
30
35
40
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1 2 3 4 5 6 7
Figure 2: Class-lengths and class-widths of the mature leaves of the Pale-brown and Pink shallot 
(Allium ascalonicum) cultivars 
 
 
 
 
 
 
 
 
Key: 1: 30.1-33.0 cm      4: 39.1-42.0 cm    7: 48.1-5.0 cm 
         2: 33.1-36.0 cm      5: 42.1-45.00 cm 
         3: 36.1-39.40 cm    6: 45.1-48.0 cm 
Key: 1: 35.1-40.0 cm      4: 55.1-60.0 cm 
         2: 40.1-45.0 cm      5: 60.1-65.0 cm 
         3: 45.1-50.0 cm      6: 65.1-70.1 cm  ( x10-3 ) 
Class Length Class Width 
67 
 
EXPERIMENT B. Elemental composition of the bulbs of the two shallot cultivar 
Six major chemicals, as indicated in Tables 3 to 8, were detected in bulbs of both the Pale- 
brown and pink shallot cultivars. The chemicals were of different quantities in the following 
descending order:  Nitrogen˃Potassium˃Phosphorus˃Sodium˃Calcium˃Magnesium. As 
shown in Table 8, the percentages of Nitrogen in the Pale-brown and Pink cultivars were 
1.456±0.007 and 1.192±0.05 respectively. The mean percentage of magnesium, in comparison, 
for the pale-brown and pink cultivars were 0.054±0.00 and 0.046, respectively. 
 
This higher level of Nitrogen and  Magnesium in the Pale-brown  cultivar than in the Pink 
cultivar was applicable to all the chemicals except for the rare occurrence of higher 
concentrations  of Sodium in Tests 2 and 3 involving bulbs purchased from the Anloga and 
Mallam Atta markets as reported in Tables 4 and 7. Even with these, the values for the two 
cultivars were extremely close. According to the data of the Anloga lot recorded in Table 4, 
values for the pale-brown cultivar for Tests 1, 2 and 3 were 0.183, 0.181 and 0.186 per cent, 
respectively and for the pink cultivar were 0.181, 0.185 and 0.186 per cent, respectively. 
Similarly, information in Table 7 on the Mallam Atta products indicated 0.183, 0.185 and 0.190 
per cent Sodium, respectively for Tests 1, 2 and 3 of the pale-brown cultivar, while the pink 
cultivar had 0.183, 0.189 and 0.192 per cent, respectively. 
It is noteworthy that the values for any chemical were different for the different tests, howbeit 
within reasonable ranges. 
 
68 
 
 
 
 
 
Table 3: Major chemical elements ( % ) of bulbs of two cultivars of shallot (Allium 
ascalonicum) purchased from Agbogbloshie Market, Accra in October 2012. 
 
       
                                                                     Test 
 
                                                  1                                   2                                      3 
                         
       Chemical        Pale                                   Pale                                  Pale 
        Elements (%) brown cv.    Pink cv.         brown cv.   Pink cv.         brown cv.  Pink cv. 
 
      Calcium            0.192           0.162             0.188            0.188            0.178          0.174 
      Magnesium       0.051           0.041             0.041            0.039            0.051          0.050 
      Nitrogen            1.643           1.245             1.595            1.432            1.621          1.319 
      Phosphorus        0.373           0.412            0.394             0.363            0.423    0.369     
      Potassium          1.183           1.134            1.178             1.123            1.180          1.134                                                
      Sodium              0.181           0.163            0.164             0.143            0.171          0.169                               
 
 
 
 
69 
 
 
 
 
 
Table 4: Major chemical elements (%) of bulbs of two cultivars of shallot (Allium 
ascalonicum) purchased from Anloga Market, in October 2012. 
 
                                                                     Test 
 
                                                  1                                     2                                      3 
                          
       Chemical        Pale                                    Pale                                 Pale 
      Elements (%)  brown cv.    Pink cv.        brown cv.   Pink             brown cv.   Pink cv. 
 
        Calcium         0.150          0.124             0.148            0.121           0.158           0.124 
        Magnesium    0.049          0.037             0.049            0.040           0.043           0.037                                                  
        Nitrogen        1.599          1.233             1.589            1.229            1.588          1.228 
        Phosphorus    0.399          0.343             0.401            0.341            0.400 0.336     
        Potassium      1.089          1.045             1.094            1.048            1.093          1.054                                                
        Sodium          0.183          0.181             0.181            0.185            0.185          0.186                               
 
 
 
70 
 
 
 
 
 
Table 5: Major chemical elements ( % ) of bulbs of the two cultivars of shallot ( Allium 
ascalonicum) purchased from Kaneshie Market, Accra in October 2012. 
 
         
                                                                                   Test 
 
                                                  1                                   2                                      3 
                          
        Chemical        Pale                                  Pale                                 Pale 
       Elements (%) brown cv.    Pink cv.        brown cv.   Pink              brown cv.   Pink cv. 
 
        Calcium        0.149           0.096             0.153          0.133            0.152           0.123 
        Magnesium   0.057           0.051             0.062          0.061            0.058           0.053 
        Nitrogen       1.245           0.882             1.452          1.250            1.352            1.084 
        Phosphorus   0.375           0.296             0.429          0.377            0.498 0.354     
        Potassium     1.096           0.738             1.017          0.873            1.025            0.934                                                
        Sodium         0.178           0.167             0.169          0.167            0.168            0.167                                       
   
 
71 
 
 
 
 
 
Table 6: Major chemical elements ( % ) of bulbs of the two cultivars of shallot ( Allium 
ascalonicum) purchased from Makola Market, Accra in October 2012. 
 
         
                                                                     Test 
 
                                                  1                                     2                                      3 
                          
      Chemical       Pale                                    Pale                                 Pale 
     Elements (%) brown c.v.    Pink c.v.        brown c.v.   Pink             brown c.v.   Pink c.v. 
 
      Calcium         0.193             0.193            0.191           0.187             0.187           0.185 
      Magnesium    0.062            0.048             0.062           0.059             0.059           0.044 
      Nitrogen        1.632             1.013            1.453           1.233              1.214           1.112 
      Phosphorus    0.401            0.394             0.367           0.345              0.389   0.383     
      Potassium      1.201            1.111             1.211           1.081              1.093           0.073                                                
      Sodium          0.182            0.173             0.193           0.188              0.185           0.171                               
 
 
72 
 
 
 
 
 
Table 7: Major chemical elements (%) of bulbs of the two cultivars of shallot ( Allium 
ascalonicum) purchased from Mallam Atta Market, Accra in October 2012. 
 
         
                                                                                    Test 
 
                                                  1                                 2                                        3 
                          
       Chemical        Pale                                   Pale                                 Pale 
      Elements (%)  brown cv.    Pink cv.         brown cv.      Pink           brown cv.    Pink cv. 
 
       Calcium        0.140          0.110             0.147             0.121            0.142            0.110  
       Magnesium   0.055          0.050             0.060             0.046            0.059            0.046                                                  
       Nitrogen        1.224          1.211             1.263             1.199            1.276            1.214 
       Phosphorus   0.389           0.395             0.379             0.400            0.411    0.393     
       Potassium     1.212           1.111             1.236             1.019            1.226            1.112                                                
       Sodium         0.183           0.183             0.185             0.189            0.190            0.192                            
 
 
73 
 
 
 
 
 
Table 8: Means of the values of the major chemical elements ( % ) of the bulbs of the two 
cultivars of shallot ( Allium ascalonicum) recorded in Tables 3 to 7 
 
.           
            Chemical                  Pale-brown cv.                                    Pink cv. 
                                        Range              Mean                  Range                     Mean 
 
             Calcium          0.149-0.193       0.164±0.01          0.096-0.215           0.145±0.02 
             Magnesium     0.041-0.062       0.054±0.00          0.037-0.061           0.046±0.00 
             Nitrogen          1.124-1.643       1.456±0.07          0.882-1.432           1.192±0.05 
             Phosphorus     0.367-0.498       0.406±0.01          0.296-0.412           0.367±0.01 
             Potassium       1.025-1.236       1.142 ±0.03         0.738-1.134           0.979±0.08 
             Sodium           0.168-0.193       0.179±0.00          0.143-0.192           0.175±0.01 
  
 
 
 
 
 
74 
 
EXPERIMENT C. Aspergillus niger growth on bulbs of fresh stocks of the two shallot 
cultivars in market stalls. 
The bulbs of the two cultivars of shallot were seemingly readily prone to A. niger infection as 
shown by the data in Tables 9. The fungus did not only invade the exposed surfaces of the bulbs 
but also, despite the cover of the scale leaves, invaded the surfaces of the swollen leaf bases 
and even the contact surfaces of the bulblets. The extent of colonization, however, varied with 
the different sites. Other factors which influenced infection were the source of bulbs and the 
type of cultivar. 
 
Remarkably, for all the sites of colonization, bulbs of the pale-brown cultivar showed higher 
number of infected bulbs than bulbs of the Pink variety. The difference in number of bulbs 
involved may be marginal as in the category of “ Contact surfaces of mini-bulbs” where the 
respective numbers of infected bulbs for the pale-brown and pink cultivars for Agbogbloshie 
market were 64 and 64,and  the Anloga market 68 and 65 out of the 100 observed bulbs in each 
case. The greatest difference was observed on the adaxial surface of the outer scale leaf with 
the corresponding values out of 100 bulbs of 80 and 68 for the Agbogbloshie bulbs, and 86 and 
73 for the Anloga bulbs.  
 
Colonization of the different parts of the bulbs showed all sorts of combinations and 
permutations. The photograph in Plate 8 shows an example of the colonization of the scale 
leaves of the bulb. 
 
75 
 
Table 9: A. niger growth on the bulbs of the two shallot cultivars at the time of purchase from 
Agbogbloshie Market, Accra ( August 2011) and Anloga market, Volta region (January 
2014). 
 
                                      Percentage, out of 100 bulbs, with A. niger growth purchased from 
 
     Site of   bulb with               Agbogbloshie Market      Anloga Market 
A. niger growth      Pale-brown cv.              Pink cv.             Pale-brown cv.    Pink cv.                                             
 
      Abaxial surface  
      of outer brown                  68                             58                       76                         69 
      papery leaf scale 
 
      Adaxial surface of  
      outer brown                      80                              68                      86                         73 
      papery leaf scale 
 
     Abaxial surface  
     of inner brown                   84                              82                      86                        80 
     papery leaf scale  
 
     Adaxial surface of  
     inner brown                      70                               68                      80                        76 
     papery leaf base 
 
     Abaxial surface of 
     Outermost fleshy  
      leaf base                          72                               69                      78                        65 
 
     Contact surfaces  
     of mini-bulbs                   64                                64                     68                        65 
 
 
        
76 
 
 
 
 
 
 
                                                                                                                                           X 2 
Plate 8. Photograph showing colonies of Aspergillus niger on the inner and outer scale leaves 
of naturally infected shallot bulb (Allium ascalonicum) of Pale-brown cultivar on sale at the 
Agbogbloshie market 
 
 
 
77 
 
EXPERIMENT D. Influence of micro-habitats of shallot bulbs on the development of 
Aspergillus niger conidiophores 
An examination of the colonies of A. niger growing at the different locations with the high 
power of the microscope revealed an influence of the microhabitat on the colonies. The 
unusually rapid transportation of the protoplast of the mature hyphae to the growing apices 
rendered the empty hyphae unstainable and, therefore, difficult to observe morphological 
details. Reliable quantitative data were provided by the thick-walled conidiophore. The length 
of the conidiophores at the different locations were measured and their means calculated. 
 
The values of the mean conidiophore lengths are tabulated in Table 10. All the four sets of 
conidiophores showed significantly different mean conidiophore lengths, ranging from 
1125.8±9.2 to 2124.8±12.5 µm on bulbs of the pale-brown cultivar and 799.8±10.2 to 1720.5± 
11.5µm on bulbs of the pink cultivar. These values clearly point to the development of larger 
conidiophores in bulbs of the pale-brown cultivar. The possible reasons for this observation 
will be discussed later in detail under “General Discussion”. 
 
 
 
 
 
 
78 
 
 
Table 10: Influence of the micro-environment of bulbs of the two shallot cultivars purchased 
from Anloga, Volta region on the development of the conidiophores of A. niger. 
         
             Site of  bulb with           Mean length of 50 Conidiophore±Standard Error  (µm)on 
A. niger growth Pale-brown cv.                           Pink cv. 
              
           Abaxial  surface of outer           2124.8±12.5 a                         1720.5±11.5 b 
           brown scale leaf 
 
           Abaxial surface of inner           1855.3±10.8 a                         1480.3±12.3 b 
           brown scale leaf 
             
           Abaxial surface of 
           outermost fleshy                      1125.8±9.2 a                            799.8±10.2 b 
            leaf base.  
 
           Contact surface of                    1024.6±8.8 a                          1000.8±11.8 b 
           mini bulbs 
                       
           
 
By the calculated Scheffe’s Confidence Limit, pairs of values in horizontal rows bearing the 
same letters are not significantly different at 5% level of probability. 
 
 
 
79 
 
Experiment E. Culture characteristics of the five test isolates of Aspergillus niger 
The isolates were obtained from bulbs of both shallot cultivars and selected on the basis of the 
three characteristics stated in Table 11. They differed by the rate of growth on Potato Dextrose 
Agar Petri plates. Isolate 1 grew fastest attaining a mean culture diameter of 6.98±0.08 cm in 7 
days. It was followed by Isolates 3, 4, 5 and 2 in descending order. The slower growing isolate 
attained a mean culture diameter of 4.35±0.04 cm only. Values for the five Isolates significantly 
different from each other are indicated in Table 11. 
 
Of the two remaining characteristics each isolate had its peculiar combination. Other 
characteristics of the Isolates will undoubtedly emerge in the course of this investigation 
 
 
 
 
 
 
 
 
 
 
80 
 
 
 
 
 
Table 11: Characteristics of A. niger Isolates 1, 2, 3, 4 and 5 as test fungi in   Experiments F-J, 
L, N, P and Q. 
 
                                 Mean Culture               Growth habit 
                                 Diameter (cm)              after of mycelium 
           Isolate           7 days at 32±2oC          on Petri plate                 Colour of mycelium 
 
               1                  6.98±0.08 a                 Flat mycelium                  Brown black 
               2    4.35±0.04 b                 Presence of Zonation       Brown 
               3                  6.40±0.08 c                 Frequent sectoring           Brown black 
               4                  6.28±0.07 c                 Flat mycelium                  Black 
               5                  5.98±0.06 d                 Fluffy growth                   Black 
               
 
 
By the calculated Scheffe’s Confidence Limit, pairs of values in horizontal rows bearing the 
same letters are not significantly different at 5% level of probability. 
 
 
 
 
 
81 
 
 
                                        1                                                                         2 
 
                                    3                                                                         4 
 
 
PLATE 9:  Photographs of cultures of 
Aspergillus niger Isolates 1, 2, 3, 4 and 5 
 X  2 3⁄ / 
 
 
82 
 
EXPERIMENT F. Germination of conidia of the five test Aspergillus niger Isolates and 
pattern of growth on the swollen leaf bases and the scale leaves and fate of the germ tubes. 
The variables which were involved in the conidia germination in this experiment were; the 
Isolate of the fungus, the cultivar of the shallot, the component part of the bulb and the media. 
Some showed a uniform pattern of the influence while other factors showed irregular effect. 
The following statements summarize the findings reported in Tables 12 to 16. 
a. Aspergillus niger conidia did not germinate in distilled water 
b. PDB dilutions of 1:40 was a very poor germination medium. For example, only 7.4 
(Table 12), 4.8 (Table 13) and 10.8 (Table 16) per cent of conidia of Aspergillus niger 
Isolates 1, 2 and 5 respectively germinated on glass slides. Other instances of low 
germination in the 1: 40 PDB dilution medium showed percentage germination below 
6.0 per cent. 
c. There was good conidial germination in the higher concentrations of the PDB solutions.  
Naturally, the best germination was attained in the 1: 10 PDB dilution medium. 
Percentage germination ranged from 32.0 (Table 15) to 48.5 (Table 16) on glass slides. 
The majority of the tests gave germination in this medium on shallot bulb tissues 
between 70 (Table 12) and 93 (Table 15) per cent. 
d. Percentage germination then decreased with increasing dilution of the medium. 
e. Also, at any PDB dilution, germination was better on suspension drops on pale-brown 
cultivar bulb tissues than on pink cultivar tissues. 
f. Interestingly better percentage was recorded in suspension drops on the scale leaves than 
on the swollen leaf bases. 
83 
 
g. Mean germ-tube lengths bore the same relationship to medium concentration as 
percentage germination. The longest germ-tubes were found in the 1:10 PDB dilution 
medium and germ-tube length decreased with increasing dilution of the medium 
h. Slides incubated for 24 hours were thoroughly examined under the microscope after 
staining with Lactophenol cotton blue. The germ-tubes grew over the epidermal peels 
without showing any discerniable relationship with the surface architecture of the 
epidermis. 
i. Comparing the performance of the five Aspergillus niger isolates, Isolate 3 as shown in 
Table 14 was stimulated best by the media and Isolate 2 as shown in Table 13 did not 
grow as well as the other Isolates, giving an indication of the range of response of A. 
niger generally. 
 
The repeat experiments which used Galactose, Glucose and Sucrose at concentrations of 1.25, 
2.5, 5.0 and 10.0 g/l gave results with unique features as recorded in Tables 17 to 31. 
(a) First, there was again no germination of the conidia of all five isolates of Aspergillus 
niger in distilled water. 
(b) The conidia did not also germinate in the media of all the three sugars of concentration 
of 1.25 g/l. 
(c)  The three sugars supported conidial germination to different degrees in media of 
concentrations of 2.5, 5.0 and 10.0 g/l. 
(d)  For all the three sugars, the concentrations of 10.0 g/l, supported the highest percentage 
germination and greatest growth of the germ-tubes, albeit to different degrees. The 
respective values then decreased with increased dilution of the media. 
84 
 
(e) Notably, in this test as with the Potato Dextrose Broth test, the conidia germinated better 
on the scale leaves than on the swollen leaf base. For instance, germination on the outer 
scale leaf, inner scale leaf and swollen leaf base in the 10 g/l galactose medium in Tables 
17 to 21 on both pale-brown and pink cultivar was 34.3-40.3, 29.2-37.2 and 18.4-23.2 
per cent, respectively. 
(f) Mean germ-tube lengths bore similar relationship to medium concentration and conidial 
percentage germination. Mean germ-tube length on the outer scale leaf, inner scale leaf 
and swollen leaf base in the 10 g/l galactose medium in Tables 17 to 21 of both the pale-
brown and pink cultivar was 35.3±3.4 – 57.8±4.0, 31.2±3.3 – 53.7±3.2 and 29.3±3.9 – 
50.4±4.9 µm respectively. 
(g) The conidia of Isolates 1, 2 and 4 germinated best in the glucose media while those of 
Isolates 3 and 5 germinated best in the sucrose media. The galactose media were the 
most unfavourable for the all the A. niger isolates. 
(h) Whereas conidia of A. niger Isolate 1 germinated better than the remaining isolates, 
performance of Isolate 2 was inferior to the rest. 
(i) The germ-tubes of all the isolates in all the media grew in all directions on the swollen 
leaf base and scale leaves. 
 
 
 
 
 
 
 
85 
 
Table 12: Germination of conidia of A. niger Isolate 1 in light of 76 lux in 12 hours in Potato 
Dextrose Broth (PDB) suspension drops on different component parts of bulbs of shallot 
(Allium ascalonicum) 
                                                           
                                                                         Percentage                           Mean Germ-tube 
                                                                        Germination                         Length (µm)  
      
                                      PDB:                          
                                      distilled              Pale-                                       Pale               
        Component           water                  brown              Pink                brown            Pink 
        part of bulb           mixture (v/v)       cv.                   cv.                   cv.                  cv. 
 
        Outer surface           1:10   76.4   78.5              89.6±5.6 a       96.3±7.0 a 
        of outer                    1:20   73.7 62.3              69.1±3.3 a       72.2±3.1 a 
        scale leaf                 1:30                64.8  23.4              62.1±4.8 a       55.2±1.4 a 
                                        1:40  4.1   0.0              35.9±3.2                - 
                                  Distilled H2O          0.0   0.0 -     - 
 
        Inner surface           1:10 88.0 88.1              94.3±6.2 a      99.3±3.2 a 
        of outer                    1:20 71.2 71.3     62.2±5.3 a     76.5±3.1 a 
        scale leaf                 1:30  51.9 42.1              60.8±5.8 a      59.7±2.9 a 
                                        1:40  1.0   0.0              26.5±4.1 - 
                                  Distilled H2O  0.0   0.0    -                    - 
 
 
 Outer surface          1:10               89.1                 74.9              102.2±4.9 a     97.8±2.1 a 
 of inner                   1:20               64.0                 58.9                65.2±5.3 a     60.3±2.7 a 
 scale leaf         1:30               68.8                  33.2               60.8±5.8 a     52.1±5.3 a 
                                1:40                 3.9 0.0                26.5±4.1            - 
                           Distilled H2O       0.0                    0.0 -                - 
 
Inner surface           1:10               87.2                 71.9                98.7±3.6 a      90.3±2.2 a 
of inner                   1:20               69.9                  63.8               55.9±3.8 a      63.2±2.3 a 
scale leaf         1:30              52.7                  47.4                42.1±2.9 a      45.4±5.3 a 
                                1:40                2.8                    1.3                25.4±2.0 a      22.2±1.3 a 
                          Distilled H2O       0.0                    0.0    -                      - 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
86 
 
 
      Table 12 continued. 
                                                                  
                                                                         Percentage                           Mean Germ-tube 
                                                                        Germination                         Length (µm)  
      
                                      PDB:                          
                                      distilled              Pale-                                       Pale               
        Component           water                  brown              Pink                brown            Pink 
        part of bulb           mixture (v/v)       cv.                   cv.                   cv.                  cv 
 
 
 Outer surface          1:10 44.1            43.9       83.9±3.6 a     80.2±1.9 a 
 of outermost           1:20                    21.6            18.9 55.3±4.0 a   63.9±2.7 a 
 swollen leaf         1:30                    22.3            12.0 27.4±3.8 a   35.2±3.8 a 
 base                        1:40                      0.0              0.0 -    - 
                           Distilled H2O            0.0              0.0 -                    -    
  
 
 
                                1:10          42.9                          55.9±2.1 
 Glass slide              1:20          23.7                          39.7±2.7 
                     1:30         13.7                          22.6±4.0 
                                1:40           7.4                            18.3±1.2 
                          Distilled H2O           0.0                                   - 
 
 
 
 
 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
87 
 
Table 13: Germination of conidia of A. niger Isolate 2 in light of 76 lux in 12 hours in Potato 
Dextrose Broth (PDB) suspension drops on different component parts of bulbs of shallot 
(Allium ascalonicum). 
                                                                 
                                                                         Percentage                           Mean Germ-tube 
                                                                        Germination                         Length (µm)  
      
                                      PDB:                          
                                      distilled              Pale-                                    Pale               
        Component           water                  brown              Pink             brown              Pink 
        part of bulb           mixture (v/v)       cv.                   cv.               cv.                  cv. 
 
        Outer surface           1:10 69.0               69.5   89.4±4.4 a  83.8±6.1 a 
        of outer                    1:20 65.3               47.3   81.9±2.4 a  72.6±4.2 a 
        scale leaf                 1:30                   42.3               22.8   71.3±3.8 a  60.0±2.0 a 
                                        1:40   0.0                 0.0        -     - 
                                    Distilled H2O          0.0                 0.0        -     - 
 
        Inner surface           1:10   77.5               72.7    86.4±2.7 a       78.9±7.1 a 
        of outer                   1:20                   62.0               58.3    85.6±3.5 a    70.0±2.7 b 
        scale leaf                 1:30                  56.8               37.4    68.6±4.2 a    62.1±2.5 a 
                                        1:40                    5.7                 0.0    34.4±2.5 a    30.2±3.8 a 
                                   Distilled H2O          0.0                 0.0        -      - 
 
 Outer surface          1:10                  82.4                 77.7            80.3±3.2 a        81.2±2.9 a 
 of inner                   1:20                  60.4                 43.9            69.4±4.3 a        70.3±3.2 a 
 scale leaf         1:30                  34.5                 22.9            55.4±4.2 a        54.3±6.0 a 
                                1:40                    4.5                   0.0              34.4±1.8            
                           Distilled H2O          0.0                   0.0  -      - 
 
Inner surface           1:10                  65.4                 76.7            77.5±5.5 a        80.2±2.5 a 
of inner                   1:20                  62.0                 40.5  64.3±3.8 a          70.2±2.1 a 
scale leaf         1:30                  30.4                 23.8  33.4±2.4 a         43.2±2.8 a 
                                1:40                    0.0                   0.0    -    - 
                           Distilled H2O          0.0                   0.0    -    - 
 
 
 Table 13 continued.   
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
 
88 
 
Table 13 continued 
                                                                  
                                                                         Percentage                           Mean Germ-tube 
                                                                        Germination                         Length (µm)  
      
                                      PDB:                          
                                      distilled              Pale-                                       Pale               
        Component           water                  brown              Pink                brown            Pink 
        part of bulb           mixture (v/v)       cv.                   cv.                  cv.                  cv. 
 
 
 Outer surface          1:10                  33.0 30.4             54.4±3.7 a     44.2±3.2 a 
 of outermost           1:20                  32.5 23.3             40.3±4.2 a       34.5±3.1 a 
 swollen leaf         1:30                    8.6    0.0             21.1±1.9 a       20.0±2.0 a 
 base                        1:40                    0.0   0.0   -                    - 
                           Distilled H2O          0.0   0.0   -          - 
 
 
 
                                1:10        37.1         32.3±3.1 
 Glass slide              1:20        18.5         34.6±2.3 
                     1:30        11.0         26.4±3.7 
                                1:40                              4.8                       21.4±2.8 
                          Distilled H2O                     0.0                                  - 
 
 
 
 
 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
89 
 
Table 14: Germination of conidia of A. niger Isolate 3 in light of 76 lux in 12 hours in Potato 
Dextrose Broth (PDB) suspension drops on different component parts of bulbs of shallot 
(Allium ascalonicum). 
                                                               
                                                                         Percentage                           Mean Germ-tube 
                                                                        Germination                         Length (µm)                       
                          PDB 
                                      distilled              Pale-                                  Pale               
        Component           water                  brown              Pink           brown              Pink 
        part of bulb           mixture (v/v)       cv.                   cv.             cv.                    cv 
 
 
        Outer surface           1:10       85.6    76.9            102.4±5.7 a 93.2±2.5 a 
        of outer                    1:20                   61.6    60.6              82.7±7.2 a 64.7±4.2 a 
        scale leaf                 1:30                   53.5    34.5              70.4±5.2 a 60.4±3.7 a 
                                        1:40                     0.0       0.0   -   - 
                                    Distilled H2O          0.0       0.0   -   - 
 
        Inner surface           1:10 77.0 61.3            112.3±7.8 a   90.3 ±4.5 a 
        of outer                   1:20                    45.1 43.0 82.7±2.6 a   78.4±5.2 a 
        scale leaf                 1:30                    35.6       22.7 54.5±6.3 a   60.4±3.6 a 
                                        1:40     0.0   0.0   -    - 
                                  Distilled H2O     0.0    0.0   -    - 
 
 
 Outer surface          1:10   90.4 76.8            109.9±7.4 a    95.4±4.7 a 
 of inner                   1:20   73.7 47.0  87.7±4.7 a    85.3±3.9 a 
 scale leaf         1:30   45.9 32.1  65.8±4.5 a    70.4±3.8 a 
                                1:40     0.0   0.0    -     - 
                           Distilled H2O     0.0   0.0    -     - 
 
Inner surface           1:10    87.9 79.0   84.9±4.3 a   80.4±5.2 a 
of inner                   1:20    56.5 45.8   80.9±4.3 a   69.9±4.7 b 
scale leaf         1:30    40.8 25.8   67.3±2.4 a   60.3±3.5 a 
                                1:40      0.0   0.0    -    - 
                           Distilled H2O          0.0   0.0    -     - 
 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal row 
bearing the same letters are not significantly different at 5% level of probability 
90 
 
    Table 14 continued 
 
                                                                 
                                                                         Percentage                           Mean Germ-tube 
                                                                        Germination                         Length (µm)  
      
                                      PDB:                          
                                      distilled              Pale-                                    Pale               
        Component           water                  brown              Pink             brown            Pink 
        part of bulb           mixture (v/v)       cv.                   cv.               cv.                  cv 
 
                     
 
 Outer surface          1:10  41.9               29.6              47.2±3.6 a     50.0±3.2 a 
 of outermost           1:20  20.5               16.6              39.5±5.6 a      41.2±4.2 a 
 swollen leaf         1:30  10.7                 9.0               20.1±1.5 a      31.2±5.1 a 
 base                        1:40    0.0                 0.0   -  - 
                           Distilled H2O    0.0                 0.0   -  - 
 
 
 
                               1:10           42.2        40.6±4.3 
 Glass slide             1:20           27.7        33.5±2.4 
                    1:30           11.9         30.1±1.8 
                               1:40             0.0              - 
                          Distilled H2O                  0.0              -  
 
 
 
 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
91 
 
Table 15: Germination of conidia of A. niger Isolate 4 in light of 76 lux in 12 hours in Potato 
Dextrose Broth (PDB) suspension drops on different component parts of bulbs of shallot 
(Allium ascalonicum). 
                                                                       
                                                                         Percentage                           Mean Germ-tube 
                                                                        Germination                         Length (µm)  
      
                                      PDB:                          
                                      distilled             Pale                                   Pale               
        Component           water                 brown           Pink              brown                  Pink 
        part of bulb           mixture (v/v)      cv.                cv.                cv.                        cv 
 
   
       Outer surface          1:10                  88.3              88.4              116.0±4.9 a      110.0±7.2 a 
        of outer                  1:20                  62.3              54.4  76.7±3.5 a   83.3±2.8 a 
        scale leaf                1:30  42.0              23.3  45.6±5.2 a   67.2±2.1 b 
                                       1:40   0.0                0.0     -    - 
                                    Distilled H2O   0.0                0.0     -    - 
 
        Inner surface           1:10                 91.8 83.6  120.5±7.9 a       99.9±4.6 a 
        of outer                   1:20                  64.4 45.9    90.5±3.9 a       87.4±4.5 a 
        scale leaf                 1:30  41.8 20.0    55.5±3.7 a       50.3±3.5 a 
                                        1:40   0.0   0.0      -       - 
                                   Distilled H2O   0.0   0.0      -       - 
 
        Outer surface          1:10              89.8               80.6             110.4±6.7 a          118.4±7.9 
        of inner                   1:20              64.9               52.4               83.2±3.4 a            98.4±5.2 
        scale leaf                1:30              44.4               32.1               67.3±4.6 a            76.5±5.3 
                                       1:40                0.0                0.0                 -                 - 
                                Distilled H2O            0.0                0.0                 -        - 
 
       Inner surface           1:10        92.9        86.4           98.4±6.4 a          100.1±3.6 a 
       of inner                   1:20        56.0        40.7              55.4±4.3 a            67.5±4.1 a 
       scale leaf               1:30        42.1        29.9           45.9±2.8 a            50.1±3.7 a 
                                      1:40          0.0         0.0             -                 - 
                                 Distilled H2O         0.0         0.0             -                 - 
 
 
     
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
92 
 
 Table 15 continued 
                                                                       
 
                                                                         Percentage                           Mean Germ-tube 
                                                                        Germination                         Length (µm)  
      
                                      PDB:                          
                                      distilled              Pale-                                       Pale               
        Component           water                  brown              Pink                brown            Pink 
        part of bulb           mixture (v/v)       cv.                   cv.                   cv.                  cv 
 
 
 Outer surface          1:10                     38.1            26.9          58.7±3.9 a          62.1±1.8 a 
 of outermost           1:20                     26.8            18.9          60.7±3.1 a          52.1±4.7 a 
 swollen leaf         1:30                     16.4            16.0          45.3±4.2 a          40.7±3.6 a 
 base                        1:40                       0.0              0.0               - - 
                           Distilled H2O             0.0              0.0               -                 - 
 
 
 
                                1:10                                 32.0                   51.2±3.7 
 Glass slide              1:20                                 21.2                   38.9±4.2 
                     1:30                       16.1                   30.3±3.3 
                                1:40                                   0.0                         -                  
                          Distilled H2O                          0.0                         -                  
 
 
 
 
 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
93 
 
Table 16: Germination of conidia of A. niger Isolate 5 in light of 76 lux in 12 hours in Potato 
Dextrose Broth (PDB) suspension drops on different component parts of bulbs of shallot 
(Allium ascalonicum). 
 
                                                                         Percentage                           Mean Germ-tube 
                                                                        Germination                         Length (µm)  
      
                                      PDB:                          
                                      distilled              Pale-                                       Pale               
        Component           water                  brown              Pink                brown            Pink 
        part of bulb           mixture (v/v)       cv.                   cv.                   cv.                  cv 
 
   
        Outer surface           1:10 72.0                 77.5 84.0±2.0 a     77.2±5.4 a 
        of outer                    1:20 55.3 57.3 77.6±6.7 a   65.9±7.2 a 
        scale leaf                 1:30  24.9 22.6 45.6±3.5 a   55.3±3.3 a 
                                        1:40   0.0   0.0      - - 
                                    Distilled H2O          0.0   0.0      - - 
        Inner surface           1:10   77.5                 70.7            92.2±5.8 a      83.3±4.9 a 
        of outer                   1:20                   54.4                 60.4            81.2±2.7 a      76.4±4.1 a 
        scale leaf                 1:30                   28.8                 27.3            60.4±4.6 a      56.7±3.5 a 
                                        1:40                     0.0                   0.0   -   - 
                                   Distilled H2O           0.0                  0.0   -   - 
 
 Outer surface          1:10                   79.8                  83.7   87.3±3.7 a    84.3±3.9 a 
 of inner                   1:20                   60.3                  63.1   88.9±4.9 a    76.3±2.9 a 
 scale leaf         1:30                   30.7                   32.9   70.5±4.7 a    65.3±2.8 a 
                                1:40 0.0  0.0      -      - 
                           Distilled H2O 0.0  0.0      -      - 
 
Inner surface           1:10                   64.6 72.7 80.4±2.6 a      76.8±8.4 a 
of inner                   1:20                   52.7 43.2 69.3±3.7 a    70.4±4.6 a 
scale leaf        1:30                   33.4 33.8 58.9±2.0 a      57.7±4.9 a 
                               1:40                     0.0   0.0   -       - 
                            Distilled H2O          0.0   0.0   -       - 
 
 
  
 By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
94 
 
 
Table 16 continued. 
                                                                           
                                                                         Percentage                           Mean Germ-tube 
                                                                        Germination                         Length (µm)  
      
                                      PDB:                          
                                      distilled              Pale                                    Pale               
        Component           water                  brown              Pink            brown            Pink 
        part of bulb           mixture (v/v)       cv.                   cv.              cv.                  cv 
 
 
 Outer surface          1:10                 43.0                38.4  56.8±4.5 a  60.4±3.8 a 
 of outermost           1:20                 32.3                33.1  50.4±4.3 a  50.9±4.2 a 
 swollen leaf         1:30                 10.6             9.9  35.9±3.1 a  42.1±5.2 a 
 base                        1:40                   0.0 0.0     -    - 
                           Distilled H2O         0.0 0.0     -    - 
 
 
 
                                1:10    48.5                    44.5±5.7        
 Glass slide              1:20    31.6                     41.7±4.8   
                     1:30    21.0                    34.2±1.9   
                                1:40                        10.8                     23.1±3.1   
                          Distilled H2O    0.0                                             - 
 
 
 
 
 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
95 
 
Table 17: Germination of conidia of A. niger Isolate 1 at 32±2oC in 12 in light of 76 lux 
hours in suspension drops of Glucose of different concentrations on parts of bulbs of the two 
cultivars of shallot (Allium ascalonicum). 
 
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of glucose (g/L)     bulb           Pale-brown cv.   Pink cv.     Pale-brown      Pink cv. 
 
                     10.0             Outer S.L.         47.3               51.2             68.5±5.3 a        61.3±4.5 a 
                                         Inner S.L.         45.3                49.3             60.3±5.0 a       53.0±4.7 a 
                                         S.L.B                40.2                38.5             62.5±4.5 a        64.3±6.6 a 
                     5.0               Outer S.L.        43.5                41.2             60.4±3.3 a       56.5±5.5 a 
                                         Inner S.L.         40.2                37.3             53.2±4.2 a       50.3±3.1 a 
                                         S.L.B                35.2                33.4             50.8±5.0 a        51.2±6.7 a 
                    2.5               Outer S.L.         18.3                12.5             33.4±4.3 a        35.8±4.0 a 
                                         Inner S.L.         12.5                15.2             30.2±4.0 a        29.3±3.5 a 
                                         S.L.B                 9.8                 10.3             26.5±3.6 a        27.3±2.8 a 
                   1.25              Outer S.L.          0.0                  0.0                    -                     - 
                                        Inner S.L.          0.0                   0.0                    -                     - 
                                        S.L.B                 0.0                   0.0                    -                     - 
                    0.0              Outer S.L.           0.0                  0.0                    -                     - 
                                        Inner S.L.          0.0                   0.0                    -                     - 
                                        S.L.B                 0.0                   0.0                    -                      - 
    * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
96 
 
Table 18: Germination of conidia of A. niger Isolate 2 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of Glucose of different concentrations on parts of bulbs of the two 
cultivars of shallot (Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of glucose (g/L)     bulb            Pale-brown cv.   Pink cv.     Pale-brown      Pink cv. 
 
                     10.0              Outer S.L.        44.2                 42.3           60.8±6.5 a        53.4±4.2 a 
                                          Inner S.L.         40.2                 37.5          56.8±4.8 a        50.1±4.0 a 
                                           S.L.B               32.3                 30.3          50.8±3.9 a        46.5±4.5 a 
                      5.0               Outer S.L.        40.5                 33.2          49.5±5.6 a        51.3±5.2 a 
                                          Inner S.L.         33.2                 30.5          46.3±4.3 a         45.2±4.0 a 
                                           S.L.B               30.3                 22.8          40.0±5.2 a         39.8±5.1 a 
                     2.5               Outer S.L.         21.3                 15.5          30.3±3.0 a         23.5±4.0 a 
                                         Inner S.L.          23.4                 17.2          26.2±2.9 a         21.2±2.8 a 
                                         S.L. B               10.2                 11.1           20.8±3.1 a         20.3±2.5 a 
                   1.25               Outer S.L.          0.0                   0.0                -                       - 
                                         Inner S.L.          0.0                    0.0                -                       - 
                                          S.L.B                0.0                    0.0                -                       - 
                   0.0                 Outer S.L.         0.0                    0.0                 -                       - 
                                         Inner S.L.          0.0                    0.0                 -                       - 
                                         S.L.B                 0.0                    0.0                -                        - 
     * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
97 
 
Table 19: Germination of conidia of A. niger Isolate 3 at 32±2oC in light of 76 lux 12 hours in 
suspension drops of Glucose of different concentrations on parts of bulbs of the two cultivars 
of shallot (Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of glucose (g/L)     bulb            Pale-brown cv.   Pink cv.     Pale-brown      Pink cv. 
 
                     10.0         Outer S.L.          44.2                   46.5          53.4±3.9 a         54.3±5.0 a 
                                     Inner S.L            43.2                   48.3          50.9±4.3 a         50.0±4.3 a 
                                     S.L.B                 32.3                    33.2          43.5±5.1 a         45.3±5.5 a 
 
                     5.0            Outer S.L.          35.3                  37.5 40.3±3.3 a        43.5±5.0 a 
                                      Inner S.L            34.5                 40.2 37.8±4.3 a        40.8±4.2 a 
                                      S.L.B                 26.7                  25.2 33.4±4.0 a         32.3±3.3 a 
 
                     2.5           Outer S.L.           28.2                 15.8    29.3±3.0 a        25.8±3.1 a 
                                     Inner S.L             26.3                 25.2    27.2±2.8 a       24.3±2.6 a 
                                      S.L.B                 10.2                   8.5    26.5±2.8 a       24.2±2.3 a 
        1.25           Outer S.L.            0.0                   0.0              -                       - 
                                     Inner S.L.            0.0                    0.0              -                       - 
                                     S.L.B                   0.0                    0.0              -                       - 
                    0.0           Outer S.L.            0.0                    0.0              -                        - 
                                    Inner S.L.             0.0                    0.0              -                        - 
                                    S.L.B                   0.0                    0.0               -                        - 
    * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
  
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
98 
 
Table 20: Germination of conidia of A. niger Isolate 4 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of Glucose of different concentrations on parts of bulbs of the two 
cultivars of shallot (Allium ascalonicum) 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of glucose (g/L)     bulb            Pale-brown cv.   Pink cv.     Pale-brown      Pink cv. 
 
                 10.0                 Outer S.L.         51.3                 45.2         63.4±5.3 a          60.8±3.2 a 
                                         Inner S.L.         47.3                  44.3         60.2±6.2 a          58.3±4.3 a 
                                         S.L.B                40.2                  35.2         53.5±4.3 a          55.4±3.2 a 
                 5.0                   Outer S.L.        42.0                  39.3          58.3±4.1 a          55.3±3.8 a 
                                         Inner S.L.         37.3                  36.3   59.2±5.0 a         52.0±4.9 a 
                                         S .L.B               29.8                  28.4   53.1±5.9 a         54.8±3.4 a 
               2.5                     Outer S.L.        25.2                   23.2         50.3±5.1 a          48.9±4.1 a 
                                          Inner S.L.        20.1                   24.2         46.3±5.0 a         50.2±3.6 a 
                                          S.L.B               16.3                   12.5         48.5±4.3 a         46.8±3.8 a 
              1.25                     Outer S.L.         0.0                     0.0              -                       - 
                                         Inner S.L.          0.0                     0.0               -                        - 
                                         S.L.B                 0.0                     0.0              -                       - 
              0.0                      Outer S.L.         0.0                      0.0              -                       - 
                                         Inner S.L.          0.0                      0.0              -                       - 
                                         S.L.B                 0.0                      0.0              -                       - 
   * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
  
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
99 
 
Table 21: Germination of conidia of A. niger Isolate 5 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of Glucose of different concentrations on parts of bulbs of the two 
cultivars of shallot ( Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of glucose (g/L)     bulb            Pale-brown cv.   Pink cv.     Pale-brown      Pink cv. 
 
                    10.0                Outer S.L         46.7                44.4           66.3±4.8 a          63.4±4.9 a 
                                           Inner S.L.         45.2                40.2           63.8±5.3 a         58.0±6.3 a 
                                            S.L.B               30.1                33.5           64.5±6.0 a         58.3±3.6 a 
                    5.0                  Outer S.L         32.3                31.5           65.8±3.8 a          67.8±3.9 a 
                                           Inner S.L.         27.5                28.9           61.2±4.1 a         62.0±5.3 a 
                                           S.L.B               18.3                 20.2           64.5±6.0 a         59.3±4.0 a 
                   2.5                  Outer S.L          21.1                18.3           43.2±2.8 a         39.2±4.1 a 
                                          Inner S.L.         18.5                 18.1           40.3±3.2 a         37.8±3.3 a 
                                          S.L.B                11.2                 10.3           38.3±2.7 a         36.3±4.6 a 
      1.25                  Outer S.L.          0.0                   0.0               -                       - 
                                          Inner S.L.          0.0                   0.0               -                       - 
                                           S.L.B                0.0                   0.0              -                        - 
                 0.0                    Outer S.L.         0.0                   0.0               -                       - 
                                          Inner S.L.          0.0                   0.0               -                       - 
                                          S.L.B                0.0                   0.0               -                        - 
         
 * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
100 
 
Table 22: Germination of conidia of A. niger Isolate 1 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of Galactose of different concentrations on parts of bulbs of the two 
cultivars of shallot ( Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
           of galactose (g/L)   bulb            Pale-brown cv.   Pink cv.     Pale-brown     Pink cv. 
 
                    10.0               Outer S.L         40.3                38.5           56.9±5.1 a        57.8±4.0 a 
                                          Inner S.L.         38.3                33.4           52.3±4.3 a       53.2±3.2 a 
                                          S.L.B                23.2                22.1           50.4±4.0 a       49.3±4.4 a 
                    5.0                Outer S.L          33.2                30.8           48.8±3.1 a        43.1±2.6 a 
                                          Inner S.L.         32.1                29.2           43.2±4.3 a       38.3±2.9 a 
                                          S.L.B                21.2                18.3           40.2±2.9 a       33.4±3.0 a 
                   2.5                  Outer S.L          23.1               19.5            27.8±2.3 a       28.3±2.3 a 
                                          Inner S.L.         21.3               15.8             30.2±3.1 a       24.2±2.5 a 
                                          S.L.B                12.3               10.2             25.2±2.9 a       20.2±3.0 a 
                 1.25                  Outer S.L.         0.0                  0.0               -                      - 
                                          Inner S.L.          0.0                  0.0              -                       - 
                                          S.L.B                 0.0                  0.0              -                       - 
                  0.0                  Outer S.L.          0.0                  0.0              -                       - 
                                         Inner S.L.           0.0                  0.0              -                       - 
                                         S.L.B                  0.0                  0.0              -                       - 
         
 * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
101 
 
Table 23: Germination of conidia of A. niger Isolate 2 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of Galactose of different concentrations on parts of bulbs of the two 
cultivars of shallot ( Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of galactose (g/L)  bulb            Pale-brown cv.    Pink cv.     Pale-brown      Pink cv. 
 
                     10.0              Outer S.L.      36.1                  35.5            44.5±5.0 a        35.3±3.4 a 
                                          Inner S.L        30.1                  34.8            40.2±5.6 a        31.2±3.3 a 
                                          S.L.B             18.4                   20.3            32.3±4.3 a        29.3±3.9 a 
                      5.0               Outer S.L.      31.2                   29.4           40.3±4.2 a         41.3±3.0 a 
                                          Inner S.L.       32.2                   27.5           36.2±4.3 a         33.2±3.1 a 
                                          S.L.B             18.0                   17.3  30.2±3.9 a        31.4±2.9 a 
                     2.5               Outer S.L.       20.8                   14.2            24.8±3.3 a        21.2±2.3 a 
                                         Inner S.L         15.5                  13.8             21.6±3.6 a        22.3±2.0 a 
                                          S.L.B               9.5                     8.3            20.2±2.0 a         18.3±1.9 a 
        1.25               Outer S.L.        0.0                      0.0                 -                      - 
                                         Inner S.L.         0.0                     0.0                 -                      - 
                                          S.L.B               0.0                      0.0                 -                      - 
                    0.0                Outer S.L.        0.0                      0.0                 -                      - 
                                         Inner S.L.         0.0                      0.0                 -                      - 
                                         S.L.B                0.0                      0.0                 -                      - 
          
* Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
102 
 
Table 24: Germination of conidia of A. niger Isolate 3 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of Galactose of different concentrations on parts of bulbs of two cultivars 
of  shallot  ( Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of galactose (g/L) bulb            Pale-brown cv.   Pink cv.     Pale-brown    Pink cv. 
 
                   10                    Outer S.L.       34.3               35.8            51.9±6.1 a        53.8±6.3 a 
                                           Inner S.L.        30.2               32.4            53.2±5.8 a       50.2±5.3 a 
                                            S.L.B              21.2               20.2            50.4±4.9 a       46.3±5.8 a 
                  5.0                     Outer S.L.      25.3                30.3            42.3±3.9 a      40.3±3.8 a 
                                            Inner S.L.      24.2                23.4            40.2±4.6 a       36.8±4.1 a 
                                             S.L.B            19.4                17.2            37.3±5.0 a       34.2±3.3 a 
                  2.5                     Outer S.L.      13.5                12.3           30.5±5.5 a       29.3±3.1 a 
                                            Inner S.L.      10.9                 12.1           28.3±4.3 a       26.2±3.1 a 
                                             S.L.B              9.3                  7.8            20.5±3.1 a      22.4±2.5 a 
                1.25                     Outer S.L.       0.0                  0.0              -                    - 
                                            Inner S.L.        0.0                  0.0              -                    - 
                                            S.L.B              0.0                  0.0              -                    - 
                  0.0                     Outer S.L.       0.0                  0.0              -                    - 
                                            Inner S.L.       0.0                  0.0             -                      - 
                                            S.L.B              0.0                  0.0              -                     - 
         
 * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
103 
 
Table 25: Germination of conidia of A. niger Isolate 4 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of Galactose of different concentrations on parts of bulbs of the two 
cultivars of shallot ( Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of galactose (g/L)  bulb            Pale-brown cv.   Pink cv.     Pale-brown      Pink cv. 
 
                    10.0               Outer S.L         37.8                  36.9          47.2±3.9 a      48.8±5.6 a 
                                          Inner S.L.         35.2                  34.3          46.3±4.1 a      45.3±4.0 a 
                                           S.L.B               22.2                  19.5          44.7±3.5 a       43.8±4.0 a 
                    5.0                 Outer S.L         29.3                  26.3          40.3±3.3 a       41.2±4.1 a 
                                          Inner S.L.         25.2                  21.3          36.2±2.8 a       35.3±3.1 a 
                                          S.L.B               17.9                  13.8           30.2±3.1 a       28.3±2.5 a 
                   2.5                 Outer S.L          14.8                  10.2           26.3±2.3 a       30.2±3.1 a 
                                         Inner S.L.         13.2                  10.5           24.2±1.9 a       22.4±2.3 a 
                                         S.L.B                  9.3                   8.8            21.3±2.1 a       19.4±2.0 a 
                 1.25                 Outer S.L.          0.0                   0.0              -                    - 
                                         Inner S.L.          0.0                    0.0              -                    - 
                                         S.L.B                 0.0                   0.0              -                    - 
                 0.0                  Outer S.L.          0.0                    0.0             -                    - 
                                        Inner S.L.           0.0                    0.0             -                    - 
                                        S.L.B                  0.0                    0.0              -                    - 
        
  * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
104 
 
Table 26: Germination of conidia of A. niger Isolate 5 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of Galactose of different concentrations on parts of bulbs of the two 
cultivars of shallot ( Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of glucose (g/L)     bulb            Pale-brown cv.   Pink cv.     Pale-brown      Pink cv. 
 
                   10.0               Outer S.L.       39.3                   37.8 50.1±4.0 a         52.3±5.0 a 
                                         Inner S.L.       37.2                    35.3          47.2±4.4 a         48.3±6.3 a 
                                          S.L.B             19.8                    20.1 44.4±4.8 a        45.0±5.5 a 
                   5.0                Outer S.L.        32.4                    31.2          41.0±3.6 a        40.3±3.3 a 
                                        Inner S.L.        31.3                     26.3     36.3±3.3 a        35.3±3.8 a 
                                        S.L.B              12.8                     17.4     38.2±4.0 a        30.3±2.5 a 
                   2.5                Outer S.L.       19.3                     18.3          33.3±2.5 a        29.3±2.6 a 
                                        Inner S.L.        18.3                     17.3          29.2±3.3 a       32.5±2.4 a 
                                        S.L.B              10.1                       9.9          25.3±2.8 a       26.8±3.0 a 
                1.25                Outer S.L.         0.0                       0.0              -                     - 
                                        Inner S.L.         0.0                       0.0              -                     - 
                                        S.L.B               0.0                       0.0             -                     - 
                 0.0                  Outer S.L.        0.0                       0.0             -                     - 
                                        Inner S.L.        0.0                        0.0             -                     - 
                                        S.L.B               0.0                        0.0             -                     - 
      
    * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
  
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
105 
 
Table 27: Germination of conidia of A. niger Isolate 1 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of sucrose of different concentrations on parts of bulbs of the two 
cultivars of  shallot (Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of sucrose (g/L)     bulb            Pale-brown cv.   Pink cv.     Pale-brown      Pink cv. 
 
                    10.0           Outer S.L.           49.9                 50.0             64.8±7.1 a        60.0±5.5 a 
                                      Inner S.L.            45.3                 47.3             60.3±6.3 a        61.3±5.7 a 
                                       S.L.B                  37.3                 40.2             56.8±6.5 a        52.4±4.3 a 
                    5.0              Outer S.L.          39.3                 43.1             50.2±5.3 a        48.5±4.1 a 
                                       Inner S.L.           40.3                 40.8             46.3±5.5 a        42.3±3.3 a 
                                       S.L.B                  30.2                 31.1     48.2±4.0 a        40.3±4.4 a 
                   2.5              Outer S.L.           20.2                 19.4             41.3±4.8 a        35.6±3.3 a 
                                      Inner S.L.            19.3                 18.3             36.2±3.9 a        37.2±2.8 a 
                                      S.L.B                   11.1                 10.9             30.3±4.4 a         27.3±3.7 a 
                 1.25              Outer S.L.            0.0                   0.0                    -                    - 
                                     Inner S.L.             0.0                    0.0                    -                   - 
                                      S.L.B                   0.0                    0.0                    -                    - 
                  0.0              Outer S.L.            0.0                    0.0                    -                    - 
                                      Inner S.L.            0.0                    0.0                    -                    - 
                                     S.L.B                    0.0                    0.0                   -                     - 
         
 * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
106 
 
Table 28: Germination of conidia of A. niger Isolate 2 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of sucrose of different concentrations on parts of bulbs of the two 
cultivars of shallot ( Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of glucose (g/L)    bulb            Pale-brown cv.   Pink cv.     Pale-brown      Pink cv. 
 
                     10.0             Outer S.L.        44.8                 43.5          43.3±4.1 a         40.3±3.3 a 
                                         Inner S.L.         40.3                 44.5          42.8±3.3 a         42.3±4.2 a 
                                         S.L.B                34.5                 33.3          40.5±3.1 a         41.3±5.0 a 
                     5.0               Outer S.L.        36.5                 35.5          33.4±4.0 a         35.3±3.4 a 
                                         Inner S.L.         32.3                 30.1          32.8±4.1 a         33.2±4.3 a 
                                         S.L.B               24.2                 26.4          31.4±2.9 a         25.4±4.4 a 
                    2.5               Outer S.L.         15.3                 12.5          26.3±3.3 a         27.3±3.2 a 
                                         Inner S.L.        14.7                 12.5           24.2±3.4 a         20.2±2.8 a 
                                         S.L.B               10.8                   9.2           20.8±2.1 a         18.3±2.5 a 
                  1.25               Outer S.L.          0.0                   0.0              -                      - 
                                        Inner S.L.           0.0                   0.0              -                      - 
                                         S.L.B                 0.0                   0.0              -                      - 
                   0.0                Outer S.L.          0.0                   0.0              -                      - 
                                        Inner S.L.           0.0                   0.0              -                      - 
                                        S.L.B                  0.0                   0.0              -                      - 
         
 * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
   
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
107 
 
Table 29: Germination of conidia of A. niger Isolate 3 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of sucrose of different concentrations on parts of bulbs of the two 
cultivars of  shallot ( Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of sucrose (g/L)    bulb            Pale-brown cv.   Pink cv.     Pale-brown        Pink cv. 
 
                    10.0              Outer S.L.         47.5                 46.5          58.2±3.9 a          55.2±3.3 a 
                                         Inner S.L.          46.3                 44.4          55.3±4.3 a         49.3±4.2 a 
                                          S.L.B                39.3                 40.4          51.2±5.0 a          44.3±5.0 a 
                    5.0                Outer S.L.         41.2                 39.2          45.4±4.5 a          40.8±4.1 a 
                                         Inner S.L.          40.5                 35.3          40.2±2.9 a          37.9±5.1 a 
                                         S.L.B                 31.3                 30.8           42.3±3.4 a         35.4±4.0 a 
                   2.5                 Outer S.L.         18.4                 17.5           33.4±4.2 a         36.3±4.4 a 
                                         Inner S.L.         17.4                 16.2            31.2±3.9 a         30.4±3.3 a 
                                         S.L.B                11.8                 10.5           30.8±2.8 a          27.8±2.5 a 
                 1.25                Outer S.L.           0.0                   0.0               -                       - 
                                        Inner S.L.            0.0                   0.0               -                       - 
                                        S.L.B                   0.0                   0.0              -                        - 
                  0.0                 Outer S.L.           0.0                   0.0              -                        - 
                                        Inner S.L.            0.0                   0.0              -                       - 
                                        S.L.B                   0.0                   0.0              -                        - 
        
  * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
  
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
108 
 
Table 30: Germination of conidia of A. niger Isolate 4 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of sucrose of different concentrations on parts of bulbs of the two 
cultivars of  shallot ( Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration       Part of  
            of sucrose (g/L)   bulb            Pale-brown cv.    Pink cv.     Pale-brown      Pink cv. 
 
                  10.0               Outer S.L.       46.1                  44.8              63.8±7.1 a         60.2±5.6 a 
                                        Inner S.L.        38.9                  40.3             52.4±6.2 a         55.3±6.7 a 
                                        S.L.B               37.7                  37.3             50.8±5.8 a         51.2±4.9 a 
                   5.0                Outer S.L.       33.8                  34.5              55.2±5.5 a        43.4±4.0 a 
                                        Inner S.L.        34.8                 33.6               54.3±5.7 a        50.5±4.3 a 
                                        S .L.B              30.2                 29.2              50.3±4.3 a         51.0±5.4 a 
                  2.5                 Outer S.L.       17.9                  18.3              51.2± 4.2 a       40.4±3.3 a 
                                        Inner S.L.       14.5                  16.5               43.3±4.7 a        36.3±2.8 a 
                                        S.L.B              10.8                    9.3               37.4±3.0 a        34.2±2.5 a 
               1.25                  Outer S.L.       0.0                     0.0                 -                     - 
                                         Inner S.L.       0.0                    0.0                 -                     - 
                                         S.L.B              0.0                    0.0                -                     - 
                0.0                   Outer S.L.       0.0                     0.0                -                     - 
                                        Inner S.L.        0.0                     0.0                -                     - 
                                        S.L.B               0.0                     0.0                -                     - 
       
   * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
109 
 
Table 31: Germination of conidia of A. niger Isolate 5 at 32±2oC in light of 76 lux in 12 hours 
in suspension drops of sucrose of different concentrations on parts of bulbs of the two 
cultivars of  shallot ( Allium ascalonicum). 
       
                                                                                               Mean germ-tube 
                                                     % Germination                   length(µm)       
           Concentration        Part of  
            of sucrose (g/L)    bulb            Pale-brown cv.   Pink cv.     Pale-brown         Pink cv. 
 
                    10.0              Outer S.L.       50.1                  48.1            49.3±5.0 a        50.5±6.1 a 
                                          Inner S.L.       48.3                  47.8            48.2±6.1 a        44.3±5.1 a 
                                          S.L.B              40.8                  39.9            47.3±5.5 a        43.3±4.3 a 
                     5.0                Outer S.L.      38.3                   41.3            50.2±4.4 a        45.2±3.8 a 
                                          Inner S.L.       38.0                   37.8           46.3±5.0 a        40.3±4.1 a 
                                          S.L.B              31.3                   26.3           44.3±4.1 a        42.3±3.3 a 
                    2.5                 Outer S.L.      20.2                   19.5             40.2±3.9 a       37.3±4.1 a 
                                           Inner S.L.      18.5                   15.4            38.3±4.2 a        35.3±3.2 a 
                                           S.L.B            11.8                   11.2             35.2±4.0 a        34.1±5.0 a 
       1.25                  Outer S.L.       0.0                     0.0                -                       - 
                                           Inner S.L.        0.0                    0.0                -                       - 
                                           S.L.B               0.0                    0.0                -                       - 
                    0.0                  Outer S.L.       0.0                     0.0                -                       - 
                                           Inner S.L.        0.0                     0.0                -                       - 
                                           S.L.B               0.0                     0.0                 -                      - 
       
   * Outer S.L. = Outer Scale Leaf    Inner S.L. = Inner Scale leaf      S.L.B = Swollen leaf base 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each horizontal 
row bearing the same letters are not significantly different at 5% level of probability 
110 
 
EXPERIMENT G. Germination of conidia of the five test Aspergillus niger isolates in 
aqueous extracts of the two shallot cultivars. 
Conidia of Aspergillus niger coming in contact with fluids exuding from wounds on shallot 
bulbs will germinate very well and the germ-tubes will be exposed to a very congenial medium 
for growth as indicated by the data in Tables 32 to 36 and Fig 3. With the exception of conidia 
of Isolate 3 shown in table 34, conidia of the remaining four Isolates registered 100 percent 
germination in some of the media. Conidia of Isolate 2, the best example ( Table 33), attained 
100 per cent  germination in the pale-brown cultivar  extracts of 1/8 dilution to the standard 
undiluted medium, and in the ¼ extract dilution to the standard  undiluted medium of the pink 
cultivar. It was evident that germination of the A. niger conidia in the undiluted extract of the 
shallot bulbs would greatly aid in initiating invasion in bruised bulbs. 
 
For both cultivars, the extract of the Pale-brown cultivar was superior to that of the Pink 
cultivar. 
 
 
 
 
 
 
 
111 
 
Table 32: Germination of conidia of A. niger Isolate 1 in fluid of bulbs of the two cultivars of 
shallot ( Allium ascalonicum) for 12 hours at 32±2oC. 
  
                            Percentage germination                   Mean length of germ tubes of germin- 
                                    in extract of                              ated conidia ± S.E in extract of 
 
  
Concentration  
of extract               Pale brown cv.        Pink cv.               Pale-brown cv.            Pink cv. 
  
 
 
        S                          100                      93.5                       ND                         237.8±1.8 
        
1
2
                            95.5                    61.9                       ND                         214.0±2.7 
        
1
4
        94.1                    64.5                       ND                         200.4±1.3 
        
1
8
                           73.3                     64.2                     270.5±6.8 a              257.5±3.3 a 
       
1
16
                          78.4                     58.7                     197.8±4.3 a               198.1±5.2 a 
         
 
ND:  Not determined because of extensive growth 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each  
horizontal row bearing the same letters are not significantly different at 5% level of probability. 
 
112 
 
Table 33: Germination of conidia of A. niger Isolate 2 in fluid of bulbs of the two cultivars of 
shallot (Allium ascalonicum) for 12 hours at 32±2oC. 
  
                            Percentage germination                   Mean length of germ tubes of germin- 
                                    in extract of                              ated conidia ± S.E in extract of 
 
  
Concentration                                                           
 of extract            Pale brown cv.        Pink cv.               Pale-brown cv.            Pink cv. 
  
 
 
      S                          100                       100                          ND                        ND 
      
1
2
                           100                      100                           ND                        ND 
      
1
4
     100                      100                           ND                        ND 
      
1
8
                           100                      93.3                          ND                       263.5±1.9 
     
1
16
                          96.6                     90.9                       289.7±5.3 a             245.1±6.2 b 
         
 
 
ND:  Not determined because of extensive growth 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each 
horizontal row bearing the same letters are not significantly different at 5% level of probability. 
 
113 
 
Table 34: Germination of conidia of A. niger Isolate 3 in fluid of bulbs of the two cultivars of 
shallot (Allium ascalonicum) for 12 hours at 32±2oC  
  
                            Percentage germination                   Mean length of germ tubes of germin- 
                                    in extract of                              ated conidia ± S.E in extract of 
 
  
Concentration                                                           
 of extract            Pale brown cv.        Pink cv.               Pale-brown cv.            Pink cv. 
  
 
 
      S                          97.2                         87.4                 244.3±2.9 a                158.9±4.6 b 
      
1
2
                           94.1                         86.0                 281.5±4.2 a                156.5±8.7 b  
      
1
4
     88.5                         84.9                 205.7±4.6 a                167.7±3.3 b 
      
1
8
                           91.2                         89.5                 188.6±6.3 a                152.1±5.6 b 
     
1
16
                          90.3                         74.1                   77.4±2.6 a                118.8±8.2 b 
       
ND:  Not determined because of extensive growth 
 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each 
horizontal row bearing the same letters are not significantly different at 5% level of probability. 
 
 
 
114 
 
Table 35: Germination of conidia of A. niger Isolate 4 in fluid of bulbs of the two cultivars of 
shallot (Allium ascalonicum) for 12 hours at 32±2oC. 
  
                            Percentage germination                   Mean length of germ tubes of germin- 
                                    in extract of                              ated conidia ± S.E in extract of 
 
  
Concentration                                                           
 of extract            Pale brown cv.        Pink cv.               Pale-brown cv.            Pink cv. 
  
 
 
     S                          100                        100                          ND                        ND 
      
1
2
                          100                         84.7                        ND                      148.1±5.4  
      
1
4
    100                         92.9                        ND                      118.1±1.4 
      
1
8
                          100                         92.3                     268.5±5.5 a            122.9±7.3 b 
     
1
16
                          87.7                       64.4                     239.5±6.8 a            106.5±4.3 b 
 
ND:  Not determined because of extensive growth. 
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each  
horizontal row bearing the same letters are not significantly different at 5% level of probability. 
 
115 
 
Table 36: Germination of conidia of A. niger Isolate 5 in fluid of bulbs of the two cultivars of 
shallot (Allium ascalonicum) for 12 hours at 32±2oC. 
  
                            Percentage germination                   Mean length of germ tubes of germin- 
                                    in extract of                              ated conidia ± S.E in extract of 
 
  
Concentration                                                           
 of extract            Pale brown cv.        Pink cv.               Pale-brown cv.            Pink cv. 
  
 
 
      S                         100                        100                         ND                         ND 
      
1
2
                          100                         96.9                   251.9±4.2 a              244.7±2.5 a 
      
1
4
     96.2                       95.4                   243.8±3.9 a              218.5±1.9 b 
      
1
8
                          100                         96.0                   248.5±3.1 a              200.9±6.4 b 
     
1
16
                          77.5                       47.7                   213.2± 2.3 a             204.9±7.2 a 
         
ND:  Not determined because of extensive growth 
       
By the calculated Scheffe’s Confidence Limit, values of Mean-Germ-tube Length in each 
horizontal row bearing the same letters are not significantly different at 5% level of probability. 
 
 
 
116 
 
    
 
      
 
 
  
 
 
0
20
40
60
80
100
120
0 0.25 0.5 0.75 1
%
 G
e
rm
in
at
io
n
 
Concentartion of extract
Isolate 1
0
20
40
60
80
100
120
0 0.25 0.5 0.75 1
%
 G
e
rm
in
at
io
n
 
Concentartion of extract
Isolate 2
0
20
40
60
80
100
120
0 0.25 0.5 0.75 1
%
 G
e
rm
in
at
io
n
 
Concentartion of extract
Isolate 3
0
20
40
60
80
100
120
0 0.25 0.5 0.75 1
%
 G
e
rm
in
at
io
n
 
Concentartion of extract
Isolate 4
0
20
40
60
80
100
120
0 0.25 0.5 0.75 1
%
 G
e
rm
in
at
io
n
 
Concentartion of extract
Isolate 5
Figure 3: Germination of conidia of the five 
test Aspergillus niger isolates in fluids 
expressed from ground bulb of the Pale-
brown (      ) and Pink (       ) shallot 
cultivars. 
 
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117 
 
EXPERIMENT H. Shallot bulb inoculation tests using the two shallot cultivars and the 
five test Aspergillus niger isolates.  
It was not possible to incite infection by surface-inoculation. The mycelia of the inocula did not 
penetrate the intact epidermis of the swollen leaf base of the bulbs of both shallot cultivars and 
no rotting ensued as reported in Table 37. 
Wounds were required for infection of the bulbs. Thus wound-inoculated bulbs were rotted by 
all A. niger isolates invading in three days bulb tissues of mean diameters of 10.4±0.4 to 
12.5±0.5mm of the pale-brown cultivar and 7.9±0.5 to 11.0±0.4mm of the pink cultivar. Rate 
of infection of the pale-brown cultivar was consistently faster than that of the pink cultivar. The 
same trend was found when the depths of the rotted tissues were studied. The invasion went 
deeper in the tissues of the pale- brown cultivar. 
 
 
 
118 
 
Table 37: Extent of rot of bulbs of shallot (Allium ascalonicum) either surface-inoculated or wound-inoculated with mycelium discs of 
different A. niger Isolates and stored at 32±2oC in humid atmosphere (100% R.H.) for 3 days. 
 
Mean diameter of rot (mm) ± S.E of    Mean depth of rot (mm) ± S.E of 
  
A. niger Surface inoculated bulbs of      Wound-inoculated bulbs of   Surface-inoculated bulbs of  Wound-inoculated bulbs
  
                Isolate           Pale-brown cv.   Pink cv. Pale-brown cv.   Pink cv.       Pale-brown cv.    Pink cv.    Pale-brown cv.   Pink cv. 
 
 1                   0.0                    0.0                  12.1±0.7 a          10.4±0.6 a           0.0                  0.0           2.0±0.0 a           1.9±0.1 a 
  
                    2                   0.0                    0.0                  12.3±0.5 a           10.8±0.5 a           0.0                  0.0           2.0±0.0 a           1.9±0.1 a 
                     
 3                   0.0                    0.0                 12.8±0.5 a          11.0±0.4 a            0.0                  0.0           2.0±0.0 a           2.0±0.1 a 
  
 4                   0.0                    0.0                 10.4±0.4 a           7.9±0.5 a             0.0                 0.0           1.9.0±0.1a         1.6±0.1a 
 
 5                   0.0                    0.0                12.5±0.5 a          11.3±0.4 a             0.0                 0.0           2.0±0.0 a            1.6±0.1 a 
 
 By the calculated Scheffe’s Confidence Limit, pairs of values for Mean Diameter of rot and mean Depth of rot 
in horizontal rows bearing the same letters are not significantly different at 5% level of probability. 
 
119 
 
EXPERIMENT I. Infection of wound-inoculated bulbs at different relative humidities 
using the two shallot cultivars and the five test Aspergillus niger isolates. 
The data in Tables 38 to 47 show the various ways in which the course of infection could be 
influenced by relative humidity. Both the Pale-brown and Pink shallot cultivars could be 
invaded by A. niger isolates at humidities from 50 to 100% R.H. The rates of invasion in 3 days 
at the two extreme relative humidities were consistently significantly different. The greatest 
differences, recorded in Table 40, showed a mean diameter of rotted tissue of 9.4±0.3 and 
15.2±0.4 mm at 50% and 100% R.H., respectively, caused by A. niger Isolate 3 in the Pale-
brown cultivar bulbs. The same isolate rotted a region of mean diameter of 13.3±1.0 mm in 3 
days in bulbs of the pink cultivar at 100% R.H. almost doubling the diameter of 7.5±0.2 mm at 
50% RH. At any storage humidity, greater rotting occurred in bulbs of the pale-brown cultivar 
than in those of the pink cultivar. 
 
The invading hyphae sporulated, if the storage humidity was favourable. Conidiophores were 
formed over the rotted areas at 80 to 100% R.H only in 3-4 days after inoculation as indicated 
in Tables 38 to 42. No conidiophores were formed at 50 to 70% R.H. by the rotted tissue which 
by the 10th day of incubation were found to have dried up. 
 
Humidity also influenced the degree of sporulation and the morphology of the conidiophores 
as shown by the information in Tables 43 to 47. The lower the humidity the sparser the crop of 
conidiophores and the shorter the conidiophores. These observations applied to both Pale-
120 
 
brown and Pink cultivars. However, conidiophores on the Pale-brown cultivar bulbs were 
slightly taller than those on the bulbs of the Pink cultivar at any particular relative humidity. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
121 
 
Table 38: Infection of bulbs of shallot (Allium ascalonicum) wound-inoculated with mycelium 
of A. niger Isolate 1 and incubated at 50-100% relative humidity at 32±2oC. 
 
                                                                    Rot development in 
 
                                        Pale brown cultivar                                            Pink cultivar 
 
                                                               Time for Time for 
              Incuba-     Mean rot                 A. niger                    Mean rot               A. niger 
               tion at      diameter (mm)        sporulation               diameter(mm)      sporulation 
              % R.H.      ± S.E in 3 days          ( days)                  ± S.E in 3 days      ( days) 
 
                 100            14.9±0.9 a                    3                           12.8±0.5 a               3 
  90             13.2±0.6 a b                 4                           11.2±0.3 b               4 
                  80             11.9±0.4 b c                 4                           10.6±0.5 b               4 
                  70             11.3±0.6 b c             No sporulation           8.3±0.4 c      No sporulation 
                  60             10.5±0.5 c d             No sporulation           8.0±0.2 c       No sporulation                
                  50              9.4±0.3 d               No sporulation            7.5±0.2 c       No sporulation             
 
By the calculated Scheffe’s Confidence Limit, values of mean Rot Diameter in each vertical 
row bearing the same letters are not significantly different at 5% level of probability. 
 
 
122 
 
Table 39: Infection of bulbs of shallot (Allium ascalonicum) wound-inoculated with mycelium 
of A. niger Isolate 2 and incubated at 50-100% relative humidity at 32±2oC 
 
                                                                 Rot development in 
 
                                        Pale brown cultivar                                            Pink cultivar 
 
                                                               Time for                                                Time for 
              Incuba-     Mean rot                 A. niger                    Mean rot               A. niger 
              tion at      diameter (mm)        sporulation               diameter (mm)       sporulation 
              % R.H.      ± S.E in 3 days          ( days)                  ± S.E in 3 days        ( days) 
 
                 100            13.8±1.4 a                  3                              12.1±1.6 a               3 
  90             12.3±1.7 a b               4                              11.8±1.3 a               4 
                  80             10.5±2.1 a b               4                              10.2±1.3 a b             4 
                  70              9.6±0.8 b c       No sporulation                  9.1±1.0 a b     No sporulation         
                  60              9.1±0.7 b c       No sporulation                  8.7±0.5 a b     No sporulation                
                  50              8.2±0.8 c         No sporulation                   8.0±0.2 b        No sporulation                
 
By the calculated Scheffe’s Confidence Limit, values of mean Rot Diameter in each vertical 
row bearing the same letters are not significantly different at 5% level of probability. 
 
123 
 
Table 40: Infection of bulbs of shallot (Allium ascalonicum) wound-inoculated with mycelium 
of A. niger Isolate 3 and incubated at 50-100% relative humidity at 32±2oC 
 
 
                                                                          Rot development in 
 
                                        Pale brown cultivar                                            Pink cultivar 
 
              Time for Time for 
              Incuba-      Mean rot                A. niger                     Mean rot               A. niger 
              tion at      diameter (mm)        sporulation                diameter (mm)      sporulation 
              % R.H.      ± S.E in 3 days          (days)                     ± S.E in 3 days      ( days) 
 
                 100            15.2±0.4 a                    3                             13.3±1.0 a              3 
  90             14.3±0.3 a                    4                             12.6±0.6 a              4 
                  80             12.2±0.5 b                    4                             11.8±0.4 a              4 
                  70             10.1±0.2 c        No sporulation                     9.3±0.6 b    No sporulation                     
 60              9.8±0.2 d         No sporulation                    9.0±0.3 b    No sporulation                       
                   50              9.4±0.3 e         No sporulation                    7.5±0.2 c     No sporulation                        
 
By the calculated Scheffe’s Confidence Limit, values of mean Rot Diameter in each vertical 
row bearing the same letters are not significantly different at 5% level of probability. 
 
124 
 
Table 41: Infection of bulbs of shallot (Allium ascalonicum) wound-inoculated with mycelium 
of A. niger Isolate 4 and incubated at 50-100% relative humidity at 32±2oC. 
 
                                                                 Rot development in 
 
                                         Pale brown cultivar                                       Pink cultivar 
 
              Incuba-     Mean rot                 Time for                    Mean rot               Time for 
               tion at      diameter (mm)        sporulation                diameter(mm)      sporulation 
              % R.H.     ± S.E in 3 days          ( days)                     ± S.E in 3 days      ( days) 
 
                 100            14.6±1.3 a                    3                           14.0±1.0 a               3 
  90             14.1±1.0 a b                 4                           13.8±0.9 a               4 
                  80             13.6±0.8 a b                 4                           12.5±0.7 a b             4 
                  70             12.1±0.6 b         No sporulation                11.3±0.5 a b    No sporulation 
                  60             11.3±0.3 c         No sporulation                10.2±0.5 b c     No sporulation      
                  50              9.4±0.3 d          No sporulation                 9.0±0.4 c        No sporulation          
 
By the calculated Scheffe’s Confidence Limit, values of mean Rot Diameter in each vertical 
row bearing the same letters are not significantly different at 5% level of probability. 
 
 
 
 
125 
 
Table 42: Infection of bulbs of shallot (Allium ascalonicum) wound-inoculated with mycelium 
of A. niger Isolate 3 and incubated at 50-100% relative humidity at 32±2oC  
 
                                                                 Rot development in 
 
                                        Pale brown cultivar                                            Pink cultivar 
 
              Incuba-     Mean rot                 Time for                    Mean rot               Time for 
               tion at      Diameter (mm)        sporulation                diameter(mm)      sporulation 
              % R.H.      ± S.E in 3 days          ( days)                     ± S.E in 3 days      ( days) 
 
                 100            13.9±0.5 a                    3                              12.4±0.9 a              3 
  90             13.3±0.5 a                    4                              11.6±0.9 a              4 
                  80             11.6±0.4 a b                  4                              10.1±0.6 a              4 
                  70             10.5±0.4 b c        No sporulation                   9.9±0.4 a     No sporulation 
                  60               9.5±0.5 c d        No sporulation                   7.9±0.4 b     No sporulation                
                  50               8.4±0.2 d          No sporulation                   7.0±0.1 b     No sporulation              
 
 
By the calculated Scheffe’s Confidence Limit, values of mean Rot Diameter in each vertical 
row bearing the same letters are not significantly different at 5% level of probability. 
 
 
126 
 
Table 43: Effect of Relative Humidity on sporulation of A. niger Isolate 1 on bulbs of shallot 
(Allium ascalonicum) at 32±2oC under 12 hour day-night cycle in 10 days 
 
                                              Density of           Mean length of conidiophore 
                                            Sporulation on                                           (µm) on                                                                                    
 
             Incubation          
              at % R.H.         Pale-brown cv.       Pink cv.              Pale-brown cv.      Pink cv.       
 
                 100                      +++ *                 +++                      702.1±6.5 a           664.3±5.5 b 
  90                        ++                       ++                      653.4±9.3 a           627.8±8.5 b                
                  80                          +                         +                      610.5±8.7 a            601.0±7.8 a 
 
*  +++ :  Dense 
      ++ :  Moderate 
        + :  Scanty     
   
By the calculated Scheffe’s Confidence Limit, pairs of values of Mean Conidiophore Lengths 
in horizontal rows bearing the same letters are not significantly different at 5% level of 
probability. 
 
 
 
 
127 
 
Table 44: Effect of Relative Humidity on sporulation of A. niger Isolate 2 on bulbs of shallot 
(Allium ascalonicum) at 32±2oC under 12 hour day-night cycle in 10 days 
 
                                              Density of           Mean length of conidiophore 
                                            Sporulation on                                           ( µm) on                                                                                    
 
               Incubation          
              at  % R.H.          Pale-brown cv.    Pink cv.                  Pale-brown cv.        Pink cv.       
 
                 100                      +++ *                 +++                      653.8±10.8 a           642.3±6.3 a 
  90                        ++                       ++                      601.1±4.3 a            589.1±7.3 a                
                  80                          +                          +                      495.8±8.8 a            479.1±6.9 a 
 
 
*  +++ :  Dense 
      ++ :  Moderate 
        + :  Scanty     
   
By the calculated Scheffe’s Confidence Limit, pairs of values of Mean Conidiophore Lengths 
in horizontal rows bearing the same letters are not significantly different at 5% level of 
probability. 
 
 
 
128 
 
Table 45: Effect of Relative Humidity on sporulation of A. niger Isolate 3 on bulbs of shallot 
(Allium ascalonicum) at 32±2oC under 12 hour day-night cycle in ten days. 
 
                                              Density of             Mean length of conidiophore 
                                            Sporulation on                                           (µm) on                                                                                    
 
               Incubation      
              at  % R.H.        Pale-brown cv.      Pink cv.                Pale-brown cv.     Pink cv. 
 
                 100                      +++ *                 +++                      724.0±6.8 a           700.1±6.1 a 
  90                        ++                       ++                      672.8±7.7 a          654.8±9.2 a               
                  80                          +                          +                      611.1±6.4 a          587.2±4.9 b 
 
*  +++ :  Dense 
      ++ :  Moderate 
        + :  Scanty     
 
By the calculated Scheffe’s Confidence Limit, pairs of values of Mean Conidiophore Lengths 
in horizontal rows bearing the same letters are not significantly different at 5% level of 
probability. 
 
 
 
 
 
129 
 
Table 46: Effect of Relative Humidity on sporulation of A. niger Isolate 4 on bulbs of shallot 
(Allium ascalonicum) at 32±2oC under 12 hour day-night cycle in 10 days  
 
                                              Density of           Mean length of conidiophore 
                                            Sporulation on                                           ( µm) on                                                                                    
 
               Incubation          
              at  % R.H.           Pale-brown cv.    Pink cv. Pale-brown cv.     Pink cv. 
 
                 100                      +++ *                 +++                      692.8±7.3 a           665.8±7.2 a 
  90                        ++                       ++                      653.1±10.2 a         620.1±6.4 a                
                  80                          +                          +                     562.3±6.3 a            529.3±6.3 b 
 
*  +++ :  Dense 
      ++ :  Moderate 
                   + :   Scanty     
 
By the calculated Scheffe’s Confidence Limit, pairs of values of Mean Conidiophore Lengths 
in horizontal rows bearing the same letters are not significantly different at 5% level of 
probability. 
 
 
 
 
 
130 
 
 
Table 47: Effect of Relative Humidity on sporulation of A. niger Isolate 5 on bulbs of shallot 
(Allium ascalonicum) at 32±2oC under 12 hour day-night cycle in 10 days 
 
                                              Density of           Mean length of conidiophore 
                                            Sporulation on                                           ( µm) on                                                                                    
 
               Incubation          
              at  % R.H.          Pale-brown cv.      Pink cv.              Pale-brown cv.      Pink cv. 
 
                 100                      +++ *                 +++                      687.1±9.2 a            662.1±4.9 a 
  90                        ++                       ++                      624.0±8.3 a           591.7±10.2 a                
                  80                          +                          +                      484.1±6.1 a           479.0±8.3 a 
                   
*  +++ :  Dense 
      ++ :  Moderate 
                   + :   Scanty     
 
By the calculated Scheffe’s Confidence Limit, pairs of values of Mean Conidiophore Lengths 
in horizontal rows bearing the same letters are not significantly different at 5% level of 
probability. 
 
 
131 
 
 
 
                                                                                                                                             X  3/4 
    Plate 10. Photographs showing wound-inoculated bulbs of the Pale-brown (TOP) and Pink 
(BOTTOM) cultivators of shallot ( Allium ascalonicum) and incubated at different relative 
humidities. ( From left: 100, 90, 80, 70, 60 and 50% R.H.) 
132 
 
Experiment J. In-vitro production of pectic enzymes by the five test Aspergillus niger 
isolates 
The details in Tables 49a-e show the enzyme activity of Czapeck Dox culture filtrate of the A. 
niger Isolates as determined by the disk maceration time method. Enzyme activities attained a 
peak on the 9th day of growth of all the five A. niger isolates. The activities ranges from 7.75 
by the enzymes of Isolate 3 and 5 culture filtrates ( Tables 49 c and e) to 8.89 by the culture 
filtrates of  A. niger Isolates 1, 2 and 4 ( Tables 49a, b and d); respectively. Coincidentally, 
mycelial growth by A. niger Isolates 3, 4 and 5 (Tables 48c, 48d and 48e) also reached the peak 
by the 9th day after inoculation. Isolate 1 (Table 48a) and 2 (Table 48b) however, grew faster 
and attained peak mycelial dry weight by the 6th day. Enzyme synthesis, therefore, reached a 
peak when mycelial autolysis had set in. 
 
The rate of production of the macerating enzyme by Isolate 5 (Table 49e) proceeded very fast 
synthesizing enzymes with enzyme activity of 6.15 in the first three days to the maximum of 
7.75 recorded on the ninth day. In contrast, next was Isolate 2 (Table 49b) producing filtrate 
with enzyme activity of 6.49 and 8.89 respectively within the corresponding 3 and 9 days 
respectively. Lesser activities were associated with the remaining three A. niger Isolates with 
Isolate 3 being the least (Table 49c) with corresponding rates of 4.52 and 7.75, respectively. In 
all cases enzyme activity declined slowly when autolysis set in. 
 
 
 
133 
 
 
 
 
 
 
 
Table 48a: Growth of A. niger Isolate 1 grown in Czapek-Dox broth medium (pH 7.0) at 
32±2oC under a 12-hour day-night cycle in 12 days. 
  
    Days after            Mycelium Dry weight (g)  
    Inoculation                            Replicates                                       Mean  ± S.E   
 
        3                        0.4511        0.4312         0.4112          0.4312±0.012 
        6                        0.4739        0.4725         0.4767                     0.4744±0.001 
        9                        0.4642        0.4567         0.4332                     0.4514±0.009 
      12                        0.4346        0.4152         0.4254                     0.4251±0.006         
 
 
 
 
 
 
134 
 
 
 
 
 
 
 
Table 48b: Growth of A. niger Isolate 2 grown in Czapek-Dox broth medium (pH 7.0) at 
32±2oC under a 12-hour day-night cycle in 12 days. 
 
    Days after              Mycelium Dry weight (g)  
    Inoculation                            Replicates                                       Mean  ± S.E   
               
             3                   0.3834         0.3683          0.3734           0.3750±0.004 
             6                   0.3765         0.3750          0.3957                    0.3824±0.067 
             9                   0.3202         0.2942          0.3512                    0.3221±0.017 
           12                   0.3112          0.2912         0.2940                    0.2988±0.006         
         
 
 
 
 
 
135 
 
 
 
 
 
 
  
 Table 48c: Growth of A. niger Isolate 3 grown in Czapek-Dox broth medium (pH 7.0) at 
32±2oC under a 12-hour day-night cycle in 12 days. 
 
      Days after              Mycelium Dry weight (g)  
     Inoculation                            Replicates                                       Mean  ± S.E   
              
              3   0.3949       0.4012       0.3952                         0.3971±0.002 
              6                  0.3994       0.4112        0.4200                        0.4102±0.006 
              9                  0.4111       0.4202        0.4359                         0.4224±0.007 
            12                  0.4018       0.3993        0.4112                         0.4041±0.004         
               
 
 
 
 
 
136 
 
 
 
 
 
 
 
:Table 48d: Growth of A. niger Isolate 4 grown in Czapek-Dox broth medium (pH 7.0) at 
32±2oC under a 12-hour day-night cycle in 12 days. 
  
      Days after              Mycelium Dry weight (g)  
     Inoculation                            Replicates                                       Mean  ± S.E   
              
            3                  0.4112         0.3923         0.4008                       0.4014±0.005 
            6                  0.4490         0.4502         0.4450                       0.4481±0.016 
            9                  0.4653         0.4328         0.4928                       0.4636±0.017 
          12                  0.4121         0.4567         0.4346                       0.4345±0.013         
 
 
 
 
 
 
137 
 
 
 
 
 
 
    
 Table 48e: Growth of A. niger Isolate 5 grown in Czapek-Dox broth medium (pH 7.0) at 
32±2oC under a 12-hour day-night cycle in 12 days. 
 
      Days after              Mycelium Dry weight (g)  
     Inoculation                            Replicates                                       Mean  ± S.E   
 
          3                      0.3892      0.4100     0.3995                   0.3996±0.006 
          6                      0.4210      0.4120     0.4019                        0.4116±0.006 
          9                      0.4520     0.4431      0.4999                        0.4650±0.018 
        12                      0.4421     0.4989      0.4210                        0.4540±0.023         
                          
 
 
 
 
 
 
138 
 
 
 
 
 
 
 
Table 49a: Mean maceration time of Potato tuber discs by Aspergillus niger Isolate 1 filtrate 
and calculated enzyme activity of the culture filtrate. 
 
    Days of growth         Maceration time of discs ( seconds) 
    of culture                  Sample 1      Sample 2              Mean                  Enzyme activity 
     
            3                           22.8             24.2                  23.50                          4.26 
           6                            11.7             12.5                  12.10                          8.26 
           9                            11.7             10.8                  11.25                          8.89 
         12                            11.7             11.7                  11.70                          8.55 
 
 
 
 
 
 
 
139 
 
 
 
 
 
 
 
Table 49b: Mean maceration time of Potato tuber discs by Aspergillus niger Isolate 2 filtrate 
and calculated enzyme activity of the culture filtrate. 
 
    Days of growth         Maceration time of discs ( seconds) 
    of culture                  Sample 1      Sample 2              Mean                  Enzyme activity 
     
            3                           15.0             15.8                  15.40                          6.49 
           6                            11.7             12.5                  12.10                          8.26 
           9                            11.7             10.8                  11.25                          8.89 
         12                            11.7             12.5                  12.10                          8.26 
 
 
 
 
 
 
  
140 
 
 
 
 
 
   
 
Table 49c: Mean maceration time of Potato tuber discs by Aspergillus niger Isolate 3 filtrate 
and calculated enzyme activity of the culture filtrate. 
 
    Days of growth         Maceration time of discs (seconds) 
    of culture                  Sample 1      Sample 2              Mean                  Enzyme activity 
     
            3                           22.5             21.7                  22.10                          4.52 
           6                            20.0             18.3                  19.50                          5.13 
           9                            13.3             12.5                  12.90                          7.75 
         12                            13.3             12.5                  12.90                          7.75 
 
 
 
 
 
 
 
141 
 
 
 
 
 
 
 
Table 49d: Mean maceration time of Potato tuber discs by Aspergillus niger Isolate 4 filtrate 
and calculated enzyme activity of the culture filtrate. 
 
    Days of growth         Maceration time of discs ( seconds) 
    of culture                  Sample 1      Sample 2              Mean                  Enzyme activity 
     
           3                            16.7             16.7                  16.70                          5.99 
           6                            12.5             12.5                  12.50                          8.00 
           9                            11.7             10.8                  11.25                          8.89 
         12                            12.5             11.7                  11.10                          8.26 
 
 
 
 
 
 
 
142 
 
 
 
 
 
 
 
Table 49e: Mean maceration time of Potato tuber discs by Aspergillus niger Isolate 5 filtrate 
and calculated enzyme activity of the culture filtrate. 
   
    Days of growth         Maceration time of discs ( seconds) 
    of culture                  Sample 1      Sample 2              Mean                  Enzyme activity 
     
           3                           16.7             15.8                   16.25                          6.15 
           6                            14.2             15.8                  15.00                          6.67 
           9                            12.5             13.3                  12.90                          7.75 
         12                            13.3             14.2                  13.10                          7.63 
 
 
 
 
 
 
143 
 
 
Although the A. niger Isolates were grown in 8 days in the different organic nutrients media, 
the mean mycelial dry weight could be reasonably compared to that of the 9 day old cultures in 
the Czapeck Dox medium. The mean mycelial dry weight of the Czapeck Dox medium reported 
earlier and those of the shallot bulb extract (Tables 54a and b) were far greater; a range of 0.298 
to 0.465g (Tables 48c and 48e) than those of the other organic media (Tables 50 to 53 and Table 
55). The mean dry weights of the fungi of the pale-brown shallot cultivar extracts ranged from 
0.334 to 0.368g, those of the pink cultivar extracts from 0.367 to 0.411g, and those of the 
Czapeck Dox media ranged from 0.322 to 0.481g. In comparison the range for the rest of the 
media was 0.095 (Table 51) to 0.135g (Tables 50 and 53). 
The initial pH’s of all the media were neutral. The pH significantly drifted to the acidic side ( 
2.67-5.68) during the growth of the cultures in the various media. 
 
 
 
 
 
 
 
 
 
144 
 
 Table 50: Growth of Aspergillus niger Isolates in Bean Meal Extract at 32±2oC under a 12-
hour day-night cycle in 8 days.  
                                                                                           
                                                                                         Dry weight of mycelium (g) 
                                             pH                                        produced in 30 ml medium                                                                                                                                                                
          
           Isolates         Initial               Final                    Replicates              Mean± S.E 
 
                                                                                       0.1336   
                 1             6.91                  3.26                       0.1331               0.1352±0.002 a 
                                                                                       0.1389 
 
                                                                                       0.1884 
    2 6.91          2.76                       0.1742                0.1805±0.004 b 
                                                                                       0.1789 
                          
                                                                                       0.1142 
               3             6.91                    2.81                       0.1383               0.1251±0.007 a 
                                      0.1229 
 
                                                                                       0.1303 
              4            6.91        2.72                       0.1092                0.1224±0.007 a    
                                      0.1276 
 
                                                                                      0.1372 
             5          6.91        2.70                      0.1301                 0.1295±0.005 a 
                                     0.1211 
By the calculated Scheffe’s Confidence Limit, values of Mean Mycelium Dry Weights bearing 
the same letters are not significantly different at 5% level of probability 
145 
 
Table 51: Growth of Aspergillus niger Isolates in Cassava Dextrose Broth at 32±2oC under a 
12-hour day-night cycle in 8 days. 
                                                                                           
                                                                                         Dry weight of mycelium (g) 
                                              pH                                       produced in 30 ml medium                                                                                                                                                                
          
           Isolates         Initial               Final                    Replicates              Mean± S.E 
 
                                                                                          0.1077   
                 1             6.99                    3.05                        0.1044              0.1057±0.001 a  
                                                                                          0.1049 
                                                                                          0.1176 
     2 6.99           2.85                         0.1155             0.1156±0.001 b  
                                           0.1136 
                                 
                                                                                          0.1054 
 3            6.99                     2.80                        0.1348              0.1170±0.009 b 
                                          0.1109 
  
                                                                                          0.0942 
                4 6.99          3.23                         0.0954             0.0958±0.001 c   
                                          0.0978 
  
                                                                                           0.1129 
                5           6.99         3.27                          0.1010             0.1046±0.004 a  
                                          0.0999 
By the calculated Scheffe’s Confidence Limit, values of Mean Mycelium Dry Weights bearing 
the same letters are not significantly different at 5% level of probability 
146 
 
Table 52: Growth of Aspergillus niger Isolates in Oat Meal Extract at 32±2oC under a 12-hour 
day-night cycle in 8 days  
 
                                                                                         Dry weight of mycelium (g) 
                                             pH                                       produced in 30 ml medium                                                                                                                                                                
          
           Isolates         Initial               Final                    Replicates              Mean± S.E 
 
                                                                                       0.0949   
                 1             6.55                    3.19                     0.0993                0.1022±0.005 a 
                                                                                       0.1123 
 
                                                                                      0.0959 
     2 6.55           2.93                     0.0849                 0.1013±0.011 a 
                                       0.1231 
                                 
                                                                                      0.1153 
 3             6.55                  2.85                      0.1101                0.1079±0.005 a 
                                     0.0983 
  
                                                                                      0.1123 
               4              6.55       3.05                       0.1159                 0.1059±0.008 a    
                                     0.0895 
  
                                                                                      0.0932 
               5          6.55       2.72                       0.1152                 0.1098±0.008 a 
                                     0.1211 
By the calculated Scheffe’s Confidence Limit, values of Mean Mycelium Dry Weights bearing 
the same letters are not significantly different at 5% level of probability 
147 
 
Table 53: Growth of Aspergillus niger Isolates in Potato Dextrose Broth at 32±2oC under a 
12-hour day-night cycle in 8 days  
 
                                                                                          Dry weight of mycelium (g) 
                                            pH                                         produced in 30 ml medium                                                                                                                                                                
          
           Isolates         Initial               Final                    Replicates              Mean± S.E 
 
                                                                                      0.1173   
                 1           6.47                   3.05                       0.1493                 0.1326±0.009 a 
                                                                                      0.1312 
                                                                                      
                                                                                       0.2014 
     2 6.47          2.67                       0.1868                0.1312±0.037 a 
                                        0.2054 
                                 
                                                                                      0.1441 
 3            6.47                  2.82                       0.1395                 0.1352±0.006 a 
                                      0.1221 
  
                                                                                       0.1339 
               4             6.47       2.73                         0.1059                0.1239±0.009 a   
                                       0.1318 
  
                                                                                       0.1348 
               5         6.47       2.74                         0.1233               0.1260±0.005 a 
                                                                                       0.1199 
By the calculated Scheffe’s Confidence Limit, values of Mean Mycelium Dry Weights bearing 
the same letters are not significantly different at 5% level of probability 
148 
 
Table 54a: Growth of Aspergillus niger Isolate 1 in aqueous extract of bulbs of shallot            
(Allium ascalonicum) at 32±2oC under a 12-hour day-night cycle in 8 days. 
                                                                                                       
                                                                                            Dry weight of mycelium (g) 
                                                       pH                                 produced in 30 ml medium                                                                                                                                                                
          
        Shallot cultivar          Initial               Final                    Replicates              Mean± S.E 
 
                                                                                                  0.3551 
         Pale-brown              6.51                    3.51                      0.4060               0.3698±0.017 a  
                                                    0.3873 
                                                                                                  0.3308 
 
                                                                                                  0.3913 
           Pink                       6.44                    3.91                       0.3790             0.3855±0.009 b 
                                                                                                  0.3654 
                                 0.4062 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean Mycelium Dry Weights bearing 
the same letters are not significantly different at 5% level of probability. 
 
 
149 
 
Table 54b: Growth of Aspergillus niger Isolate 2 in aqueous extract of bulbs of shallot                       
( Allium ascalonicum) at 32±2oC under a 12-hour day-night cycle in 8 days. 
                                                                                                       
                                                                                            Dry weight of mycelium (g) 
                                                       pH                                 produced in 30 ml medium                                                                                                                                                                
          
        Shallot cultivar          Initial               Final                    Replicates           Mean± S.E 
 
                                                                                                 0.3536 
          Pale-brown              6.24                  2.87                       0.3157               0.3674±0.021 a   
                                                   0.3866 
                                                                                                 0.4137 
                                                                     
                                                                                                 0.4064 
           Pink                       6.28                  2.88                        0.5298              0.4176±0.043 a 
                                                                                                 0.3211 
                                0.4134 
 
By the calculated Scheffe’s Confidence Limit, values of Mean Mycelium Dry Weights bearing 
the same letters are not significantly different at 5% level of probability. 
 
 
 
 
 
150 
 
Table 54c: Growth of Aspergillus niger Isolate 3 in aqueous extract of bulbs of shallot              
(Allium ascalonicum) at 32±2oC under a 12-hour day-night cycle in 8 days  
                                                                                                      
                                                                                            Dry weight of mycelium (g) 
                                                       pH                                 produced in 30 ml medium                                                                                                                                                                
          
        Shallot cultivar          Initial               Final                    Replicates              Mean± S.E 
 
                                                                                                 0.3267 
         Pale-brown              6.24                    5.41                      0.3241               0.3345±0.005 a   
                                                    0.3398 
                                                                                                  0.3473 
                                                                  
                                                           0.3820 
           Pink                        6.28                 5.68                        0.3682              0.3812±0.005 b 
                                                                                                 0.3946 
                                0.3801 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean Mycelium Dry Weights bearing 
the same letters are not significantly different at 5% level of probability. 
 
 
 
 
151 
 
Table 54d: Growth of Aspergillus niger Isolate 4 in aqueous extract of bulbs of shallot            
(Allium ascalonicum) at 32±2oC under a 12-hour day-night cycle in 8 days  
 
                                                                                                       
                                                                                            Dry weight of mycelium (g) 
                                                       pH                                 produced in 30 ml medium                                                                                                                                                                
          
        Shallot cultivar          Initial               Final                    Replicates              Mean± S.E 
 
                                                                                                  0.3549 
             Pale-brown            6.24                 4.49                        0.3241              0.35880±0.001 a   
                                                    0.3398 
                                                                                                  0.3473 
                                                                  
                                                                                                   0.4016 
             Pink                       6.28                 4.65                        0.3854             0.4018±0.007 b 
                                                                                                   0.4216 
                                  0.3984 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean Mycelium Dry Weights bearing 
the same letters are not significantly different at 5% level of probability. 
 
 
 
 
152 
 
 Table 54e:  Growth of Aspergillus niger Isolate 5 in aqueous extract of bulbs of shallot         
(Allium ascalonicum) at 32±2oC under a 12-hour day-night cycle in 8 days. 
                                                                                                       
                                                                                            Dry weight of mycelium (g) 
                                                       pH                                 produced in 30 ml medium                                                                                                                                                                
          
        Shallot cultivar        Initial               Final                    Replicates              Mean± S.E 
 
                                                                                              0.3559 
 
        Pale-brown              6.21                  3.51                      0.3324                   0.3350±0.007 a   
                                                0.3217 
                                                                                              0.3299 
                                                                                              
                                                                                              0.3615 
        Pink                       6.24                   3.40                       0.3411                  0.3669±0.011 b 
                                                                                              0.3924 
                             0.3727 
 
 
 
By the calculated Scheffe’s Confidence Limit, values of Mean Mycelium Dry Weights bearing 
the same letters are not significantly different at 5% level of probability. 
 
 
 
 
153 
 
Table 55: Growth of Aspergillus niger isolates in Sweet Potato Dextrose Broth medium at 
32±2oC under a 12-hour day-night cycle in 8 days.                                                                                        
  
                                                                                         Dry weight of mycelium (g) 
                                             pH                                       produced in 30 ml medium                                                                                                                                                                
          
           Isolates         Initial               Final                    Replicates                   Mean± S.E 
 
                                                                                      0.1160   
                 1             6.81                  3.62                      0.1013                     0.1101±0.004 a 
                                                                                      0.1129 
 
                                                                                      0.1314 
   2 6.81         3.48                       0.1239                     0.1231±0.005 a 
                                       0.1139 
                                 
                                                                                      0.1339 
              3              6.81                  3.52                        0.1248                    0.1300±0.003 a 
                                      0.1312 
  
                                                                                      0.1129 
            4            6.81      2.73                         0.1324                    0.1164±0.008 a    
                                      0.1039 
  
                                                                                      0.0995 
            5         6.81     2.95                          0.1215                    0.1136±0.007 a 
                                                                           0.1198 
By the calculated Scheffe’s Confidence Limit, values of Mean Mycelium Dry Weights bearing 
the same letters are not significantly different at 5% level of probability. 
154 
 
EXPERIMENT K. Mycoflora of the bulbs and the rhizosphere of shallot plants raised in 
soil of Anloga farms amended with Urea fertilizer.  
The characteristics of soil of shallot plots in Anloga are shown in Tables 56 to 58. The main 
features are: 
(a) The resident fungus species; 
(b) Very high sand content, and 
(c)  Predominance of nitrogen over other chemical elements. 
When Potato Dextrose Agar and Sabouraud Agar were used to isolate fungi from the same soil 
sample, different results were obtained. More species (9) together with  Mycelia streilia were 
isolated with Sabouraud Agar than with Potato Dextrose Agar(6) as indicated in Tables 56 and 
57. Aspergillus was the dominant genus in both lists of fungi and Aspergillus niger the dominant 
species. The genera of fungi encountered in the soil were Aspergillus, Curvularia, Penicillium 
and Rhizopus. 
 
The soil was very sandy with 98.8 per cent sand content and very low 3.48per cent humus 
content (Table 58). Comparing the component chemicals, the level of nitrogen was far higher 
than the other chemical elements. Unexpectedly, it was followed by iron which was 
substantially superior to calcium, magnesium, phosphorus and potassium.   
 
 
 
155 
 
 
Table 56: Fungal species isolated with Potato Dextrose Agar from freshly prepared plots at 
Anloga for cultivation of shallots ( Allium ascalonicum) immediately before planting. 
 
 
              Fungus species                           CFU ml-1 x 103                     % Frequency 
 
               Aspergillus flavus                          8.0                                         6.4 
               Aspergillus niger                         60.2                                       47.9 
               Curvularia lunata                        15.2                                       12.1 
               Fusarium spp.                              18.6                                       14.8 
                Penicillium spp.                          16.0                                       12.7 
               Trichoderma viride                       5.0                                         3.9 
                Mycelia  sterilia                           2.8                                         2.2 
 
 
 
 
 
 
 
 
156 
 
 
Table 57: Fungal species isolated with Sabouraud Agar from freshly prepared plots at Anloga 
for cultivation of shallot ( Allium ascalonicum) immediately before planting. 
           
 
              Fungus species                           CFU ml-1 x 103                     % Frequency 
 
               Aspergillus flavus                           6.0                                        5.1 
               Aspergillus fumigatus                     5.0                                        4.2 
               Aspergillus niger                           45.8                                     38.6 
               Curvularia lunata                           8.0                                        6.7 
               Fusarium spp                                15.4                                      13.0 
               Penicillium sp. 1                             9.8                                        8.3 
               Penicillium sp. 2                           13.0                                      10.9 
               Rhizopus spp.                                  3.0                                        2.5       
               Trichoderma viride                         4.0                                        3.4 
               Mycelia   sterilia                             8.6                                        7.3 
 
 
 
 
 
157 
 
Table 58: Mineral and chemical composition of soil used for cultivation of shallot from 
Anloga. 
 
 Plot 
    Physico-chemical 
    parameters    1                      2                 3           Mean 
 
    pH 6.98                7.00               7.01            7.00 
 
Mineral        Clay                    4.8481              4.7440           4.7333          4.7751 
Matter 
(%) 
          Sand                 95.1519             92.8829   92.8999       93.3116 
           Silt                    2.0000               2.3724            2.3667         2.2797 
    Organic                              
    Matter (%)           Humus              3.4401                3.5120            3.4813         3.4778 
Chemical             Calcium             0.0046               0.0036             0.0047       0.0043 
Constitu-              Iron                   0.0820               0.0757              0.0819      0.0799 
  ent (%)               Magnesium      0.0076                0.0064              0.0062       0.0067 
                             Nitrogen           0.1008               0.1005               0.0979       0.0997 
                             Phosphorus      0.0160               0.0147               0.0164       0.0157 
                             Potassium        0.0160               0.0135               0.0154       0.0150 
 
 
158 
 
 
 
 
 
 
                                                                                                                                                 X 5/9 
 
Plate 11. Photograph showing fungi isolated from freshly prepared soil of shallot farm at   
Anloga using Sabouraud Agar at 32±2oC for 8 days. 
 
 
159 
 
Measurement of the bulbs at 20, 40 and 60 days after planting showed that both the length and 
width ( at widest point) of the bulblets, recorded in Table 59, increased with age of  both 
cultivars. The rate of growth of the bulblets was faster from 20 to 40 days than from 40 to 60 
day after planting. Also at each measurement time, the lengths and widths of the Pale-brown 
cultivar were greater than those of the Pink cultivar. Therefore the Pink cultivar bulbs were 
comparatively smaller than bulbs of Pale-brown cultivar. 
 
Finally, pigmentation of the scale leaves developed faster in the Pale-brown cultivar (first 
observed on the 40th day) than in the Pink cultivar observed on the 60th day. 
 
The addition of Urea to the soil only slightly improved growth. The increase in the values were, 
however, not statistically significant at 5% level of probability. For instance, the bulbs in soils 
with 0.00, 1.94 and 3.88 g per 1200 g of soil measured 31.8±1.4 x 13.3±0.8, 31.8±1.8 x 14.8±0.9 
and 32.4±2.0 x 15.1±1.2mm, respectively, in the Pale-brown cultivar.  
 
 
 
 
 
 
160 
 
 Table 59: Morphology of bulblets of the two shallot cultivars developing in the greenhouse 
under a 12- hour day-night cycle in Anloga shallot farm soil containing different quantities of 
Urea fertilizer. 
 
         Concentrat- 
         ion of                   Days            Features                                              Cultivar 
         Urea      after       of                                            
         (g)      planting       bulblets                            Pale-brown                    Pink 
  
        0.00                        20              Mean length (mm)              17.7±1.8                 16.6±1.1 
         Mean width (mm)                 5.6±0.7                   5.1±0.5 
         Colour                                   white                       white 
                           40               Mean length (mm)              28.8±1.5                 22.4±2.3 
         Mean width ( mm)              10.2±0.8                   6.8±0.9 
         Colour                                  light brown               white 
                                 60               Mean length (mm)              31.3±1.4                 29.2±2.0 
         Mean width ( mm)              13.3±0.8                 11.2±0.7 
         Colour                                  Pale-brown                 Pink 
 
         1.94                      20               Mean length (mm)              20.3±1.9                 18.3±2.0 
         Mean width ( mm)                 7.2±0.3                  6.8±0.7 
         Colour                                  white                         white 
                                     40                Mean length (mm)               29.2±1.4                24.2±2.1  
         Mean width ( mm)               11.2±1.0                  7.2±1.0  
                                                         Colour      light brown                white 
                                     60                Mean length (mm)               31.8±1.8                28.2±1.8 
          Mean width ( mm)              14.8±0.9                12.2±1.0 
          Colour                                  Pale-brown                 Pink  
161 
 
 Table 59 continued 
 
.    
         Concentrat- 
         ion of                   Days            Features                                              Cultivar 
         Urea      after       of                                            
         (g)      planting       bulblets                            Pale-brown                      Pink 
  
        3.88                        20              Mean length (mm)              17.9±2.3                 17.2±1.8 
         Mean width ( mm)                4.7±0.7                    5.2±0.5 
         Colour                                    white                        white 
                           40               Mean length (mm)              30.2±1.4                 26.2±1.9 
         Mean width ( mm)              10.8±1.0                   7.9±0.5 
         Colour                                  light- brown               white 
                                 60               Mean length (mm)               32.4±2.0                30.3±2.3 
          Mean width ( mm)              15.1±1.2                13.2±0.8 
          Colour                                  Pale-brown                 Pink 
 
 
                              
                          
   
 
162 
 
Tables 60 to 62 contain certain pertinent data on mycoflora of the subterranean organs of the 
pale-brown and pink cultivar shallot plants growing in Anloga farm soils, with or without urea 
fertilizer. There were members of four genera. The few species isolated from the plants reflected 
the species of the Anloga farm soil indicated in Tables 56 and 57. It is unusual that a single 
species, in this investigation Aspergillus niger, would so consistently predominate the fungal 
flora of the rhizosphere. Since other species appeared so tardily and in very low numbers, A. 
niger alone could justifiably be used to define the effect of either bulb age or urea treatment or 
specific plant organ. 
(a) Far greater fungal populations were present in the phyllosphere and rhizosphere than in 
the non-rhizosphere soil. 
(b) Clearly greater fungal population was associated with the bulb than with the roots. 
(c) In the majority of cases  the population of mycoflora was higher on the Pale-brown 
plants than on the Pink cultivars 
(d) Colonization of the bulbs and the roots proceeded so quickly in the first 20 days of 
growth that very little change occurred thereafter. By the 40th day a stable dense 
community had been established as can be deduced by the crowded dilution of Plates 
12 and 13. 
(e) Notably, Aspergillus terreus was isolated from the non-rhizosphere soil on many 
occasions but not from the phyllosphere soil (Tables 60-62)  
 
163 
 
Table 60: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR) and rhizosphere (RH) soils, and from soil 
particles adhering to bulbs (BS) of the two shallot cultivars grown in unamended Anloga shallot farm soil for 60 days. 
 
                                                        Source                                                     C.F.U’s x 103 /g soil 
                                                          of 
           Days after        Shalllot        fungal      Aspergillus   Aspergillus     Aspergillus   Fusarium     Penicillium    Rhizopus      Mycelia     
            planting          cultivar         species     flavus           niger               terreus           spp spp spp.            sterilia 
                 20          Pale-brown        BS             -           27.3                -           -       1.0        0.3             0.3 
                                                         RH             -            13.0             1.0          -             0.7    -  0.7 
                                   NR             -             4.0             1.3           -             0.3         -  0.3 
                                Pink                  BS          0.7                30.0            1.0          -       0.7                 -   - 
                                                         RH          0.7          11.0              -          -         -         -  1.3 
                                                         NR          0.7            4.3            0.7          -       1.0              -  0.3 
                 40          Pale-brown        BS          1.0          35.0              -          -             2.7       1.7  2.0 
                                                         RH          1.0          16.0            1.7          -       2.7         -  0.3 
                                 NR          1.3          11.0            0.3          -       1.7         -  0.7 
                              Pink                    BS 1.0          37.3             -          -              1.3       1.0  1.0 
                                                         RH  1.0          14.7           1.3          -       2.0         -  1.0 
                                                         NR 1.3         11.7           1.0          -       2.3         -  0.7 
164 
 
              
     Table 60 continued 
     
                                                      Source                                                  C.F.U’s x 103 /g soil 
                                                       of 
            Days after   Shallot           fungal     Aspergillus   Aspergillus    Aspergillus    Fusarium    Penicillium   Rhizopus   Mycelia 
            planting      cultivar          species     flavus           niger              terreus           spp             spp spp.          sterilia 
                  60         Pale-brown     BS  1.3        30.0  1.0        -       1.3  1.0       0.3  
                                                      RH  1.0        14.3  -      1.0       1.7    -       0.3 
                     NR  0.7        10.0  0.3      1.0       0.7    -       0.3 
                                Pink              BS  1.0       25.0  1.0      1.3       1.7  1.3       1.0 
                                                     RH   1.0       13.3  0.7      1.0       1.0    -       0.3 
                                                     NR  1.0       11.7  0.3      0.7       1.0    -       0.7 
 
 
 
 
165 
 
Table 61: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR) and rhizosphere (RH) soils, and from soil 
particles adhering to bulbs (BS) of the two shallot cultivars grown in Anloga shallot farm soil treated with 1.94g of Urea for 60 days. 
 
                                                        Source                                                         C.F.U’s x 103 /g soil 
                                                          of 
           Days after        Shalllot        fungal      Aspergillus   Aspergillus     Aspergillus   Fusarium     Penicillium    Rhizopus      Mycelia     
            planting          cultivar         species     flavus           niger               terreus           spp spp spp.             sterilia 
                 20          Pale-brown        BS             -          36.3                -           -       4.0          -             2.0 
                                                         RH          0.3            6.7             1.3          -             1.0    -  1.0 
                                   NR             -            3.3             0.7           -             0.7         -  0.3 
                                Pink                  BS          0.3               40.0            1.7          -       1.7                 -   - 
                                                         RH          0.7           7.3              -         0.7        -         -  0.3 
                                                         NR          0.3           3.0            0.7         1.0                0.3              -  1.0 
                 40          Pale-brown        BS          1.3         40.0              -          -             2.7        1.7  0.7 
                                                         RH          1.0         19.3            1.3          -       2.3         -  0.3 
                                 NR          1.0         15.3            0.3          -       2.3         -  1.0 
                              Pink                    BS 1.0         36.7             -          -              1.3        1.3  0.3 
                                                         RH  1.0         21.7            1.0          -       1.7         -  0.3 
                                                         NR 1.0         13.7            1.0          -       2.3         -  1.0 
               
166 
 
Table 61 continued 
     
                                                      Source                                                              C.F.U’s x 103 /g soil 
                                                       of 
            Days after   Shallot           fungal     Aspergillus   Aspergillus    Aspergillus    Fusarium    Penicillium   Rhizopus  Mycelia 
            Planting      cultivar          species     flavus           niger              terreus           spp             spp spp.           sterilia 
                  60         Pale-brown     BS  1.0  31.0  1.0        -       1.3  -         - 
                                                      RH  1.0  16.7    -      1.3       1.3  -         - 
                     NR  0.3  13.3  0.3      1.0       1.0  -       1.0 
                                Pink              BS  1.0  26.7    -      1.0       2.0  -       0.3 
                                                     RH   1.0  15.0    -      1.0       1.3  -       0.3 
                                                     NR  0.3  12.0  0.3      1.0       1.0  -       1.0 
 
 
 
 
 
167 
 
Table 62: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR) and rhizosphere (RH) soils, and from soil 
particles adhering to bulbs (BS) of the two Shallot cultivars grown in Anloga shallot farm soil treated with 3.88 g of Urea for 60 days.  
 
                                                        Source                                                                C.F.U’s x 103 /g soil 
                                                          of 
           Days after        Shalllot        fungal      Aspergillus   Aspergillus     Aspergillus   Fusarium     Penicillium    Rhizopus      Mycelia     
            planting          cultivar         species     flavus           niger               terreus           spp spp    spp.          sterilia 
                 20          Pale-brown        BS             -           42.7                -           -       3.3            -               0.7 
                                                         RH            -            15.0             1.7          -             1.3      -    0.7 
                                   NR           0.7              3.3             1.3           -               -           -    0.3 
                                Pink                  BS           0.7  46.0            1.0          -       1.7                   -   1.0 
                                                         RH          1.0           13.3              -         0.7        -           -    0.3 
                                                         NR            -             3.0            0.7           -                1.0                -    1.0 
                 40          Pale-brown        BS          1.3           44.0              -          -             2.7          1.7   0.3 
                                                         RH          1.3           16.7            1.3          -       3.3           -    1.3 
                                 NR          1.3           13.3            1.3          -       3.3           -    0.7 
                              Pink                    BS 1.0           40.0             -          -              1.3           -    1.3 
                                                         RH  1.3           20.0            1.0          -       2.0           -    1.0 
                                                         NR 1.0           11.0            1.0          -       2.0           -    0.3 
168 
 
               
     Table 62 continued 
     
                                                      Source                                                                  C.F.U’s x 103 /g soil 
                                                       of 
            Days after   Shallot           fungal     Aspergillus   Aspergillus    Aspergillus    Fusarium    Penicillium   Rhizopus   Mycelia 
            planting      cultivar          species     flavus             niger              terreus           spp             spp spp.           sterilia 
                  60         Pale-brown     BS  1.0  33.3  1.0        -       1.3  1.0       1.0 
                                                      RH  1.3  12.3    -      1.3       1.3  -       0.3 
                     NR  0.3  11.3    -      0.7       0.7  -       03 
                                Pink              BS  0.7  35.3    -      1.0       1.7  -        - 
                                                     RH   0.3  11.0    -        -       1.3  -       0.7 
                                                     NR  0.7  10.0  0.3      0.7       1.0  -       0.3 
 
 
 
 
 
 
169 
 
 
 
 
 
                                                                                                                                               X 5/9 
 
Plate 12. Photograph showing fungi isolated from the 60 days old bulb surface of  Pale-brown 
cultivar of shallot ( Allium ascalonicum) using Sabouraud agar ( BOTTOM) and Potato 
Dextrose Agar (TOP). Note carbon-black colonies of A. niger. 
 
 
170 
 
 
 
 
 
                                                                                                                                               X 5/9 
 
Plate 13. Photograph showing fungi isolated from the 60 days old bulb surface of Pink cultivar 
of shallot        ( Allium  ascalonicum) using Sabouraud agar ( BOTTOM) and Potato Dextrose 
Agar (TOP). 
Note carbon-black colonies of A. niger. 
 
171 
 
 
EXPERIMENT L. Response of bulbs of the two shallot cultivars formed in Anloga farm 
soils amended with Urea fertilizer to Aspergillus niger inoculation using A. niger Isolates 
1 and 2. 
Urea was meant to enrich the soil. It was assumed that plants benefiting from the enhanced 
robust growth would be less susceptible to A. niger attack. The bulbs of shallot plants which 
received urea fertilizer in the preceding experiment (Experiment K) were, indeed, bigger than 
bulbs of the control plants as shown in Table 59. However, the data in Tables 63a and b indicate 
differently. Plants in soils amended with 1.94 g/l urea fertilizer were either rotted at the same 
rate as the control bulbs or at even a greater rate. The rate, however, declined with the higher 
urea concentration of 3.88 g/l as shown in Table 63c.  
 
Bulbs of all treatments were invaded after experimental inoculation at 50 to 100% R.H. And as 
observed in the earlier tests in Experiment I and recorded in Tables 38 to 47, the diameter of 
the rotted areas of the bulbs increased with increasing storage humidity. Secondly, the Pale-
brown cultivar was uniformly more susceptible to A. niger attack than the Pink cultivar at all 
relative humidities. Thirdly, A. niger Isolate 1 was consistently more virulent than Isolate 2. 
 
 
 
 
172 
 
 
 
Table 63a: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in unamended soil, 
wound-inoculated with mycelium of A. niger Isolates 1 and 2 and incubated at 50-100% 
relative humidity at 32±2oC for 3 days.  
 
                                       Mean diameter of rot (mm) in shallot cultivars caused by A. niger 
 
                                                    Isolate 1                                                   Isolate 2 
              
        Storage                  Pale-brown                Pink                       Pale-brown            Pink   
        Humidity (%)         cv.                            cv. cv.                          cv. 
 
         100                       15.0±1.0 a                  14.1±1.1 a                13.9±1.0 a           12.3±0.9 a 
           90                       14.3±1.1 a                  13.0±1.0 a                13.5±0.9 a           12.0±0.9 a 
           80                       13.6±0.9 a                  12.0±1.0 a                12.9±0.7 a           11.0±1.0 a 
          70                 12.5±0.7 a                  11.1±0.6 a                11.3±0.8 a           11.3±0.6 a 
          60                        11.1±0.8 a                  10.0±0.7 a                10.6±0.6 a            10.1±0.5 a 
          50                        10.6±0.5 a                    9.6±0.7 a                10.3±0.6 a             9.0±0.6 a 
 
  
By the calculated Scheffe’s Confidence Limit, pairs of Values for each A. niger Isolate in 
horizontal rows bearing the same letters are not significantly different at 5% level of probability. 
173 
 
Table 63b: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in soil amended with, 
1.94 g of Urea fertilizer, wound-inoculated with mycelium of A. niger Isolates 1 and 2 and 
incubated at 50-100% relative humidity at 32±2oC for 3 days. 
 
                                       Mean diameter of rot (mm) in shallot cultivars caused by A. niger 
 
                                                    Isolate 1                                                   Isolate 2 
              
        Storage                  Pale-brown                Pink                       Pale-brown            Pink   
        Humidity (%)         cv.                            cv. cv.                          cv. 
 
         100                       15.9±1.3 a                  14.3±0.9 a                  14.6±1.0 a           14.0±0.9 a 
           90                       14.5±1.3 a                  13.5±0.9 a                  13.9±0.9 a           13.1±0.8 a 
           80                       14.0±1.1 a                  13.0±1.0 a                  13.6±1.0 a           12.6±0.8 a 
          70                 13.1±0.9 a                  12.1±0.7 a                  12.8±0.8 a           11.1±0.9 a 
          60                        12.3±0.9 a                  11.0±0.8 a                  11.7±0.8 a            10.3±0.5 a 
          50                        11.2±0.6 a                  10.0±0.6 a                  10.3±0.8 a            10.0±0.7 a 
 
  
By the calculated Scheffe’s Confidence Limit, pairs of Values for each A. niger Isolate in 
horizontal rows bearing the same letters are not significantly different at 5% level of probability. 
 
174 
 
Table 63c: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in soil amended with 
3.88g of Urea fertilizer wound-inoculated with mycelium of A. niger Isolates 1 and 2 and 
incubated at 50-100% relative humidity at 32±2oC for 3 days. 
 
                                       Mean diameter of rot (mm) in shallot cultivars caused by A. niger 
 
                                                    Isolate 1                                                   Isolate 2 
              
        Storage                  Pale-brown                Pink                       Pale-brown            Pink   
        Humidity (%)         cv.                            cv. cv.                          cv. 
 
         100                       14.4±1.5 a                  13.0±1.2 a                  13.0±0.9 a           11.8±0.9 a 
           90                       14.0±0.9 a                  12.1±1.0 a                  12.0±0.7 a           11.0±0.9 a 
           80                       13.1±0.9 a                  11.2±1.0 a                  11.1±0.8 a           10.1±0.7 a 
           70                 11.3±0.6 a                  10.5±0.7 a                 10.2±0.5 a             9.3±0.6 a 
           60                       10.5±0.6 a                     9.3±0.8 a                   9.1±0.7 a             8.8±0.6 a 
           50                         9.3±0.7 a                     9.0±0.7 a                   9.0±0.6 a             8.3±0.7 a 
 
  
By the calculated Scheffe’s Confidence Limit, pairs of Values for each A. niger Isolate in 
horizontal rows bearing the same letters are not significantly different at 5% level of probability. 
 
        
. 
 
175 
 
EXPERIMENT M:  Mycoflora of the bulbs and the rhizosphere of shallot plants raised 
in soil of Anloga farms amended with Potassium fertilizer. 
The data in Tables 64 to 69 highlight the effect of Potassium, a component of the indispensable 
plant nutrient complex, Nitrogen-Phosphorus-Potassium (NPK), on growth of the two shallot 
cultivars and on shallot-fungus association in soil. In addition, on pathogenicity of Aspergillus 
niger on the bulbs through its influence on permeability of plant membranes which would be a 
key objective of tests in the next exercise. 
 
The results in Tables 64 show clearly that over the range of potassium concentration used, 6.67 
g/l was the optimum. All parameters assessed peaked at this concentration and then declined 
thereafter. The sizes of the Pale-brown bulbs at 60 days   in soils with 0.0, 3.33. 6.67, 10.0 and 
13.33 g/l were 33.3±2.0 x 19.8±1.2, 37.5±3.1 x 22.4±1.8, 41.5±1.5x 25.3±0.9, 38.9±1.1x 
27.3±1.1 and 32.8±1.1 x 21.3±0.9mm, respectively. Values for the Pink cultivar followed the 
same trend. The Pale-brown cultivar bulbs at 20, 40 and 60 days were bigger than their 
counterpart Pink cultivar bulbs. 
 
Presence of potassium in greater amounts produced a unique shallot plant-fungus association. 
Although A. niger again crowded the shallot plants, the bulb was a more favourable organ than 
the roots. Some other aspects were notable. 
 
176 
 
The rhizosphere of A. niger population in many instances was only feebly superior as can be 
seen in Table 65. A. flavus and A. terreus were not as eminent as in the urea tests. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
177 
 
Table 64: Morphology of bulblets of the two shallot cultivars developing in the greenhouse 
under a 12- hour day-night cycle in Anloga shallot farm soil containing different quantities of 
Potassium fertilizer. 
 
         Concentrat- Characteristic 
         ion of                   Days            Features                                              Cultivar 
         Potassium      after       of                                            
         (g/L)      planting       bulblets                            Pale-brown                      Pink 
  
                                      20              Mean length (mm)          25.8±2.3                   24.3±2.1 
         Mean width ( mm)            7.8±0.3                     6.6±0.5 
         Colour                              White                         White 
                           40               Mean length (mm)          30.2±2.6                   28.2±1.9        
        0.00         Mean width ( mm)          14.5±1.1                   13.0±0.6 
         Colour                             Pale-brown               White 
                                 60               Mean length (mm)          33.3±2.0                   32.0±2.6 
         Mean width ( mm)          19.8±1.2                   18.3±1.6 
         Colour                              Pale-brown                 Pink 
 
                                     20               Mean length (mm)            29.5±2.1                  26.5±2.5 
         Mean width ( mm)            10.3±0.6                    9.5±0.4 
         Colour                                White                         White 
                                     40                Mean length (mm)            33.5±3.0                  29.0±2.3 
         3.33         Mean width ( mm)            18.5±1.0                  15.3±0.9 
                                                         Colour                                Pale-brown             White 
                                     60                Mean length (mm)            37.5±3.1                  34.2±2.2 
          Mean width ( mm)           22.4±1.8                  17.2±1.1 
 
          Colour                                Pale-brown              Pink 
178 
 
       
 Table 64continued 
.    
         Concentrat- Characteristic 
         ion of                   Days            Features                                              Cultivar 
         Potassium      after       of                                            
         (g/L)      planting       bulblets                            Pale-brown                      Pink 
  
                                      20              Mean length (mm)            31.2±1.3                   27.7±1.1 
         Mean width ( mm)            10.5±0.5                   10.0±0.6 
         Colour                               White                         White 
                           40               Mean length (mm)            37.5±1.3                   25.3±1.0 
        6.67         Mean width ( mm)            21.5±1.0                   16.3±0.6 
         Colour                               Pale-brown               White 
                                 60               Mean length (mm)            41.5±1.5                   32.3±1.4 
          Mean width ( mm)           25.3±0.9                   20.2±1.1 
          Colour                               Pale-brown                Pink 
  
                                     20               Mean length (mm)            29.2±1.1                   24.0±0.9 
         Mean width ( mm)              9.5±0.7                     7.6±0.4 
         Colour                                 White                      White 
                                     40                Mean length (mm)             34.3±1.3                   31.3±1.2 
    10.00         Mean width ( mm)             22.5±1.0                   17.3±0.8 
                                                         Colour                                 Pale-brown              White 
                                     60                Mean length (mm)              38.9±1.1                   36.2±1.3 
          Mean width ( mm)             27.3±1.1                   23.5±1.0 
          Colour                                 Pale-brown               Pink 
  
 
179 
 
Table 64 continued 
    
         Concentrat- Characteristic 
         ion of                   Days            Features                                              Cultivar 
         Potassium      after       of                                            
         (g/L)      planting       bulblets                            Pale-brown                      Pink 
  
                                      20              Mean length (mm)            23.5±0.9                   17.5±0.9 
         Mean width ( mm)              8.9±0.2                     7.2±0.5 
         Colour                                White                          White 
                           40               Mean length (mm)            30.5±1.4                   27.0±1.1 
 13.33         Mean width ( mm)            18.4±0.7                   16.8±0.9 
         Colour                                White                         White 
                                 60               Mean length (mm)            32.8±1.1                   29.3±1.0 
         Mean width ( mm)            21.3±0.9                   20.8±0.8 
         Colour                                Pale-brown                  Pink 
 
  
  
 
180 
 
Table 65: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR) and rhizosphere (RH) soils, and from soil 
particles adhering to bulbs (BS) of the two shallot cultivars grown in unamended Anloga shallot farm soil for 60 days. 
     
                                                        Source                                                  C.F.U’s x 103 /g soil 
                                                          of 
           Days after        Shallot        fungal      Aspergillus   Aspergillus     Aspergillus   Fusarium     Penicillium         Mycelia 
            planting          cultivar         species     flavus           niger               terreus           spp spp    sterilia 
                 20          Pale-brown       BS      4.3   50.3      -    -    -         0.3 
                                                         RH        -   13.3      -    -  2.3         0.7 
                        NR       2.0   13.7    1.0    -    -         0.7 
                                Pink                  BS      5.0   47.3      -    -  2.7         0.7 
                                                         RH       1.3   15.7      -    -  4.0         0.3 
                                                         NR      1.7   16.7    1.3    -  2.7         0.3 
                 40          Pale-brown        BS        -   38.3      -  1.3  1.7         1.0 
                                                         RH        -   11.7    1.7    -  1.3         1.0 
                        NR         -   26.7      -    -  2.7         0.7 
                              Pink                    BS        -   33.0    1.7    -  2.7         1.3 
                                                         RH       0.7   13.3      -    -  1.0         2.0 
                                                         NR        -   28.0      -    -  2.3         1.0  
181 
 
 
            Table 65 continued 
          
                                                      Source                                                  C.F.U’s x 103 /g soil 
                                                       of 
            Days after   Shallot           fungal        Aspergillus     Aspergillus     Aspergillus   Fusarium    Penicillium   Mycelia 
            planting      cultivar          species       flavus             niger               terreus            spp.                spp.     sterilia 
                  60         Pale-brown     BS      0.3  36.7    -          0.7        0.3   0.3          
                                                      RH      0.7  17.7  0.3            -        0.3   2.0 
                     NR        -  24.0    -          0.3        1.0   1.0 
                                Pink              BS        -  28.3    -            -        1.0   1.3 
                                                     RH         -  14.3  0.7            -        0.7   1.7 
                                                     NR        -  23.3    -          0.3        0.7   1.3 
 
 
 
 
 
182 
 
Table 66: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR) and rhizosphere (RH) soils, and from soil 
particles adhering to bulbs (BS) of the two shallot cultivars grown in Anloga shallot farm soil treated with 3.33g of Potassium for 60 
days.  
     
                                                        Source                                                 C.F.U’s x 103 /g soil 
                                                          of 
           Days after        Shallot        fungal         Aspergillus     Aspergillus   Aspergillus   Fusarium   Penicillium     Mycelia 
           planting          cultivar         species        flavus              niger             terreus           spp.   spp. sterilia 
                 20          Pale-brown       BS       4.0      54.3      -    -          6.0        1.3 
                                                         RH       2.7      40.0      -    -          3.3        1.0 
                        NR        3.0      16.0    1.7    -            -          - 
                                Pink                  BS         -      50.7      -    -          8.0        1.0 
                                                         RH          -      38.7    0.7    -          4.5        1.0 
                                                         NR       1.7      19.3      -    -          4.0        0.7 
                 40          Pale-brown        BS         -      46.7      -  1.7          1.3        0.3 
                                                         RH         -      14.3      -    -          0.7        1.0 
                        NR          -      23.3      -    -          1.0          - 
                              Pink                    BS         -      44.3    0.7    -          2.7        0.7 
                                                         RH          -      26.7    1.0    -          1.0        1.0 
                                                         NR         -      30.7      -    -          1.0        1.0 
183 
 
  
            Table 66 continued 
     
                                                      Source                                              C.F.U’s x 103 /g soil 
                                                       of 
            Days after   Shallot           fungal        Aspergillus   Aspergillus    Aspergillus    Fusarium      Penicillium   Mycelia 
            planting      cultivar          species        flavus           niger              terreus           spp.               spp.     sterilia 
                  60         Pale-brown     BS     0.3   40.3  -         0.7        1.7   0.3 
                                                      RH     2.3  20.0  -           -         1.0   1.0 
                     NR       -  22.7  -           -         0.7   0.3 
                                Pink              BS       -  37.7  -         1.0        1.3   0.7 
                                                     RH       -  16.0           1.0           -         1.0   0.3 
                                                     NR       -  25.0  -           -         1.0   0.3 
 
 
  
 
 
184 
 
Table 67: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR) and rhizosphere (RH) soils, and from soil 
particles adhering to bulbs (BS) of the two shallot cultivars grown in Anloga shallot farm soil treated with 6.67g/l of Potassium for 60 
days. 
     
                                                        Source                                                        C.F.U’s x 103 /g soil 
                                                          of 
           Days after        Shallot        fungal      Aspergillus   Aspergillus     Aspergillus   Fusarium     Penicillium      Mycelia 
            planting          cultivar         species     flavus           niger               terreus           spp. spp. sterilia 
                 20          Pale-brown       BS       -     76.7       -  -           4.3         2.0 
                                                         RH     2.7     57.0       -  -           1.7         0.7 
                        NR       -      49.3       -  -          10.0         2.0 
                                Pink                  BS       -     72.3       -  -          11.0         1.3 
                                                         RH      1.0     51.3       -  -           2.3           - 
                                                         NR       -     51.7       -  -           7.3 `       1.0 
                 40          Pale-brown        BS       -     66.7     2.0           0.7  -         0.3 
                                                         RH     0.7     37.3     1.0  -          1.3         0.3 
                        NR       -     40.3       -           2.0          2.7         0.3 
                              Pink                    BS       -     46.0     0.7  -          2.7         0.3 
                                                         RH        -           32.7       -  -          0.7         1.0 
                                                         NR       -     33.3     0.7  -          1.0           - 
 
185 
 
 
 
            Table 67 continued 
     
                                                      Source                                               C.F.U’s x 103 /g soil 
                                                       of 
            Days after   Shallot           fungal     Aspergillus   Aspergillus    Aspergillus    Fusarium    Penicillium    Mycelia 
            planting      cultivar          species     flavus           niger              terreus           spp.             spp. sterilia 
                  60         Pale-brown     BS      -  56.7  -         0.7       0.7  0.3 
                                                      RH          -  30.0  -           -        1.0  0.7 
                     NR      -  36.0  -         1.3       1.3  0.3 
                                Pink              BS    1.0  43.3  -         1.7       1.0    - 
                                                     RH       -  34.3           0.3           -        0.7  0.3 
                                                     NR      -  30.0  -           -        2.7  0.3 
 
 
 
 
 
186 
 
Table 68: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR) and rhizosphere (RH) soils, and from soil 
particles adhering to bulbs (BS) of the two shallot cultivars grown in Anloga shallot farm soil treated with 10.00 g/l of Potassium for 
60 days. 
     
                                                        Source                                                       C.F.U’s x 103 /g soil 
                                                          of 
           Days after        Shalllot        fungal      Aspergillus   Aspergillus     Aspergillus   Fusarium     Penicillium      Mycelia 
            planting          cultivar         species     flavus           niger               terreus           spp spp sterilia 
                 20          Pale-brown       BS         -     70.0       -  -  5.0          1.0   
                                                         RH        1.7    47.7         -  -  1.7          0.3 
                        NR         1.0    30.0       -  -  3.7          1.0 
                                Pink                  BS         -     7.5       -  -  6.0          1.0 
                                                         RH          -     45.0       -  -  3.3          0.3 
                                                         NR       2.7     33.3     0.7  -  3.3          0.3 
                 40          Pale-brown        BS         -     62.7       -  1.0  1.7          0.3 
                                                         RH       0.7     20.2       -  -  1.3          0.3 
                        NR          -     19.3       -  -  1.7          0.7 
                              Pink                    BS         -     59.3     1.0  -  1.3          0.3 
                                                         RH         0.7    19.0       -  1.0  -            - 
                                                         NR          -     16.3     1.0  -  0.7          0.3 
 
187 
 
            Table 68  continued 
     
                                                      Source                                                C.F.U’s x 103 /g soil 
                                                       of 
            Days after   Shallot           fungal     Aspergillus   Aspergillus    Aspergillus    Fusarium    Penicillium    Mycelia 
            planting      cultivar          species     flavus           niger              terreus           spp             spp  sterilia 
                  60         Pale-brown     BS       -  50.0  -           0.3       0.7  1.3 
                                                      RH     1.0  23.0           0.7            -       1.7  1.3 
                     NR       -  21.7  -          1.0       1.0  1.0 
                                Pink              BS     1.0  51.0  -          1.7       0.7  2.0 
                                                     RH        -  23.3  -            -       0.3  1.0 
                                                     NR       -  18.3  -          1.7       0.7  1.0 
 
 
 
 
 
 
 
188 
 
Table 69: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR) and rhizosphere (RH) soils, and from soil 
particles adhering to bulbs (BS) of the two shallot cultivars grown in Anloga shallot farm soil treated with 13.33 g/l of Potassium for 
60 days. 
 
                                                        Source                                                       C.F.U’s x 103 /g soil 
                                                          of 
           Days after        Shallot        fungal      Aspergillus   Aspergillus     Aspergillus   Fusarium     Penicillium      Mycelia 
            planting          cultivar         species     flavus           niger               terreus           spp. spp. sterilia 
                 20          Pale-brown       BS           -     66.7      -   -  6.7         2.0 
                                                         RH         1.7    40.7      -   -  2.0         0.3 
                        NR          1.0    29.0      -   -  5.3           - 
                                Pink                  BS           -     67.3      -   -  8.3         2.0 
                                                         RH            -     43.3      -   -  6.7         1.3 
                                                         NR          1.7    26.7      -   -    -         0.3 
                 40          Pale-brown        BS           -     47.0      -  0.7  1.7           - 
                                                         RH          1.0    14.0      -   -    -         0.3 
                        NR            -     20.0      -   -  3.0         0.3 
                              Pink                    BS           -     50.0    0.3   -  1.0         1.0 
                                                         RH          0.7    16.7      -   -  0.3         0.7 
                                                         NR           -     21.3      -   -  1.7         1.3 
 
189 
 
            Table 69 continued 
     
                                                      Source                                                       C.F.U’s x 103 /g soil 
                                                       of 
            Days after   Shallot           fungal     Aspergillus      Aspergillus    Aspergillus    Fusarium    Penicillium   Mycelia 
            planting      cultivar          species     flavus              niger              terreus           spp             spp   sterilia 
                  60         Pale-brown     BS      -  33.3  -         0.3      0.7  0.3 
                                                      RH      -  16.3  0.3           -       1.3  0.3 
                     NR      -  20.0  -         1.0      0.7  1.0 
                                Pink              BS    0.7  38.3  -         1.3        -  0.3 
                                                     RH       -  17.7  -         1.3        -  1.0 
                                                     NR      -  17.0  -         0.7      0.3  0.3 
 
 
 
 
 
190 
 
EXPERIMENT N. Response of bulbs of the two shallot cultivars formed in Anloga soils 
amended with Potassium fertilizer to inoculation with Aspergillus niger  using A. niger   
Isolates 1 and 2 
In the earlier tests described in Experiment I (Tables 38-42) and L (Table 63), the rate of rotting 
following wound inoculation was slowest at 50% R.H. The rate increased with increasing 
humidity to achieve the fastest rate at 100% R.H. Secondly, the pale-brown cultivar bulbs were 
invaded at a faster rate by Aspergillus niger than bulbs of the Pink cultivar at all storage 
humidities. That was also what happened in this experiment when shallot bulbs formed by 
plants grown in the Anloga farm soil amended with potassium fertilizer were wound-inoculated 
with Aspergillus niger Isolates 1 and 2 according to data in Tables 70a-e. 
 
In addition, the rate of rotting was affected by the potassium fertilizer. It predisposed the bulbs 
formed to A. niger attack. Bulbs formed in soils treated with 6.67 g/l potassium fertilizer were 
invaded fastest, and the rate decreased as the potassium concentration decreased as the 
potassium concentration decreased or increased. For example at 100% R.H. the mean diameters 
of the rotted areas of bulbs of the pale-brown cultivar which developed in soils amended with 
potassium fertilizer of 0.0, 3.33, 6.67, 10.00 and 13.33 g/l and inoculated with A. niger Isolate 
1 were 15.3±0.8, 16.2±1.0, 17.0±1.9, 16.5±1.3 and 16.0±1.3 mm, respectively. 
 
 
 
191 
 
Table 70a: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in unamended soil, 
wound-inoculated with mycelium of A. niger Isolates 1 and 2 and incubated at 50-100% 
relative humidity at 32±2oC for 3 days. 
 
                                       Mean diameter of rot (mm) in shallot cultivars caused by A. niger 
 
                                                    Isolate 1                                                   Isolate 2 
              
        Storage                  Pale-brown                Pink                       Pale-brown            Pink   
        Humidity (%)         cv.                            cv. cv.                          cv. 
 
         100                       15.3±0.8 a                  13.4±0.9 a                  14.1±1.3 a           12.8±1.2 a 
           90                       14.7±0.5 a                  12.8±0.8 a                  13.8±1.3 a           11.5±0.9 a 
           80                       13.3±0.6 a                  12.5±0.7 a                  13.0±1.0 a           11.0±1.0 a 
          70                11.5±0.6 a                  10.0±0.4 a                   12.0±0.9 a           10.7±0.6 a 
          60                       10.8±0.4 a                    9.5±0.2 b                   11.5±0.6 a           10.0±0.8 a 
          50                       10.0±0.7 a                    9.0±0.5 a                   10.2±0.8 a             9.2±0.5 a 
 
  
By the calculated Scheffe’s Confidence Limit, pairs of Values for each A. niger Isolate in 
horizontal rows bearing the same letters are not significantly different at 5% level of probability. 
 
 
192 
 
 Table 70b: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in soil amended with, 
3.33 g/l of Potassium fertilizer wound-inoculated with mycelium of A. niger Isolates 1 and 2 
and incubated at 50-100% relative humidity at 32±2oC for 3 days. 
 
                                       Mean diameter of rot (mm) in shallot cultivars caused by A. niger 
 
                                                    Isolate 1                                                   Isolate 2 
              
        Storage                  Pale-brown                Pink                       Pale-brown              Pink  
        Humidity (%)         cv.                            cv. cv.                          cv. 
 
         100                       16.2±1.0 a                  15.3±1.3 a                  15.1±1.3 a           13.7±1.5 a 
           90                       15.2±1.2 a                  14.8±1.0 a                  14.3±0.9 a           12.6±1.1 a 
           80                       14.3±0.8 a                  13.0±1.0 a                  13.3±0.8 a           12.0±0.8 a 
          70                 13.2±0.6 a                  12.3±0.8 a                  12.5±1.0 a          10.8±0.8 a 
          60                        12.8±0.4 a                  12.0±0.7 a                   12.0±1.0 a          10.1±0.5 a 
          50                        10.2±0.4 a                    9.5±0.7 a                   10.0±0.8 a            9.0±0.6 a 
 
  
By the calculated Scheffe’s Confidence Limit, pairs of Values for each A. niger Isolate in 
horizontal rows bearing the same letters are not significantly different at 5% level of probability. 
 
193 
 
Table 70 c: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in soil amended with 
6.67 g/l of Potassium fertilizer wound-inoculated with mycelium of A. niger Isolates 1and 2 
and incubated at 50-100% relative humidity at 32±2oC for 3 days. 
 
                                       Mean diameter of rot (mm) in shallot cultivars caused by A. niger 
 
                                                    Isolate 1                                                   Isolate 2 
              
        Storage                  Pale-brown                Pink                       Pale-brown            Pink   
        Humidity (%)         cv.                            cv. cv.                          cv. 
 
         100                       17.0±1.9 a                  16.5±1.2 a                  14.9±1.2 a          15.0±1.1 a 
           90                       16.0±1.5 a                  16.1±1.3 a                  14.1±1.2 a          14.3±0.9 a 
           80                       15.5±1.0 a                  15.1±0.9 a                  13.0±0.8 a          13.1±1.1 a 
          70                 14.8±1.2 a                  14.0±0.9 a                  12.7±0.9 a          12.3±0.8 a 
          60                        13.2±0.9 a                  13.0±0.8 a                  12.5±0.6 a          11.1±0.6 a 
          50                        12.0±0.8 a                 11.2±0.5 a                  11.3±0.6 a           10.8±0.7 a 
 
  
By the calculated Scheffe’s Confidence Limit, pairs of Values for each A. niger Isolate in 
horizontal rows bearing the same letters are not significantly different at 5% level of probability. 
 
 
194 
 
 Table 70d: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in soil amended 
with, 10.00 g/l of Potassium fertilizer wound-inoculated with mycelium of A. niger Isolates 1 
and 2 and incubated at 50-100% relative humidity at 32±2oC for 3 days. 
 
                                       Mean diameter of rot (mm) in shallot cultivars caused by A. niger 
 
                                                    Isolate 1                                                   Isolate 2 
              
        Storage                  Pale-brown                Pink                       Pale-brown            Pink   
        Humidity (%)         cv.                            cv. cv.                          cv. 
 
         100                       16.5±1.3 a                  15.8±2.1 a                  15.5±0.9 a          14.1±1.0 a 
           90                       15.2±1.1 a                  14.8±1.3 a                  15.1±1.0 a          13.8±0.8 a 
           80                       14.0±1.1 a                  13.3±1.1 a                  14.3±0.8 a          13.0±0.9 a 
          70                 13.2±0.7 a                  12.5±0.9 a                  13.3±0.7 a          12.3±0.7 a 
          60                        12.5±0.8 a                  12.0±0.9 a                  12.5±0.5 a           11.5±0.7 a 
          50                        11.3±0.5 a                  11.0±1.0 a                  11.8±0.5 a           11.0±0.5 a 
 
  
By the calculated Scheffe’s Confidence Limit, pairs of Values for each A. niger Isolate in 
horizontal rows bearing the same letters are not significantly different at 5% level of probability. 
 
195 
 
Table 70e: Diameter of rot of shallot bulbs (Allium ascalonicum) grown in soil amended 
13.33 g/l of Potassium fertilizer wound-inoculated with mycelium of A. niger Isolates 1 and 2 
and incubated at 50-100% relative humidity at 32±2oC for 3 days. 
 
                                       Mean diameter of rot (mm) in shallot cultivars caused by A. niger 
 
                                                    Isolate 1                                                   Isolate 2 
              
        Storage                  Pale-brown                Pink                       Pale-brown            Pink   
        Humidity (%)         cv.                            cv. cv.                          cv. 
 
         100                       16.0±1.8 a                  14.8±1.5 a                  14.3±1.1 a           13.2±0.9 a 
           90                       15.3±1.2 a                  13.9±1.0 a                  13.8±0.7 a           13.0±0.8 a 
           80                       14.0±1.1 a                  12.5±1.1 a                  13.2±0.9 a           12.2±0.5 a 
          70                 12.0±0.9 a                  11.0±1.0 a                  11.5±0.8 a           11.5±0.7 a 
          60                        11.5±1.0 a                  10.8±0.7 a                  11.0±0.5 a           10.7±0.5 a 
          50                        11.2±0.8 a                  10.0±0.8 a                  11.0±0.6 a           10.2±0.6 a 
 
  
By the calculated Scheffe’s Confidence Limit, pairs of Values for each A. niger Isolate in 
horizontal rows bearing the same letters are not significantly different at 5% level of probability. 
 
 
 
196 
 
EXPERIMENT O.  Mycoflora of the bulbs and rhizosphere of plants of the two shallots 
cultivars grown in soils of farms of non-alliaceous crops 
The bulb is the propagation body for raising shallots. It was clear from the data in Experiment 
C (Table 9) that most of the planting material might have been inoculated in the soil with 
Aspergillus niger. The repeated planting of the plots in Anloga region could have probably built 
a reservoir of A. niger, putting the shallots in danger. The data in Tables 71 to 74 revealed that 
the non-rhizosphere soils of cassava, groundnut and maize carried low levels of A. niger 
populations. Nevertheless the young shallot bulbs of both cultivars secreted exudates that 
stimulated growth of A. niger in their vicinity. Although the bulbs developed at apparently the 
same rate into practically the same size in 20 days as indicated in Table 71; A. niger in the maize 
farm was stimulated to a higher degree than those in the other two soils. 
Remarkably, the roots were less supportive and almost the same A. niger population was 
uniformly recorded in the rhizosphere and non-rhizosphere soils. 
 
 
 
 
 
 
 
197 
 
Table 71: Some morphological and colour characteristics of 20-day old bulblets of the two 
shallot cultivars developing in soils of indicated non-alliaceous crop farms in the greenhouse 
under 12-hour day-night cycle. 
 
        Source               Cultivar 
            of            Features 
          Soil                  of bulblets                               Pale-brown                         Pink 
 
     Cassava              Mean length ( mm)                   25.5±2.0                          24.6±3.1      
     ( Manihot           Mean width ( mm)                      7.0±0.3                            7.1±0.3 
     esculenta)           Colour                                        White                              White 
      farm 
 
     Groundnut          Mean length (mm)                   26.7±1.9                         25.3±1.6 
     ( Arachis  Mean width (mm)                     7.9±0.4                            7.7±0.3 
     hypogea) Colour                                       White                              White 
     farm 
 
     Maize                 Mean length (mm)                   25.9±2.3                        25.4±1.5 
     (Zea mays)         Mean width (mm)                      7.9±0.9                          7.6±0.5 
     farm                   Colour                                        White                             White 
 
 
 
198 
 
   
Table 72: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR) and rhizosphere (RH) soils, and from soil 
particles adhering to bulbs (BS) of the two shallot cultivars grown in cassava ( Manihot esculenta) farm soil for 20 days 
 
                                                      Source                                           C.F.U’s x 103 /g soil 
                                                       of 
           Days after        Shallot      fungal   Aspergillus Aspergillus     Aspergillus   Aspergillus   Fusarium  Penicillium Rhzopus Mycelia 
            planting          cultivar      species   flavus           niger               tamarii         terreus           spp.         spp.         spp.      sterilia 
                 20          Pale-brown     BS           -                43.3                  -         -    2.3          2.7     -      - 
                                                      RH        1.0     5.7                  -       2.3      -            -   1.0    0.3 
                     NR          -                 4.7                 2.0         -      -          2.3   1.0      - 
                                Pink               BS        2.3   40.7                 1.0         -     1.0          1.0     -      - 
                                                      RH       1.0                 5.3                  -         -    2.3            -     -      - 
                                                      NR         -                 4.0                  -         -    1.0           2.3     -    0.3 
                  
 
199 
 
 
Table 73: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR) and rhizosphere (RH) soils, and from soil 
particles adhering to bulbs (BS) of the two shallot cultivars grown in groundnut ( Arachis hypogea) farm soils for 20 days.  
 
                                                      Source                                                          C.F.U’s x 103 /g soil 
                                                      of 
           Days after        Shallot     fungal    Aspergillus  Aspergillus   Aspergillus Aspergillus Fusarium  Penicillium  Rhizopus    Mycelia 
            planting          cultivar      species   flavus            niger            tamarii      terreus         spp.          spp.    spp.           sterilia 
                 20          Pale-brown    BS  -         33.3       -    -        0.7    1.0           -    1.0 
                                                     RH  1.7          2.0       -  1.0          -       -           -    0.3 
                    NR   1.0          3.3       -  0.7          -       -           -    0.3 
                                Pink              BS  -        36.3     1.0  0.7        0.7     2.3           -    0.7 
                                                     RH   0.3         2.0       -  0.3          -       -          1.0   0.3 
                                                     NR  -         3.0    1.0  1.0        1.0      -           -      - 
 
 
200 
 
 
Table 74: Fungal species, isolated with Sabouraud Agar from non-rhizosphere (NR) and rhizosphere (RH) soils, and from soil 
particles adhering to bulbs (BS) of the two shallot cultivars grown in maize ( Zea mays) farm soils for 20 days. 
 
                                                        Source                                                       C.F.U’s x 103 /g soil 
                                                          of 
           Days after        Shallot        fungal      Aspergillus   Aspergillus     Aspergillus   Fusarium     Penicillium       Mycelia 
            planting          cultivar         species     flavus           niger               terreus           spp. spp. sterilia 
                 20          Pale-brown       BS         -     56.7       -  0.7  1.0        0.3 
                                                         RH       1.0      6.7     2.7  -  1.7        0.3 
                        NR        0.7      5.3     0.7  -  1.7        1.0 
                                Pink                  BS         -    54.3       -  2.3  2.3        1.0 
                                                         RH        1.0     6.3     1.0  -  1.0          - 
                                                         NR       2.0     5.6     1.0  -  -         0.3 
 
 
201 
 
EXPERIMENT P. Conidiation of Aspergillus niger Isolates 1, 3 and 5 on ground bulb 
tissue of the two shallot cultivars and the germination capacity of the conidia. 
The blended bulb tissue of shallot supported vigorous vegetative growth of the three test A. 
niger isolates according to the data in Tables 75 to 77. The isolates, however, showed variable 
relationship with the substrates. Isolate 3 grew better on the Pink cultivar tissue than on the 
Pale-brown cultivar tissue. Thus, the mean diameters of the Isolate 3 on the Pink cultivar tissue 
in Table 76 were on each day statistically significantly higher at 5 percent level of probability. 
Cultures of Isolates 1 (Table 75) and 5 (Table 77), on the other hand grew faster in the first 
three days of incubation on the pale-brown cultivar tissue and lagged behind thereafter. 
The results in Table 78, however, showed that sporulation on the two types of tissues by each 
A. niger isolate was practically the same. 
 
A. niger Isolates 1, 3 and 5 produced equally healthy conidia on bulb tissue of both shallot 
cultivars, which germinated to the same degree in the different organic nutrients (Tables 79 to 
81). There was 100 percent germination in undiluted Potato Dextrose Broth and Sweet Potato 
Broth by all isolates. Percentage germination was slightly lower in the undiluted Bean Meal 
Broth of 90.8 to 94.8 percent (Tables 79 and 80). Percentage germination then declined with 
increasing dilution of the media to eventually zero percent germination in the 1: 30 dilution 
media. Mean germ-tube length followed the same pattern of response to medium concentration. 
Potato Dextrose broth was the most suitable conidium germination medium. 
202 
 
Table 75: Growth of Aspergillus niger Isolate 1 on blended bulbs of the two cultivars of 
shallot (Allium ascalonicum) under a 12-hour day-night cycle at 32±2oC. 
 
                                                                  Mean diameter of culture ( mm) ±S.E on 
 
      Day after                  Pale-brown  cv. Medium                    Pink cv. Medium 
                Inoculation  
 
                        1                                 18.5±2.0 a                                          20.0±1.6 a 
                        2                                 22.3±3.7 a                                          21.3±2.0 a 
                        3                                 34.0±3.4 a                                          33.3±2.4 a 
                        4                                 42.7±2.7 a                                          43.0±2.5 a 
                        5                                 49.8±3.0 a                                          59.8±1.4 b 
                        6                                 58.3±2.2 a                                          63.5±3.1 a 
                        7                                 67.0±1.9 a                                          73.2±2.1 a 
 
By calculated Scheffe’s Confidence Limits, values of Mean Culture Diameters in the same 
horizontal row bearing the same letters are not significantly different at 5% level of probability. 
 
     
 
 
 
 
203 
 
Table 76: Growth of Aspergillus niger Isolate 3 on blended bulbs of the two cultivars of 
shallot (Allium ascalonicum) under a 12-hour day-night cycle at 32±2oC. 
.   
 
                                                                  Mean diameter of culture (mm) ±S.E on 
 
      Day after                   
                Inoculation  Pale-brown cv. Medium                    Pink cv. Medium 
 
       1                                      25.5±2.6 a                                       30.0±0.6 a 
                     2                                      32.3±3.7 a                                       41.3±2.3 a 
                     3                                      48.0±3.8 a                                       53.3±2.4 a 
                     4                                      52.7±3.7 a                                       63.0±2.9 a 
                     5                                      59.8±4.0 a                                       69.8±2.4 a 
                     6                                      70.3±4.2 a                                       76.5±3.1 a 
                     7                                      77.0±4.9 a                                       83.2±3.1 a 
 
By calculated Scheffe’s Confidence Limits, values of Mean Culture Diameters in the same 
horizontal row bearing the same letters are not significantly different at 5% level of probability. 
 
 
             
 
 
204 
 
Table 77: Growth of Aspergillus niger Isolate 5 on blended bulbs of the two cultivars of 
shallot (Allium ascalonicum) under a 12-hour day-night cycle at 32±2oC. 
 
 
                                                                  Mean diameter of culture (mm) ±S.E on 
 
      Day after                   
                Inoculation  Pale-brown cv. Medium                    Pink cv. Medium 
 
       1                                      22.7±1.3 a                                          21.5 ±0.0 a                               
       2                                      26.3±0.2 a                                          25.6±0.1 a 
       3                                      31.5±0.2 a                                          30.8±0.1 a 
       4                                      47.7±0.2 a                                          45.8±0.1 a 
       5                                      65.8±0.2 a                                          69.8±0.1 b 
       6                                      77.3±0.3 a                                          78.5±3.1 a 
       7                                      84.3±0.2 a                                          88.0±0.0 b 
  
                   
 
By calculated Scheffe’s Confidence Limits, values of Mean Culture Diameters in the same 
horizontal row bearing the same letters are not significantly different at 5% level of probability. 
 
 
 
205 
 
 
 
 
 
 
 Table 78: Degree of sporulation of Aspergillus niger Isolates 1, 3 and 5 grown on blended 
tissue of bulbs of the two shallot cultivars under a 12-hour day-night cycle at 32±2oC for 7 days. 
          
                                                          Number of conidia per ml of suspension of the 
                                                          tissues of the cultivar ( x 104 ) 
        A. niger Isolate                        Pale- brown                                Pink 
 
               1                                            6.51                                         5.63 
 
              3                                             4.64                                         4.48 
 
              5                                             6.12                                         6.09 
 
 
 
 
  
 
 
206 
 
Table 79: Germination of conidia produced by 7-day old Aspergillus niger Isolate 1 incubated 
in liquid organic nutrients at 32±2oC for12 hours.  
     
                                                                Percentage                           Mean Gem tube 
        Type of            Medium                  Germination                         length ± S.E. (µm) 
         Medium         dilution*         Pale-brown cv.   Pink cv.         Pale-brown cv.   Pink cv.           
 
        Bean             Undiluted             94.8                  90.8                210.3 ±7.3 a       190.3±4.9 b        
        Meal                 1:1                    74.8                  69.9                173.8±6.3 a        150.3±4.3 b 
        Broth                1:10                  23.2                  18.3                 59.9±3.8 a           48.4±2.5 a 
        (BMB)             1:20                   14.8                  12.4                 15.9±1.7 a           12.3±1.2 a 
                                 1:30                    0.0                    0.0                       -                          - 
               
         Potato          Undiluted             100                  100                  240.3±9. 2 a       233.4±7.8 a 
         Dextrose          1:1                     86.7                83.4                 220.3±7.3 a        210.9±10.2a 
         Broth               1:10                   25.8                20.5                  78.2±5.4 a           82.4±8.8 a 
        (PDB)               1:20                  19.2                 14.0                  25.3±4.3 a           21.3±3.9 a 
                                 1:30                    0.0                   0.0                        -                        - 
 
        Sweet           Undiluted            100                   100                  228.4±7.9 a          220.4±6.9 a 
        Potato               1:1                    80.2                 78.3                 200.9±8.8 a         189.3±8.3 a 
        Broth                1:10                  24.4                  21.3                  82.3±4.8 a           74.4±2.3 a 
        (SB)                 1:20                  15.8                  13.2                   20.2±3.4 a           23.8±4.9 a 
                                 1:30                  0.0                     0.0                          -                     - 
1. *Undiluted medium : Distilled water. 
2.   By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube 
Lengths in each each medium bearing the same letters are not significantly different at 
5% level of probability. 
207 
 
Table 80: Germination of conidia produced by 7-day old Aspergillus niger Isolate 3 incubated 
in liquid organic nutrients at 32±2oC for12 hours. 
 
                                                                            Percentage                             Mean Gem-tube 
        Types of         Medium                    Germination                         length ± S.E. (µm) 
         Medium        dilution*          Pale-brown cv.   Pink cv.         Pale-brown cv.   Pink cv.           
 
        Bean             Undiluted             95.2                  94.8                198.4±6.8 a       200.2±7.7 a        
        Meal                 1:1                    78.3                  75.3                190.3±6.2 a       185.3±6.3 a 
        Broth                1:10                  25.3                  24.2                  54.8±4.4 a         49.3±4.8 a 
        (BMB)             1:20                   18.2                 15.2                   24.3±4.8 a         20.3±3.3 a 
                                 1:30                    0.0                   0.0                       -                          - 
               
         Potato          Undiluted             100                  100                  232.4±6.9 a       230.0±7.3 a 
         Dextrose          1:1                     92.8                 90.5                218.5±5.5 a       200.3±4.5 a 
         Broth               1:10                   32.4                 30.4                  80.3±4.3 a         82.5±5.1 a 
        (PDB)              1:20                   22.3                 15.3                  30.2±4.0 a          27.3±4.8 a 
                                 1:30                    0.0                   0.0                        -                        - 
 
        Sweet           Undiluted            100                   100                    235.5±9.9 a      231.4±8.8 a 
        Potato               1:1                    89.3                 82.5                   220.3±7.9 a      210.1±9.3 a 
        Broth                1:10                 30.4                  24.3                     78.5±6.3 a       79.3±3.9 a 
        (SB)                 1:20                  18.3                 16.2                     28.3±3.3 a       24.2±4.8 a 
                                 1:30                   0.0                    0.0                          -                     - 
 
1. *Undiluted medium : Distilled water. 
2.   By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube 
Lengths in each each medium bearing the same letters are not significantly different at 
5% level of probability. 
208 
 
Table 81: Germination of conidia produced by 7-day old Aspergillus niger Isolate 5 incubated 
in liquid organic nutrients at 32±oC for12 hours. 
      
                                                                Percentage                           Mean Gem tube 
        Types of          Medium                  Germination                         length ± S.E. (µm) 
         Medium         dilution*         Pale-brown c.v.   Pink c.v.         Pale-brown c.v.   Pink c.v.           
 
        Bean             Undiluted             92.5                  90.3                195.3±6.3 a       195.3±7.2 a        
        Meal                 1:1                    80.2                  75.3                183.4±4.6 a       177.4±6.2 a 
        Broth                1:10                  23.8                  21.5                 60.3±3.8 a          58.9±6.0 a 
        (BMB)             1:20                  20.5                  18.7                  28.3±3.4 a         24.3±3.3 a 
                                1:30                    0.0                    0.0                       -                          - 
               
         Potato          Undiluted             100                  100                  238.8±8.2 a       230.4±10.3 a 
         Dextrose         1:1                     93.5                 90.3                218.2±4.3 a        220.5±6.3 a 
         Broth              1:10                   34.3                 35.3                  83.4±3.9 a         79.3±4.8 a 
        (PDB)             1:20                   26.2                  27.8                  32.5±5.1 a         26.5±5.9 a 
                               1:30                     0.0                   0.0                        -                        - 
 
        Sweet           Undiluted            100                   100                   213.3±6.3 a       208.8±8.2 a 
        Potato              1:1                    89.9                 86.5                  208.3±4.2 a        198.1±6.3 a 
        Broth               1:10                  30.3                 29.1                     85.5±4.3 a         84.4±4.9 a 
        (SB)                1:20                  23.2                 20.2                     30.4±3.9 a          31.8±5.5 a 
                               1:30                    0.0                    0.0                          -                     - 
1. *Undiluted medium : Distilled water. 
2.   By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube 
Lengths in each each medium bearing the same letters are not significantly different at 
5% level of probability. 
209 
 
EXERCISE Q. Longevity of conidia formed on the two shallot substrates by Aspergillus 
niger Isolates 1, 3 and 5 at different relative humidities 
The results of the lengthy survival tests in Fig. 4 and Appendices B-V indicate that conidia of 
the three A. niger isolates (Isolate 1, 3 and 5) exhibited many similar characteristics. To 
summarize: 
(a) The conidia retained vitality at the favourable relative humidities for 200 days 
(b) The conidia remained viable for a longer time at higher relative humidities than at lower 
relative humidities 
(c) At 100% R.H., the most favourable relative humidity for survival, at least 43.8, 42.1 
and 32.3 per cent of the conidia of Isolates 1 (Appendix H ), 3 (Appendix O) and 5 
(Appendix V) respectively, were alive after 200 days’ storage on the Pink cultivar. 
(d) The higher the percentage survival, the longer the germ-tubes. 
(e) Percentage viability of conidia of the three A. niger isolates at any particular relative 
humidity was relatively the same. 
(f) For any A. niger isolate there was no significant difference between viabilities of conidia 
formed on bulb tissues of the Pale-brown and Pink shallot cultivars. 
 
 
 
 
                   
210 
 
           Conidia of A. niger Isolate 1              Conidia of A. niger Isolate 3              Conidia of A. niger Isolate 5 
     
       
 
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Figure 4a: Viability of conidia of three isolates of Aspergillus niger formed on ground Pale-brown (  ) and Pink  
(  ) shallot bulbs and stored at different Relative Humidities at 32±2oC under 12-hour Day-Night light cycle. 
211 
 
          Conidia of A. niger Isolate 1                 Conidia of A. niger Isolate 3                 Conidia of A. niger Isolate 5 
       
       
 
 
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Figure 4b: Viability of conidia of three isolates of Aspergillus niger formed on ground Pale-brown (  ) and Pink 
(  ) shallot bulbs and stored at different Relative Humidities at 32±2oC under 12-hour Day-Night light cycle. 
212 
 
          Conidia of A. niger Isolate 1                 Conidia of A. niger Isolate 3                 Conidia of A. niger Isolate 5 
      
      
   
 
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Figure 4c: Viability of conidia of three isolates of Aspergillus niger formed on ground Pale-brown (  ) and 
Pink (  ) shallot bulbs and stored at different Relative Humidities at 32±2oC under 12-hour Day-Night cycle 
regime 
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213 
 
V. DISCUSSION 
In his book, Useful plants of Ghana, West African use of wild and cultivated plants, Abbiw 
(1990) stated that, there are about six varieties of shallots based on size of bulbs, colour and 
degree of scent’. The six varieties were not named. Neither did Abbiw (1990) state the source 
of information and further relevant particulars could, not, therefore, be obtained. During the 
three years of this study, persistent and wide survey for shallot cultivars in Ghana revealed only 
two cultivars, viz., Pale-brown and Pink cultivars (see Figs. 1 and 2, and Plates 5,6 and 7) exist 
which became the subject of the present investigation 
 
The large onions (Allium cepa), have three major cultivars-Bawku, Red Creole and Texas 
Grano being cultivated extensively in the country (Teye, 1994). The Bawku variety is medium-
sized, globular in shape and has a firm flesh and purple-coloured scale leaves. The Red Creole 
bulbs are ovalish, small to medium in size with a firm flesh and red scale leaves. Texas Grana 
bulbs are large in size, flask-shaped with elongated neck and have a comparatively softer flesh 
and straw-yellow scale leaves. The size of the onion market far exceeds that of shallots and this 
reduces the interest in shallots. Diminished interest in shallots would discourage their 
cultivation. It was prudent to find ways to support continuing shallot production to maintain a 
flourishing shallot market because of the valuable contribution of shallot to human health. One 
of the ways by which this can be done is by identifying the shallot cultivar that can be 
recommend as the preferred choice for higher productivity. This cultivar should significantly, 
among other attributes, be able to withstand the ubiquitous Asperigillus niger, the cause of the 
black spot of onions and shallots, and be able to able to discourage its persistence in the 
ecosystem. 
214 
 
The results of the present experiment show the significance of the Pink cultivar for shallot 
production in Ghana. Bulbs of both cultivars suffer from high A. niger natural infection, but 
more so by those of the pale-brown cultivar. The shallot bulb, either of a single unit or either of 
a major bulb and an attached mini- bulb, is firmly covered by a double –layer of pigmented 
scale leaves. Yet, the fungus colonizes not only the exposed parts but the covered parts as well. 
From the data in Table 9, the mean percentages of bulbs, bearing A. niger growth on the abaxial 
surface of the outer scale leaf , adaxial surface of the outer scale leaf, abaxial surface of the 
inner scale leaf, adaxial surface of the inner scale leaf, abaxial surface of the outermost fleshy 
leaf base and the contact surfaces of the major bulb and mini- bulb of the Pale – brown cultivar 
was 72 , 83, 85, 75, 66 and 75 percent, respectively, and of the Pink cultivar, lower values of64, 
71, 81, 72, 65 and 67 percent, respectively. 
 
A significant feature was the colonization of hidden areas under the cover of the scale leaves in 
both cultivars. One possibility was that, insects and mites might have transported conidia and 
fragments of hyphae to those parts. In her report on Fusarium solani infection of shallot bulbs, 
Halm (1971) included photomicrographs of sections of the disc invaded by the fungus, A. niger 
and showed that invading hyphae could enter the bulb by the same route. 
 
The colonies on the swollen leaf bases were not as wide spreading as those on the scale leaves 
( Plate 8). The conidiophores on the scale leaves were also longer than those on the swollen leaf 
bases, that is the hidden parts (Table 10). The conidiophores on both cultivars on the abaxial 
surface of the outer scale leaf were almost twice the length of conidiophores on the abaxial 
215 
 
surface of the outermost fleshy leaf base. Greater development on the scale leaves was certainly 
due to the higher levels of oxygen in that micro-habitat. There is no information in the pertinent 
literature to suggest the role of substances from the different organs in the growth of the 
conidiophores. Conidiophores on the Pink cultivar were approximately 2/3rd the length of those 
on Pale-brown cultivar. Many substances are exuded by leaves through the cuticle including 
cations such as calcium, magnesium, potassium, sodium; amino acids; ammonia; free sugars; 
pectic substances; organic acids; sugar alcohols; vitamins and growth-regulating substances ( 
gibberellin) ( Preece and Dickinson, 1971). The shorter length of conidiophores on the pink 
cultivar suggests that the exudate of that cultivar restrained growth. 
 
Many plant pathogenic fungi produce cutinolytic enzymes (Albersheim, et. al., 1969; Brown, 
1965; Van den Ende and Linskens,1974). Products of decomposed cuticle are added to the 
exudates augmenting both stimulatory and toxic substances in the exudates. Ensuing higher 
level of nutrients will promote fungal growth and higher levels of toxic compounds will inhibit 
growth. The ability of A. niger to decompose the cuticular layers of the bulbs of the pale-brown 
and pink cultivars should be examined in any future studies to provide information that would 
explain the lesser growth on the pink cultivar. 
 
In addition, such a study will explain why A. niger was unable to attack the bulbs in the surface- 
inoculation tests (Table 37). Hashioka et al (1967) reported that germ tubes and appressoria of 
Pyricularia oryzae form liquid droplets, and the cutin just beneath them was dissolved. Also, 
the cuticle of apple fruits breaks down just at the penetration point of Venturia inagegualis. 
216 
 
Nicholson et al. (1972) demonstrated the presence of a membrane bound ‘infections sac’ in 
contact with the host plant cuticle at the penetration point. The present study did not extend to 
these details but could form the basis for future studies. 
 
In general, the shorter the conidiophore the smaller the vesicle and the fewer the number of 
sterigmata that will be require to cover the vesicle, thereby reducing the productiveness of the 
conidiophores. The Pink cultivar under this circumstances will prove useful in limiting the 
amount of infective conidia that will be released into the ecosystem.  
 
The data in Table 43 to 47 concern another aspects of pathogenesis of A. niger . Experimentally 
wound inoculated bulbs of both cultivars using the five isolates of   A. niger and incubated at 
50-100% R.H. were rotted to varying degrees depending on the storage humidity. The isolates 
produced conidiophores on the rotted tissues at 80 - 100 % R. H. only, no doubt determined by 
available moisture. The conidiophores in all tests and at corresponding relative humidities were 
shorter on the pink cultivar bulbs than the pale-brown cultivars. 
 
It was not surprising that there were longer conidiophores on the intact structures of the bulb ( 
Table 10) and shorter conidiophores on the rotted tissues ( Tables 43 to 47). The probability 
that factors in infected tissues could reduce the quality of the substratum has been suggested by 
some workers. Cole and Wood (1961), for example, found that there were more compounds in 
apple fruits rotted by Botrytis cinerea, Penicillium expansum, Pyrenochaeta furfuracea and 
Sclerotina  fructigena than in uninfected apples. There were also greater number of these 
217 
 
compounds in extracts of apple rotted by P. expansum and P. furfuracea than in extracts of rots 
produced by B. cinerea. 
A. niger conidia did not germinate in distilled water ( Tables 12 - 31). This confirms pertinent 
findings of earlier workers (e.g. Amewowor 1980; Kesse, 1995). Amewowor (1980) and Kesse 
(1995) could not obtain A. niger conidium germination even in complex media which could be 
judged to be of very low concentration. Amewowor (1980) prepared root exudates of  5-day 
old seedlings of bambara groundnut (Voandzeia subterranean Thouars) analysed chemically to 
identify the component amino acid and ammonia, and determine their concentrations. The 
amino acids were alanine, ammonia, aspartic acid, glutamic acid, glycine, histidine, iso-leucine, 
leucine, lysine, serine, threonine, tryptophan and valine of the respective concentrations of 
0.015, 0.004, 0.021, 0.074, 0.029, 0.013, 0.002, 0.004, 0.002, 0.036, 0.007, 0.025, and 0.004 
µmols/ml.The conidia did not germinate in this complex medium. 
 
Kesse (1995) also obtained no germination of A. niger conidia in solution of 0.1 per cent 
peptone, 1.0 percent dextrose, 1.0 per cent sucrose and exudates of grains of three maize 
cultivars - Mixed white, Obaatanpa and Yellow. 
 
The two shallot cultivars will apparently not contribute to conidium germination on the bulbs 
under normal conditions under the influence of the exudates. If the metabolism of the plant 
organ is likely to be related to the composition of the exudate, this is not manifested in the 
shallot bulbs. The data in Tables 3 to 8 indicate significantly higher levels of the chemical 
substances ( Calcium, Magnesium, Nitrogen, Phosphorus, Potassium and Sodium) in the Pale-
218 
 
brown cultivar bulbs. Citing three important examples, the mean respective percentages for the 
Pale-brown and Pink cultivars shown in Table 8 were 1.456 ± 0.07 and 1.192 ± 0.05 for 
nitrogen, 0.406 ± 0.01 and 0.367 ± 0.01 for phosphorus  and 1.142 ± 0.03 and 0.979 ± 0.08 for 
potassium. Present data cannot explain why both did not influence conidium germination in 
distilled water droplets. Future studies should endeavour to identify the constituents of the 
exudates of the scale leaves and swollen leaf bases. The results of such investigations may also 
help to explain the consistent higher conidium germination and longer germ-tubes on the scale 
leaves. 
 
Nutrient droplets splash on the bulbs must be of significantly high concentration in order to be 
able to stimulate satisfactory germination of any A. niger conidia on the bulbs. For, good 
germination occurred only in the 1:10 and 1:20 dilutions of Potato Dextrose Broth (Tables 12- 
16), undiluted and 1:1 dilution of Bean Meal Broth and Sweet Potato Broth (Tables 79 to 81), 
and 5.0 and 10.0g/l solutions of galactose, glucose and sucrose ( Tables 17 to 31). Percentage 
conidium germination in these media were accompanied by vigorous germ-tube growth. The 
germination percentages recorded on the Pale-brown and Pink cultivars were closely similar, 
showing no recognizable trend. On some occasions, the percentage germination and mean 
germ-tube length were slightly higher on the Pale-brown cultivar, and on other occasions it was 
the reverse.  
 
The conidia will play a significant role in infection in the market stalls if they settle in wounds, 
and so will the mycelium, for, the conidia germinated extremely well in extracted fluid of the 
219 
 
bulbs ( Table 32). The mycelium also failed to incite infection in bulbs which were surface-
inoculated (see Table 37) while the bulbs were readily invaded by the mycelium following 
wound inoculation (Tables 37 to 42 and Plate 10). Conidia of A. niger Isolate 1, for example,  
also attained 100 and 93.5 per cent germination, respectively in the fluids of Pale-brown and 
Pink cultivars. The corresponding germination percentages by Isolates 2, 4 and 5 were 100 and 
100 percent (Tables 33, 35 and 36), and for Isolate 3 were 97.2 and 87.4 per cent (Table 34). 
With reference to the diluted fluids, lower percentage germination was obtained with the fluids 
of the Pink cultivar. This is an indication that the Pink would be less vulnerable to A. niger 
attack. 
 
Crops have their pests as well, which create the wounds that serve as entrances for pathogens. 
Onions and shallots have common pests. Onion trips (Thrips tabaci Lindeman) are the principal 
insect pests of onions and shallots ( Hill, 1983; Hill and Waller, 1988).They are polyphagous 
and cosmopolitan pests of onions and shallots and many other vegetables. Both the nymphs and 
adults penetrate the epidermis of leaves and swollen leaf bases, and suck the sap that exudes. 
These insects, which measure about 1.0mm in length, hide during daytime in between the leaf 
bases. The cotton leaf worm, Spodoptera littoralis Boisd, is another polyphagous pest attacking 
a wide range of crops. The larvae feed on the leaves and severe attacks can cause reduction in 
bulb weight and size (Hill, 1983; Hill and Waller, 1988).The bulb nematode, Ditylenchus 
dipsaci invades the leaf bases of seedlings and older plants which swell up with loosening of 
the cells. The disease is aptly called “bloat” because of this symptom (Hill and Waller, 1988). 
 
220 
 
A. niger introduced into the wounds creaked by these invertebrates will invade bulbs under 
fairly wide range of relative humidity with invasion proceeding faster in the Pale-brown cultivar 
bulbs as the data in Tables 38 to 42. A. niger is a notable member of the xerophilic species 
which are prominent among storage fungi in “dry seeds and grains”. It was not surprising, 
therefore, that it invaded the bulbs at the low relatice humidity of 50% R.H. The relative 
humidities which permit growth of some prevailing storage fungi are 65% for Aspergillius 
halophilicus, 70-73% for Aspergillus restrictus and Aspergillus repens, 80% for Aspergillus 
candidus and Aspergillus ochraceus, and 85% for Aspergillus flavus (Christensen, 1973). 
 
Growth of A. niger from 100%R.H down to 50%R.H. will inflict considerable loss on products 
in storage and will limit productivity. Damage by A. niger was accentuated at 80 to 100% R.H. 
where the fungus went further to sporulate and to contribute to its survival. Kesse (1995) had 
earlier also recorded the following mean culture diameters: 58.0±1.3, 60.7±0.4, 62.0±0.8, 
63.0±0.9, 63.3±0.2 and 63.0± 0.8mm on maize meal agar plate plates in 7days at 62.6, 65.0, 
73.4, 85.2, 92.8 and 100% R.H., respectively. The fungus in the rotted shallot bulb tissue in this 
investigation was unable to sporulate at 50 to 70% due naturally to deficient water supply. Tests 
were not made below 50% R.H. to establish the lower percentage relative humidity limit. This 
should be studied in a future research as it will be of practical value. 
 
The seemingly limitless profligacy of fungi in the production of spores is impressive. Of 
important, also, is the development of mechanisms or devices that serve to provide maximum 
distribution of these spores. In some species of fungi special mechanisms are lacking, and hence 
221 
 
distribution appears to be largely fortuitous. This might appear to be the case in the dispersal of 
A. niger conidia. The dispersal can be considered successful if the spore lands on a suitable 
substratum in a viable condition. 
 
The conidia of A. niger are long-lived. In a longevity test which employed conidia of   A. niger 
isolates 1, 3 and 5 formed by mycelia growing on ground bulb tissue of Pale-brown and Pink 
cultivars, at least 43.8% ( Appendix H), 42.1% ( Table Appendix O) and 32.3% (  Appendix V) 
of the conidia were viable after storage for 200 days at the best storage humidity of 100% R.H. 
The conidia were stored at zero to 100% R.H. at 32±2oC under day\night light cycle. They died 
quickest at zero to 30% R.H. ( Appendix N 94 and U) and live longest at the higher humidities 
of 70 to 100% R.H.( Appendix H and O). 
 
A. niger conidia exhibited one of the many ways in which fungal spores respond to storage 
humidity. Other species whose spores have been found to be killed more rapidly by low 
humidities include Phytophthora infestans (Glendenning et. al., 1963). Phytopthora meadii 
(Pereis and Fernado, 1966) and Trachysphaera fructigena (Maramba and Clerk, 1974). In this 
group of fungi, the desiccation of the spores may disrupt the essential minimal metabolic 
processes which sustain life of the spore. Gottlieb (1966) wrote, "The one general physiological 
characteristic that separates the spore phase from the vegetative phase of a fungus is the low 
metabolic activity of spores compared with that of mature hyphae. Yet, some respiration occurs 
even in resting spores and their weight decreases, even synthesis may occur though at a very 
low rate. This low-level metabolism in resting spores keeps the metabolic machinery in repair". 
222 
 
As the A. niger conidia live long, their concentration in the air rises because there is a continuous 
production by bulbs in the stores. The high concentration of the living conidia increases the 
incidence of ontomycosis of the outer ear of the workers in that environment. In order to 
safeguard their health, regular monitoring of the levels of the airspora and the disinfection of 
the premises when necessary should be adopted. Such monitoring has been done under similar 
circumstance. For example, Gregory et. al.(1953) were concerned with concentration of 
basidiospores of the dry rot fungus (Merulius lacrymans) in the air of buildings, and, Lacey and 
Lacey (1964) worked on spores, generated by saprophytic fungi of moulding hay, in the air of 
farm buildings. 
 
A phenomenon, termed Iterative germination, in Aspergillus niger was described by Anderson 
and Smith (1971). The term is used to describe those spores that produce the sporogenous 
apparatus and do not produce any mycelium. This definition serves to distinguish a 
morphological variation from the usual microcycle conidia where hyphae, considerably reduced 
are produced. This phenomenon has been seen in diverse species such as, Penicillum digitatum 
(Zeidler and Margalith, 1973), Neurospora crassa (Cortat and Turian, 1974), Penicillium 
italicum (Van Gertel, 1983) and Zoophthora radicans (Van Roermund et al., 1984). The 
process is induced by a heat period and other specific cultural conditions. Iterative germination 
was induced in A. niger conidia exposed to 44oC for 48 hours (Smith and Anderson, 1973). The 
phenomenon occurs under conditions too severe for normal vegetative growth and thereby 
reinforces the survival value derived from the sporulation process. This potential should be 
investigated in A. niger conidia formed on bulbs of the two shallots cultivars in future studies 
which may be related to the persistence of the fungus in the ecosystem. 
223 
 
Substrates for microbial growth in soils vary not only in kind but also in amount and this 
variability affects some groups of microbes more than others. Warcup (1967) pointed out that, 
a source of energy and nutrients sufficient for the growth and multiplication of a bacterium may 
be barely adequate for a germinating fungus spore and quite inadequate to sustain extensive 
mycelial development. 
 
The growth of A. niger on so many parts of the shallot bulb, indicated in Table 9 and in Plate 
8, including effectively protected areas suggested a close association that could have begun in 
the soil where A. niger is ever present. In their manual 'A manual of the Aspergilli', Thom and 
Raper (1945) stated that, Aspergillus niger, the black aspergerlli  are abundant in all soils 
examined, and from studies which have been made by the  authors and other investigators, it 
would appear that they are particularly abundant in soils from tropical and sub-tropical areas'. 
In this research work every sample of non-rhizosphere soil contained A. niger. It was really the 
level of the A. niger population in the non-rhizosphere by which the effects of exudates of the 
plants, and, applied fertilizers could be assessed. It is well known that if the exudates are 
stimulating, the population close to the plant will increase. 
 
The results of the investigation indicated that, in unamended Anloga farm soils and unamended 
soils of non-alliaceous farms at Legon, the region  around the bulb and roots contained 
stimulatory substances which increased growth and multiplication of A. niger (see Tables 60, 
65, 72, 73 and 74). For example the A. niger population in the unamended Anloga farm soil 
planted with the Pale-brown shallot cultivar of 4.0 x 103 C.F.U’s /g of soil in the non-
224 
 
rhizosphere soil rose to 27.3 x 103 C.F.U’s/g of soil around the young bulb and 13.0 x 103  
C.F.U’s/ml around the roots in 20 days ( Table 60).  In the cassava farm soil, from 4.7 x 103 to 
43.3 x 103 C.F.U’s/g  of soil around the bulbs and only 5.7 x 103 C.F.U's/g of soil around the 
roots ( Table 72). 
The response of A. niger to the shallot plants fall into two categories represented by the   
examples cited above.  
a. There was stimulation of A. niger populations around both the bulb and the roots, but 
significantly higher around the bulbs. 
b. Stimulation was very high around the bulb and very negligible around the roots. This 
event concerned both cultivars and hence influenced, similarly, colonization of the bulbs 
of both cultivars. The results can only be fully explained if the nature and quantity of 
the exudates from the bulbs and roots are known. 
  
Table 59 shows that the plants did not seem to benefit from the urea fertilizer at the 
concentrations used judging from the sizes of the bulbs after 40 and 60 days. At least, the urea 
concentration was adequate. For in a preliminary test, urea concentration beyond 3.88g per 
1200g of soil killed the plants. Woldetsadik (2003) observed that the nitrogen fertilizers, either 
ammonium nitrate or ammonium sulphate or urea at rates of  50-200kg /ha to rain-fed shallot 
plants ( DZ- Sht- 78”, “ Dz- Sht -91” and “Fedis” cultivars) in Ethiopia, induced more 
vegetative growth, delayed maturing and reduced bulblet sizes. He could, however, obtain 
marketable bulbs per plant when he added supplemental irrigation. It sees advisable to consider 
nitrogen fertilization on the basis of soil fertility and moisture availability. 
 
225 
 
In contrast, potassium fertilization had a favourable effect on the shallot plants of both cultivars. 
Thus, at 20 days, the mean lengths of bulbs of 0.00 g/l potassium treatment of the Pale –brown 
and Pink cultivars were 25.5±1.3 and 24.3±2.1 mm, respectively. The corresponding values for 
the bulbs at the optimum potassium concentration of 6.67g/l were 31.2±1.3 and 27.7±1.1mm, 
respectively. The mean widths of the bulbs at 20 days for 0.00 g/l of potassium concentration 
were 7.8±0.3 and 6.6±2.1mm were respectively and for the 6.67g/l potassium treatment were 
10.5±0.5 and 10.0±0.6mm, respectively (see Table 64). 
 
The more important reason for applying the urea and potassium fertilizers was to determine 
their effect on infection of bulbs by A. niger. All plant parasitic diseases are the result of the 
interaction of a virulent pathogen, a favourable environment, genetic constitution, light, 
moisture, nutrients, non-nutrients chemicals, pH, and temperature which determine the growth 
of plants and affect their susceptibility to disease. These factors affect form and rate of growth 
of plants, chemical composition of the cell walls, distribution of water within the plant, rates of 
metabolism, and, production of metabolites. 
 
The pertinent literature contains extensive information on the effects of nutrients on disease 
(e.g. Hesterberg and Jurgensen, 1972; Yarwood 1976). Heavy generalized fertilization, for 
example, usually increases disease, especially those caused by powdery mildews and rusts. Of 
the separate major nutrients, nitrogen usually increases disease incidense. Potassium may 
decrease it, and phosphorus is intermediate. With both fertilizers, inoculated bulbs of both 
shallot cultivars were rotted by two test A. niger isolates at 50- 100% R.H. The diameters of the 
226 
 
rotted tissues at each incubation relative humidity were greater in bulbs of the pale-brown 
cultivar than the pink cultivar bulbs (Tables 64 and 71). The addition of fertilizers did not 
change the results of the previous experiments. The Pale-brown cultivar apparently became 
more susceptible with the potassium fertilization than the Pink cultivar. It must be emphasized 
that the fertilizers did not impose significant resistance to A. niger. A fact that the results suggest 
is that, urea, and potassium treatment especially, were good for both shallot plants ( Table 64) 
and the fungus. From the point of view of agricultural productivity, the major objective should 
be predisposition to resistance with no loss in crop productivity. 
 
In conclusion, both the Pale-brown and Pink shallot cultivars are not resistant to A. niger and 
that condition was not attained by fertilizer application. The two cultivars supported abundant 
sporulation by A. niger and the conidia produced had high germination capacity and they 
equally lived long. Nevertheless, it will be reasonable to suggest that because of the slightly 
lesser level of natural infection, depressed conidiophore development, lesser support of 
conidium germination, slower rate of rot of bulbs by A. niger growth, and lesser stimulation of 
A. niger growth in the phyllosphere and rhizosphere, the pink cultivar should be considered the 
preferable choice for the shallot industry, and, for future shallot programmes. 
 
 
 
 
227 
 
RECOMMENDATIONS 
a. On the basis of the conclusion of the Discussion, the Pink cultivar should be adopted 
for expanded cultivation of shallot. 
b. The Pink cultivar should be adopted for shallot breeding programmes to produce 
improved stock. 
c. Crop rotation should be adopted in shallot farms to prevent building up of A. niger 
inoculum in the soils due to inevitable stimulation of A. niger by the exudates of the 
bulbs and roots. 
d. The normal humidity in the shallot stalls will support sporulation of contaminant  
A. niger. The load of these long-lived spores will build up to the detriment of the health 
of the handlers. Regular disinfection of the atmosphere of the stores should be carried 
out to destroy the air-borne living conidia which cause ontomycosis. 
e.  The appropriate, amount of fertilizer soil-moisture level should be determined through 
trials to improve shallot productivity and minimize A. niger colonization. 
f. Planting materials should be disinfected to prevent inoculation of the soil 
g. There should be careful selection of crops for interplanting to avoid using alternative 
host plants or plants with exudates which are very favourable to A. niger such as 
bambara groundnut, groundnut and tomato 
h. Monitor levels of invertebrate pests that feed on the bulbs and create wounds in both the 
field and store houses and spray appropriate pesticides when they reach dangerous 
threshold. 
i.  The A. niger attack was so pervasive and the infection of even the Pink cultivar is so 
high that its adoption for expanded cultivation is not likely to yield much appreciable 
228 
 
relief. The District Assembly of Anloga should organize and manage any expansion of 
the cultivation of the crop and establish a shallot pickling industry to preserve the 
harvested product and to transform a local trade into an export enterprise. 
 
 
229 
 
VI. SUMMARY 
1. Two cultivars of shallot (Allium ascalonicum), namely, Pale-brown and Pink, used in 
this investigation are named for the pigmentation of their scale leaves. 
 
2. Both cultivars are commercially cultivated in Anloga, a coastal district in the Volta 
Region, and bulbs used in the experiments were purchased periodically from Anloga. 
 
3. The bulbs of the Pale-brown cultivar were larger, with a mean length of 3.62cm, a mean 
width of 2.91cm and a mean fresh weight of 8.79 g. 
 
4.  The smaller bulbs of the Pink cultivar had a mean length of 3.26cm, a mean width of 
2.08cm and a mean fresh weight of 5.98g. 
 
5. The growing plants of the Pale-brown cultivar in the field were larger, greener and more 
lush with longer, averagely 40.96cm long and averagely 5.38mm wide hollow 
cylindrical leaves. 
 
6. The hollow cylindrical leaves of the Pink cultivar in the field were smaller in the, Pale 
green and averagely 38.67cm long and averagely 4.84mm wide. 
 
 
230 
 
7. Chemical analysis showed higher concentrations of calcium, magnesium, nitrogen, 
phosphorus, potassium and sodium in bulbs of the Pale-brown than in those of the Pink 
cultivar. 
 
8. The abaxial and adaxial surfaces of the outer scale leaves, abaxial and adaxial surfaces 
of the inner scale leaves, abaxial surfaces of the outermost fleshy leaf base and the 
contact surfaces of the mini-bulbs of not less than 64% of bulbs of both cultivars in 
stores were extensively colonized by A. niger. 
 
9. In bulbs of the pale-brown cultivar, A. niger colonies at the abaxial surface of the outer 
scale leaf, abaxial surface of the outermost fleshy leaf base and the contact surfaces of 
the mini-bulb bore 2,124. 1,855.3, 1125.8 and 1524.6 µm long conidiophores, 
respectively. 
 
10. The corresponding sites of infection of bulbs of the Pink cultivar bore significantly 
shorter conidiophores of 1,720.5, 1,480.3, 799.8 and 1,000.8 µm, respectively. 
 
11. Five distinctively identifiable Aspergillus niger isolates, designated, Isolate 1, Isolate 2, 
Isolate 3, Isolate 4 and Isolate 5, growing on naturally infected shallot bulbs were 
selected and used in all the tests of the investigation. 
 
12. The five A. niger Isolates were employed to provide, in particular, a spectrum of 
response in the tests and not to compare the potential of the isolates. 
231 
 
13. The experiments were carried out, in the absence of incubators in the laboratory, at room 
temperature of 32±2oC and under normal day-night light conditions. 
 
14. A. niger conidia did not germinate in drops of distilled water on glass slides, and on 
parts of the bulbs. 
 
15. Any exudate that might have been released by the scale leaves and swollen leaf bases 
of the bulbs of both shallot cultivars did not also induce germination of the conidia of 
the five A. niger isolates in drops of very dilute ( 1:40) Potato Dextrose Broth except 
conidia of Isolate 1 which germinated tardily ( 1.0 – 3.9 per cent). 
 
16. Conidium percentage germination declined with increasing Potato Dextrose Broth 
dilution on the different parts of the bulbs. Taken together, the highest germination 
percentages recorded were: 
a. 92.4 and 88.4 percent in 1:10 dilution on pale-brown and pink cultivars, 
respectively. 
b. 73.7 and 68.8 per cent in 1:20 dilution on pale-brown and pink cultivars, 
respectively. 
c. 68.8 and 47.4 per cent in 1:30 dilution on pale-brown and pink cultivars, 
respectively. 
 
17. Percentage germination was mostly higher in Potato Dextrose Broth suspension drops 
on parts of the Pale-brown cultivar bulbs than on the Pink cultivar bulbs. 
232 
 
18. Germ-tubes also grew longer in the Potato Dextrose Broth suspension drops on the pale-
brown cultivar than in corresponding drops on the pink cultivar bulbs. 
 
19. Germination of conidia of the five A. niger isolates was far better on scale leaves than 
on the swollen leaf bases. 
 
20. Conidium germination on the scale leaves and swollen leaf bases of the two shallot 
cultivars, in suspension drops of galactose, glucose and sucrose of concentrations of 2.5, 
5.0 and 10.0 g/l followed the same trend which were recorded in the Potato Dextrose 
media. However, the levels of percentage germination were considerably lower. A 
concentration of 1.25g/ l of all the three sugars did not support germination. 
 
21.  Considering all the tests together, the highest percentage of conidia of the five  A. niger 
isolates in the three series of tests were: 
a. 51.3 and 51.2 per cent in 2.5 g/l media on pale-brown and pink cultivars, 
respectively. 
b. 43.5 and 43.1 per cent in 5.0 g/l media on pale-brown and pink cultivars, 
respectively. 
c. 25.2 and 28.2 per cent in 10.0 g/l media on pale-brown and pink cultivars, 
respectively. 
 
22. The different effects of the shallot cultivars on A. niger were also shown by conidium 
germination in aqueous extracts of the bulbs, tested at concentrations S (undiluted) and 
233 
 
at dilutions of   
1
2
  ,   
1
4
  ,   
1
8
, and  
1
16
. The differences in the influence of the extracts were 
manifested at particularly the lower concentrations. At  
1
8
 and  
1
16
  dilutions, germination 
in the extracts of the bulbs of the pink cultivar were much lower than that of the pale-
brown cultivar. 
Respective conidium germination percentages in the pale-brown cultivar and pink 
cultivar extracts by, 
a. Isolate 1 at dilution- 
 
1
8 
 were 73.3 and 64.2 per cent. 
1
16
 were 78.4 and 28.7 per cent. 
b. Isolate 2 at dilution 
1
8
  were 100 and 93.3 per cent. 
1
16
  were 96.6 and 90.9 per cent. 
c. Isolate 3 at dilution 
1
8
 were 91.2 and 89.5 per cent. 
1
16
  were 90.3 and 74.1 per cent. 
d. Isolate 4 at dilution 
1
8
   were 100 and 92.3 per cent. 
1
16
  were 87.7 and 64.4 per cent. 
e. Isolate 5 at dilution 
1
8
  were 100 and 96.0 per cent. 
1
16
  were at 77.5 and 47.7 per cent. 
234 
 
23. But vegetative growth response showed a reverse trend. Statistically significant 
mycelial dry weight was recorded in aqueous extract of bulbs of the Pink cultivars than 
in that of the Pale-brown cultivar in all the five A. niger Isolates. 
 
24. The germinated conidia, especially those at the higher concentrations of the media, 
produced long germ-tubes which grew in all directions on the surface of the scale leaves 
and the swollen leaf bases. They did not enter the stomata and there was no direct 
penetration of the epidermal cells. 
 
25. Growing A. niger isolates altered markedly the pH of the medium. 
a. Initial pH 6.99 of Cassava Dextrose Broth drifted to a final pH 3.04. 
b. Initial pH 6.47 of Potato Dextrose Broth drifted to a final pH 3.00. 
c. Initial pH 6.91 of Bean Meal Extract drifted  to a final pH 3.93 
d. Initial pH 6.81 of Sweet Potato Dextrose Broth drifted to a final pH 3.23. 
e. Initial pH 6.55 of Oat Meal Extract drifted to a final pH 3.14 
 
26. The mycelium of 3mm culture disc of A. niger placed on surface-sterilized bulbs of the 
two shallot cultivars and held at 100% R.H. withered and did not infect the bulbs. 
 
27. Wound-inoculated bulbs of the two shallot cultivars with 3mm culture disc of A. niger 
were rotted by the fungus at 50-100% R.H. 
 
 
235 
 
28.  Rate of developments of rot was greatest at 100% R.H. and decreased with decreasing 
storage relative humidity. 
 
29. Humidities of 50-100% R.H. affected invasion of the bulbs of the two shallot cultivars 
in many specific ways. 
a. The surface diameters of the rotted tissues were greatest at 100% R.H. 
b. The bulbs were rotted at all the relative humidities. 
c. After 10 days, the rotted tissues were greatest at 100% R.H. 
d. Mycelium in the rotted tissues of bulbs at 80 to 100% R.H. produced conidiophores 
and conidia by the 10th day after inoculation. 
e. The conidiophores were longest at 100% R.H. and shortest at 80% R.H. 
 
30.  With regard to shallot cultivar- A. niger relationship: 
a. The rate of rotting was faster in bulbs of the pale-brown cultivar. 
b. The mean diameters of the rots caused by A. niger Isolates 1, 2, 3, 4, and 5 in bulbs 
of the pale-brown cultivar held at 100% R.H. were 12.1, 12.3, 12.8, 10.4 and 12.6 
mm, respectively, and of the pink cultivar, 10.4, 10.3, 11.0, 7.9 and 11.3, 
respectively. 
c. Mean lengths of the conidiophores formed on the diseased tissues at 100, 90 and 
80% R.H. on the Pale-brown cultivar bulbs were 702.1, 653.4 and 610.0µm, 
respectively, and on the pink cultivar were 664.3, 627.8 and 601.5µm, respectively. 
d. Sporulation density at each % R.H. was the same on bulbs of both cultivars. 
 
236 
 
31. Plants of both cultivars of shallot had a definite effect on fungi in the neighbourhood of 
the bulbs and the roots. 
 
32. The soil of Anloga shallot farms was very sandy and contain very low humus content 
of 3. 48%. 
 
33. Fungal genera occurring in phyllosphere, rhizosphere and non- rhizosphere soils were 
Aspergillus, Fusarium, Penicillium and Rhizopus with Aspergillus as the predominant 
genus in all the three soil zones. 
 
34. Aspergillus niger was the most abundant Aspergillus species in all the three soil zones. 
For each type of soil it constituted more than half of the population. 
 
35. Aspergillus species isolated from the phyllosphere and rhizosphere of both shallot 
cultivars were A. flavus, A. niger and A. terreus. 
 
36.  Stimulation of the fungal species in the phyllosphere and rhizosphere of both shallot 
cultivars was maintained over 60 days. 
 
37. A. niger was also the dominant fungus species in the similar studies using cassava, 
groundnut and maize farm soils at Legon. 
 
237 
 
38.  The shallot plants and fungi around the bulbs and roots did not respond to addition of 
urea (1.94 and 3.88 g) to the soil of Anloga farms. 
 
39. Addition of Potassium (3.33, 6.67. 10.00 and 13.33 g/l) increased sizes of the shallot 
plants and population in all the three zones of the soil. The optimum Potassium 
concentration was 6.67 g/l. 
 
40.  Bulbs of Pale-brown cultivar which grew in soils with added Urea and Potassium still 
rotted faster after wound-inoculation with A. niger. 
 
41. Conidia of three selected  A. niger Isolates 1, 3 and 5 which earlier had proved equally 
viable by attaining 100 percent germination in Potato Dextrose Broth and Sweet Potato 
Broth, also showed similar  longevity. 
 
42. The conidia survived longest at the higher humidities of 60- 100% R.H. and lost viability 
quickest at lower humidities of 0.0- 50 % R.H. 
 
43. Conidia formed by mycelium on bulb tissue of the Pale-brown cultivar showed slightly 
higher percentage viability on each assessment occasion than those formed on tissues 
of the Pink cultivar. 
 
44. Because of the many occasions on which the Pink cultivar depressed conidiation by 
suppressing length of conidiophores, depressed conidium germination, depressed length 
238 
 
of germ-tubes produced by germinated conidia, was less rapidly rotted following 
wound-inoculation, and was colonized to a lesser extent in storage by A. niger. It was 
concluded that the Pink cultivar should be adopted for expansion of shallot cultivation 
in Ghana and be regarded as a suitable cultivar for future shallot programmes in attempts 
to produce improve stock that would reduce A. niger threat to the shallot industry.    
 
45. Among the many salient recommendations, the highly important proposition is the call 
on the Anloga District Assembly to establish a shallot pickling industry that will 
effectively preserve the harvested bulbs and transform a local trade into an export 
enterprise. 
 
 
 
239 
 
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                  APPENDICES 
 APPENDIX A: Lengths, diameter and weights of bulbs of shallot ( Allium ascalonicum)  
              
 
          Length ( cm)               Diameter  (cm)                 Weight (g) 
 
            Pale- brown  cv.    Pink cv.  Pale- brown cv.      Pink cv.       Pale- brown cv.   Pink cv. 
  
3.48                       2.93                   2.45              2.06              10.00                    6.03 
3.13   3.74                    2.52             1.87                7.58                    8.69 
3.53   2.66                    2.87             2.20                9.89                    4.76 
3.23   3.15                    3.04             2.08                7.45                    7.82 
3.52   3.14                    2.79             2.87                9.33                    5.26 
3.73   3.73                    2.50             2.04                9.71                    5.05 
3.34   3.37                    2.43             2.51              10.95                    4.52 
3.44   3.42                    2.60             3.11                6.48                    8.81 
4.01   3.23                    2.89             2.34                9.90                    4.43 
3.66   2.83                    2.94             2.07                7.16                    5.09 
3.38  2.94                     2.38             1.97                9.21                    5.05 
3.43  3.83                     2.52             1.88                 9.42                   4.87 
3.77  2.77                     3.00             2.01                8.89                    6.12 
4.00  3.45                     2.72             1.89                7.89                    7.23 
3.33  3.78                     2.65             1.65                6.95                    6.19 
3.51  2.85                     2.35             1.83                8.59                    4.84 
3.45 3.34                      2.83             2.01                7.96                    6.05 
3.83 3.33                      2.63             1.68              10.01                    5.89 
3.92 3.75                      3.12             1.72               9.37                     5.99 
4.18                    2.99                      3.08             1.88                8.99                     6.89 
264 
 
                  APPENDIX B: Percentage of conidia of Aspergillus niger Isolate 1 formed on ground bulbs 
of the two cultivars of Allium ascalonicum viable after storage for 10 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia). 
 
                                                 Pale-brown cultivar                             Pink cultivar 
       Storage                      Percen-              Mean           Percen-         Mean 
       Percentage                 tage                   germ-tube           tage                  germ-tube 
       Relative                     Germ-                length            Germ-              length 
       Humidity                   ination               ±S.E (µm)                  ination        ±S.E (µm) 
 
              0                             54.9                120.1±4.6 a                     53.6               110.3±4.2 a 
      10                             62.7                122.0±3.1 a                     60.7               118.9±3.3 a 
      20                             63.4                143.4±4.2 a                     65.6               140.0±5.7 a 
      30                             69.2                152.7±6.1 a                     71.0               158.1±4.0 a 
      40                             72.4                160.3±3.7 a                     71.7               163.8±3.7 a 
      50                             75.9                175.9±5.2 a                     74.3               160.3±6.0 a 
      60                             79.4                180.2±3.0 a                     77.6               172.0±3.6 a 
      70                             81.8                187.3±3.1 a                     78.9               177.0±4.3 a 
      80                             84.4                183.4±4.6 a                     85.6               187.2±4.3 a 
      90                             88.6                190.0±3.2 a                     88.9               185.2±3.6 a 
    100                             91.6                190.3±4.3 a                     89.6               188.3±4.2 a  
                   By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths 
in each horizontal row bearing the same letters are not significantly different at 5% level of 
probability. 
265 
 
APPENDIX C : Percentage of conidia of Aspergillus niger Isolate 1 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 20 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia). 
      
                                                 Pale-brown cultivar                              Pink cultivar 
       Storage                      Percen-              Mean           Percen-             Mean 
       Percentage                 tage                   germ-tube           tage                      germ-tube 
       Relative                     Germ-                length            Germ-                  length 
       Humidity                   ination               ±S.E (µm)                  ination            ±S.E (µm) 
 
             0                              52.4                 118.4±4.2 a                    50.2                121.4±1.7 a 
           10                              60.4                124.1±3.1 a                     58.3                123.3±1.9 a   
           20                              62.4                129.3±2.1 a                     61.1                127.4±2.8 a 
           30                              68.3                138.1±6.2 a                     67.2                130.8±4.6 a 
           40                              70.5                130.3±5.2 a                     68.2                134.3±3.4 a 
           50                              72.3                133.4±4.2 a                     70.5                148.4±4.2 a 
           60                              73.4                147.4±3.2 a                     71.3                140.2±2.3 a 
           70                              74.9                148.3±2.4 a                     74.5                143.4±6.3 a 
           80                              76.3                159.8±3.1 a                     80.3                158.3±1.3 a 
           90                              80.3                173.4±2.3 a                     83.7                163.4±3.4 a 
         100                              83.2                163.2±3.1 a                     81.2                172.4±3.4 a 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube 
Lengths in each horizontal row bearing the same letters are not significantly different at 
5% level of probability. 
266 
 
                   APPENDIX D: Percentage of conidia of Aspergillus niger Isolate 1 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 40 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle.(Percentage germination 
based on 400-500 observed conidia). 
           
                                                  Pale-brown cultivar                               Pink cultivar 
       Storage                      Percen-              Mean           Percen-             Mean 
       Percentage                 tage                   germ-tube           tage                      germ-tube 
       Relative                     Germ-                length            Germ-                   length 
       Humidity                   ination               ±S.E (µm)                  ination            ±S.E (µm) 
 
                              0                         51.4                 115.4±1.8 a                    49.9                  113.4±1.9 a 
              10                         58.3                 120.3±3.2 a                    55.3                  120.8±2.5 a 
              20                         60.5                 123.5±2.4 a                    59.4                  126.4±2.8 a 
              30                         62.3                 130.4±6.3 a                    63.2                  132.4±4.2 a 
              40                         65.8                 135.4±1.7 a                    64.2                  133.7±6.3 a 
              50                         69.4                 141.2±2.3 a                    65.4                  143.7±4.1 a 
              60                         69.3                 150.3±5.1 a                    67.1                  150.1±2.3 a 
             70                          70.1                 156.4±3.2 a                    66.5                  149.3±4.3 a 
             80                          70.5                 168.3±2.4 a                    69.4                  150.3±5.2 b 
             90                          72.4                 170.2±2.3 a                    74.5                  152.4±2.6 b 
           100                          78.3                 173.4±3.1 a                    76.4                  168.2±1.3 a 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths 
in each horizontal row bearing the same letters are not significantly different at 5% level of 
probability. 
267 
 
 APPENDIX E: Percentage of conidia of Aspergillus niger isolate 1 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 80 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle.(Percentage germination based 
on 400-500 observed conidia). 
                                               Pale-brown cultivar                               Pink cultivar 
       Storage                      Percen-              Mean           Percen-         Mean 
       Percentage                 tage                   germ-tube           tage                  germ-tube 
       Relative                     Germ-                length            Germ-              length 
       Humidity                   ination               ±S.E (µm)                  ination        ±S.E (µm) 
 
              0                          46.9                 103.4±4.8 a                     45.3                 100.2±4.3 a 
            10                          52.4                 112.5±3.2 a                     51.1                 110.4±1.6 a 
            20                          55.3                 116.3±2.7 a                     54.9                 112.5±1.8 a 
           30                           58.4                 118.5±1.8 a                     55.5                 114.3±2.4 a 
           40                           60.2                 120.4±3.4 a                     58.5                 121.5±2.3 a 
           50                           61.9                 121.1±2.8 a                     58.9                 130.4±1.9 b 
           60                           62.4                 136.3±3.7 a                     61.3                 133.4±5.0 a 
           70                           63.5                 148.2±6.2 a                     62.4                 139.8±4.2 a 
           80                           68.3                 147.1±5.3 a                     65.4                 144.4±5.3 a 
           90                           70.1                 158.2±2.4 a                    68.9                 154.3±2.2 a 
         100                           75.4                 163.4±1.3 a                     72.1                 158.2±1.8 a 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths in 
each horizontal row bearing the same letters are not significantly different at 5% level of 
probability. 
268 
 
APPENDIX F: Percentage of conidia of Aspergillus niger isolate 1 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 120 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle.(Percentage germination based 
on 400-500 observed conidia). 
                                                Pale-brown cultivar                              Pink cultivar 
       Storage                      Percen-              Mean           Percen-             Mean 
       Percentage                 tage                   germ-tube           tage                      germ-tube 
       Relative                     Germ-                length            Germ-                   length 
       Humidity                   ination               ±S.E (µm)                  ination            ±S.E (µm)
  
 
             0                          45.3                  121.2±5.4 a                      41.2                  120.4±5.5 a 
            10                         48.4                  131.4±4.3 a                      44.8                  123.2±4.5 a  
            20                         49.3                  132.8±3.2 a                      45.4                  124.5±3.2 a 
            30                         51.9                  143.1±2.5 a                      48.3                  129.4±2.9 b 
            40                         53.2                  144.3±1.9 a                      50.3                  128.4±3.7 b 
            50                         54.7                  146.4±2.8 a                      52.4                  127.5±3.2 b 
            60                         58.3                  150.2±4.3 a                      56.7                  128.4±4.0 b 
            70                         60.9                  148.3±5.3 a                      58.4                  132.6±4.2 a 
            80                         61.8                  154.2±4.2 a                      60.3                  130.4±5.3 b 
            90                         63.7                  153.1±2.3 a                      64.7                  134.5±4.3 b 
          100                         70.5                  160.2±1.9 a                      68.3                  138.4±3.2 b 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths in 
each horizontal row bearing the same letters are not significantly different at 5% level of 
probability. 
269 
 
 APPENDIX G: Percentage of conidia of Aspergillus niger Isolate 1 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 160 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle . (Percentage germination based 
on 400-500 observed conidia). 
 
                                                  Pale-brown cultivar                                  Pink cultivar 
       Storage                      Percen-              Mean             Percen-                 Mean 
       Percentage                 tage                   germ-tube             tage                         germ-tube 
       Relative                     Germ-                length              Germ-                     length 
       Humidity                   ination               ±S.E (µm)                    ination               ±S.E (µm) 
             0                            33.2                 100.8±2.8 a                       31.4                 96.1±5.8 a 
           10                            39.4                 110.3±5.1 a                       38.2                 99.8±4.1 a 
           20                            40.3                 102.4±4.1 a                       41.3               108.4±4.2 a 
           30                            45.5                 117.3±4.2 a                       44.2               110.4±3.8 a 
           40                           47.3                  118.5±3.7 a                       47.5               113.1±3.3 a   
           50                           50.9                  124.3±2.8 a                       48.4               130.4±2.8 a 
           60                           52.4                  132.4±1.9 a                       51.3               133.5±1.8 a 
          70                            55.3                  143.8±3.4 a                       54.1               139.4±4.2 a 
          80                            58.4                  142.1±2.4 a                       57.2               140.4±5.2 a 
          90                            60.1                  144.5±6.3 a                       58.3               150.3±3.4 a 
        100                            63.2                  147.2±1.2 a                       62.1               153.4±2.1 a 
 
 By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths 
in each horizontal row bearing the same letters are not significantly different at 5% level of 
probability. 
270 
 
  APPENDIX H: Percentage of conidia of Aspergillus niger Isolate 1 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 200 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia).88 
 
                                                  Pale-brown cultivar                               Pink cultivar 
       Storage                      Percen-              Mean           Percen-         Mean 
       Percentage                 tage                   germ-tube           tage                  germ-tube 
       Relative                     Germ-                length            Germ-              length 
       Humidity                   ination               ±S.E (µm)                  ination        ±S.E (µm) 
              0                            0.0                         -                              0.0                       - 
            10                            0.0                         -                              0.0                        - 
            20                            0.0                         -                              0.0                        - 
            30                            0.0                         -                              0.0                        - 
            40                            0.0                         -                              0.0                        - 
            50                            0.0                         -                              0.0                        - 
            60                          28.7                80.2±1.8 a                      25.3                78.2±3.2 a 
            70                          32.4                88.3±3.1 a                      28.1                82.4±1.9 a 
            80                          40.3                93.4±3.2 a                      33.1                90.2±4.3 a 
            90                          43.7               103.1±4.3 a                     37.2                98.1±2.8 a 
           100                         52.1               112.1±6.3 a                     43.8              102.3±4.1 a  
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths in 
each horizontal row bearing the same letters are not significantly different at 5% level of 
probability. 
271 
 
  APPENDIX I: Percentage of conidia of Aspergillus niger Isolate 3 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 10 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia). 
 
                                                 Pale-brown cultivar                                 Pink cultivar 
       Storage                      Percen-              Mean           Percen-               Mean 
       Percentage                 tage                   germ-tube           tage                        germ-tube 
       Relative                     Germ-                length            Germ-                    length 
       Humidity                   ination               ±S.E (µm)                  ination              ±S.E (µm) 
             0                            50.2                    58.1±3.2 a                     55.0                 55.4±3.5 b 
           10                            61.2                    67.8±2.7 a                     60.4                 66.6±3.1 b 
           20                            66.4                    97.4±6.2 a                     63.7                 78.8±3.8 a 
           30                            67.6                    93.8±4.2 a                     64.7                 80.1±4.7 a 
          40                             68.9                  101.2±5.4 a                     66.3                 91.4±4.0 a 
          50                             70.9                  124.2±5.5 a                     73.0                119.2±6.0 a 
          60                             73.1                  143.1±7.2 a                    74.7                 112.7±6.4 b 
          70                             74.1                  136.6±4.2 a                    75.8                 121.1±4.4 a 
          80                             81.6                  151.2±6.0 a                    78.2                 146.1±7.3 a 
         90                              90.0                  175.1±5.6 a                    90.8                 188.1±10.2 a 
        100                             93.6                  197.4±4.1 a                    91.1                 192.4±9.9 a                             
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths in 
each horizontal row bearing the same letters are not significantly different at 5% level of 
probability 
272 
 
  APPENDIX J: Percentage of conidia of Aspergillus niger Isolate 3 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 20 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia). 
                                                 Pale-brown cultivar                                Pink cultivar 
       Storage                      Percen-              Mean           Percen-           Mean 
       Percentage                 tage                   germ-tube           tage                     germ-tube 
       Relative                     Germ-                length            Germ-                  length 
       Humidity                   ination               ±S.E (µm)                  ination           ±S.E (µm)
  
             0                            44.9                 108.8±2.6 a                     41.9                    91.4±4.0 b 
           10                            47.4                 112.5±3.1 a                     48.9                    91.9±4.5 b 
           20                           53.8                  119.7±2.7 a                     52.5                 108.8±3.0 a 
          30                             60.5                 120.8±3.5 a                     54.5                  113.4±3.6 a 
          40                             63.5                 127.1±3.0 a                     59.6                  122.5±3.1 a 
          50                             66.3                 127.7±3.6 a                     64.2                  119.1±2.2 a 
          60                             70.3                 135.0±4.5 a                     68.6                  131.1±3.4 a 
         70                              71.8                 136.1±4.2 a                     72.5                  134.1±4.1 a 
         80                              78.8                 139.8±4.1 a                     76.7                  139.5±4.9 a 
         90                              85.3                 140.1±3.4 a                     80.2                  143.3±4.8 a 
       100                              87.2                 143.0±5.2 a                     85.7                  141.3±4.7 a 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths 
in each horizontal row bearing the same letters are not significantly different at 5% level of 
probability 
273 
 
  APPENDIX K: Percentage of conidia of Aspergillus niger Isolate 3 formed on macerated 
bulbs of two cultivars of Allium ascalonicum viable after storage for 40 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle (Percentage germination based 
on 400-500 observed conidia). 
 
                                                Pale-brown cultivar                                Pink cultivar 
       Storage                      Percen-              Mean           Percen-               Mean 
       Percentage                 tage                   germ-tube           tage                        germ-tube 
       Relative                     Germ-                length            Germ-                     length 
       Humidity                   ination               ±S.E (µm)                  ination               ±S.E (µm) 
              0                          37.6                   87.6±3.7 a                     35.3                     86.0±3.2 a 
            10                          41.6                   92.3±2.5 a                     40.8                     91.7±3.3 a 
            20                          43.6                   93.2±3.7 a                     46.1                     93.8±2.9 a 
            30                          51.4                  108.6±3.1 a                    54.7                   111.1±2.4 a 
           40                           56.3                  115.2±3.2 a                    56.8                   117.6±3.4 a 
           50                           60.0                  122.7±4.0 a                    58.3                   123.8±3.5 a 
           60                           63.2                  128.4±3.2 a                    61.0                   127.3±4.0 a 
           70                           66.3                  129.2±3.1 a                    64.0                   126.8±2.8 a 
           80                           70.6                  130.0±4.1 a                    69.2                   127.7±3.7 a 
           90                           74.9                  136.0±4.7 a                    73.1                   129.9±4.0 a 
          100                          76.4                  131.4±3.3 a                    77.2                   130.8±2.6 a 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube 
Lengths in each horizontal row bearing the same letters are not significantly different at 
5% level of probability. 
274 
 
  APPENDIX L: Percentage of conidia of Aspergillus niger Isolate 3 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 80 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia). 
                                                 Pale-brown cultivar                                Pink cultivar 
       Storage                      Percen-              Mean           Percen-            Mean 
       Percentage                 tage                   germ-tube           tage                     germ-tube 
       Relative                     Germ-                length            Germ-                  length 
       Humidity                   ination               ±S.E (µm)                  ination           ±S.E (µm)
  
            0                           34.6                  77.0±3.1 a                       33.2                   78.6±3.2 a 
          10                           43.1                  83.0±3.0 a                       39.3                   79.1±3.3 a 
          20                           48.7                  81.4±3.1 a                       44.0                   80.7±2.5 a 
          30                           52.0                  87.3±3.1 a                       52.4                   92.9±3.1 a 
          40                           53.1                  89.4±3.0 a                       56.3                   93.5±2.6 a 
          50                           55.8                  94.5±3.0 a                       55.5                 101.4±2.3 a 
          60                           56.7                 104.4±3.7 a                      57.2                 103.4±2.9 a 
         70                            59.9                 104.0±3.3 a                      58.9                 106.3±3.5 a 
         80                            61.1                 100.3±5.4 a                      59.6                 106.1±3.2 a 
         90                            64.2                 104.7±3.5 a                      63.9                 109.4±2.6 a 
        100                           73.7                 107.9±3.3 a                      70.0                 108.8±2.9 a 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths 
in each horizontal row bearing the same letters are not significantly different at 5% level of 
probability 
 
275 
 
 APPENDIX M: Percentage of conidia of Aspergillus niger Isolate 3 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 120 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia). 
 
                                             Pale-brown cultivar                                 Pink cultivar 
       Storage                      Percen-              Mean           Percen-            Mean 
       Percentage                 tage                   germ-tube           tage                     germ-tube 
       Relative                     Germ-                length            Germ-                  length 
       Humidity                   ination               ±S.E (µm)                  ination            ±S.E (µm) 
            0                           32.1                  70.3±5.1 a                      30.8                  65.3±2.5 a 
          10                           43.5                  76.1±2.3 a                      40.9                  73.3±2.3 a 
          20                           47.2                  80.2±4.2 a                      44.7                  83.2±4.3 a 
          30                           49.5                  85.3±3.3 a                      46.2                  87.3±3.2 a 
          40                           50.8                  93.2±1.5 a                      53.4                  90.5±2.4 a 
          50                           54.2                102.8±2.8 a                      56.7                  94.3±1.9 a 
          60                           57.9                100.4±4.3 a                      58.1                  98.9±2.8 a 
          70                           59.2                105.3±2.9 a                      61.1                103.5±2.3 a 
          80                           59.8                118.5±4.3 a                      62.6                107.2±5.5 a 
          90                           65.2                112.3±5.5 a                      64.8                118.3±3.4 a 
        100                           69.2                123.4±2.3 a                      66.7                120.2±2.8 a 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths in 
each horizontal row bearing the same letters are not significantly different at 5% level of 
probability. 
276 
 
APPENDIX N: Percentage of conidia of Aspergillus niger isolate 3 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 160 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle.( Percentage germination based 
on 400-500 observed conidia). 
 
                                               Pale-brown cultivar                               Pink cultivar 
       Storage                      Percen-              Mean           Percen-         Mean 
       Percentage                 tage                   germ-tube           tage                  germ-tube 
       Relative                     Germ-                length            Germ-              length 
       Humidity                   ination               ±S.E (µm)                  ination        ±S.E (µm) 
             0                            0.0                       -                                   0.0                       - 
           10                            0.0                       -                                   0.0                       - 
           20                            0.0                       -                                   0.0                       - 
           30                            0.0                       -                                   0.0                       - 
           40                          50.3                100.0±2.1 a                        47.3                  99.3±2.0 a 
           50                          52.4                106.1±3.8 a                        49.3                108.1±2.3 a 
           60                          53.9                110.3±2.4 a                        50.3                112.3±1.4 a 
          70                           57.4                118.0±2.3 a                        54.4                115.1±1.3 a 
          80                           59.3                115.3±4.3 a                        58.3                121.4±1.4 a 
          90                           60.1                120.1±1.9 a                        61.4                116.3±2.3 a 
        100                           64.3                122.7±3.2 a                        63.2                118.4±3.1 a                                           
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths in 
each horizontal row bearing the same letters are not significantly different at 5% level of 
probability. 
 
277 
 
APPENDIX O: Percentage of conidia of Aspergillus niger Isolate 3 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 200 days at different 
relative humidities at 30oC 32±2oC under 12-hour day-night cycle (Percentage germination 
based on 400-500 observed conidia). 
 
                                                Pale-brown cultivar                               Pink cultivar 
       Storage                      Percen-              Mean           Percen-         Mean 
       Percentage                 tage                   germ-tube           tage                  germ-tube 
       Relative                     Germ-                length            Germ-              length 
       Humidity                   ination               ±S.E (µm)                  ination        ±S.E (µm) 
            0                              0.0                         -                               0.0                       - 
          10                              0.0                         -                               0.0                       - 
          20                              0.0                         -                               0.0                       - 
          30                              0.0                         -                               0.0                       - 
          40                              0.0                         -                               0.0                       - 
          50                              0.0                         -                               0.0                       - 
         60                               0.0                         -                               0.0                        - 
          70                             33.4                95.4±2.8 a                      28.9                92.3±3.2 a 
         80                             45.2                 98.3±3.2 a                      35.4                93.8±4.1 a 
         90                             48.3                103.2±1.3 a                      40.3               90.4±2.6 b 
       100                             51.2                113.4±2.1 a                      42.1             102.4±4.3 a 
 By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths 
in each horizontal row bearing the same letters are not significantly different at 5% level of 
probability. 
 
278 
 
APPENDIX P: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 10 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia). 
                                               Pale-brown cultivar                               Pink cultivar 
       Storage                      Percen-              Mean           Percen-         Mean 
       Percentage                 tage                   germ-tube           tage                  germ-tube 
       Relative                     Germ-                length            Germ-              length 
       Humidity                   ination               ±S.E (µm)                  ination        ±S.E (µm) 
           0                             53.8                  113.7±3.5 a                  55.6               105.5±3.0 a 
         10                             59.5                 127.4±4.1 a                   57.8               118.2±3.6 a 
         20                             65.5                  128.7±3.8 a                  63.4               125.9±4.2 a 
         30                             69.5                 130.7±3.2 a                   64.9               135.8±4.9 a 
        40                              71.9                 142.2±4.6 a                  73.5                136.1±4.1 a 
        50                              77.3                  146.6±4.8 a                 77.1                137.7±4.0 a 
        60                              81.0                  156.3±5.2 a                 79.1                151.2±6.4 a 
        70                              84.7                  159.0±4.0 a                  85.3                165.6±4.8 a 
        80                              89.7                  164.6±4.9 a                  86.4                154.1±6.2 a 
        90                              91.1                  184.2±3.5 a                  87.5                175.4±6.7 a 
      100                             92.3                   195.2±3.4 a                  93.1                184.2±7.8 a 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths in 
each horizontal row bearing the same letters are not significantly different at 5% level of 
probability 
 
279 
 
APPENDIX Q: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 20 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia). 
                                              Pale-brown cultivar                               Pink cultivar 
       Storage                      Percen-              Mean          Percen-           Mean 
       Percentage                 tage                   germ-tube          tage                      germ-tube 
       Relative                     Germ-                length           Germ-                   length 
       Humidity                   ination               ±S.E (µm)                 ination                 ±S.E (µm)
  
               0                           48.4                  115.6±3.5 a                  43.8                  105.5±3.7 a 
             10                           52.3                  125.9±3.1 a                  45.2                  111.9±3.2 b 
             20                           57.3                  127.2±3.6 a                  49.1                   121.7±3.6 a 
             30                           62.6                  132.8±4.1 a                  53.7                   129.9±3.1 a 
             40                           65.1                  134.7±3.9 a                  64.9                   140.9±4.4 a 
             50                           69.1                  140.0±4.8 a                  68.9                   139.2±6.1 a 
             60                          72.0                  143.3±4.8 a                   70.7                   140.3±5.3 a 
            70                           77.9                  147.7±4.6 a                   76.5                   150.2±5.3 a 
             80                          78.9                  148.5±4.7 a                   78.1                   152.0±4.9 a 
             90                          81.2                  153.8±5.0 a                   81.9                   148.7±4.7 a 
           100                          87.5                  154.3±4.7 a                   85.1                   156.2±5.0 a 
       By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube 
Lengths in each horizontal row bearing the same letters are not significantly different at 
5% level of probability. 
280 
 
APPENDIX R: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 40 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia). 
 
                                               Pale-brown cultivar                               Pink cultivar 
       Storage                      Percen-              Mean             Percen-                 Mean 
       Percentage                 tage                   germ-tube             tage                         germ-tube 
       Relative                     Germ-                length              Germ-                     length 
       Humidity                   ination               ±S.E (µm)                    ination               ±S.E (µm) 
        0                               39.9                   85.8±3.3 a                        37.9                   82.7±3.0 a 
       10                               44.0                   88.8±2.9 a                        40.0                   90.2±3.8 a 
       20                               45.6                 102.3±3.7 a                        45.7                   92.0±4.0 a 
       30                               48.3                 109.8±3.6 a                        47.2                 104.1±2.7 a 
       40                               52.5                 117.5±3.0 a                        50.4                 109.2±3.0 a 
       50                               56.1                 120.3±2.5 a                        53.3                 111.0±2.0 b 
       60                               58.6                 120.8±4.5 a                        57.9                 116.7±3.6 a 
      70                                65.9                 124.5±3.2 a                        64.2                 116.3±4.0 a 
      80                                67.9                 125.3±3.1 a                        66.0                 116.1±3.4 a 
      90                                71.9                 132.2±3.4 a                        69.3                 126.8±3.8 a 
    100                                79.6                 130.7±3.6 a                        73.5                 139.5±4.5 a 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths in 
each horizontal row bearing the same letters are not significantly different at 5% level of 
probability. 
281 
 
APPENDIX S: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 80 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia). 
                                                Pale-brown cultivar                               Pink cultivar 
       Storage                      Percen-              Mean            Percen-               Mean 
       Percentage                 tage                   germ-tube            tage                       germ-tube 
       Relative                     Germ-                length             Germ-                    length 
       Humidity                   ination               ±S.E (µm)                   ination              ±S.E (µm) 
              0                           34.3                  73.4±5.1 a                        30.5                  62.3±3.4 a 
            10                           38.3                  75.3±4.2 a                        33.8                  70.4±4.3 a 
            20                           40.5                  83.4±3.7 a                        36.5                  79.3±1.8 a 
            30                           43.8                  87.8±2.3 a                        40.8                  80.7±2.5 a 
            40                           47.5                  85.3±1.9 a                        43.5                  83.5±3.3 a 
            50                           53.4                  90.8±2.7 a                        51.4                  90.3±4.2 a 
           60                            56.8                  94.3±2.3 a                        59.2                  95.4±5.4 a 
           70                            62.3                  98.5±3.8 a                        61.3                  97.8±3.3 a 
           80                            65.8                103.8±4.0 a                        63.2                  94.3±2.6 a 
           90                            70.3                100.4±3.7 a                        64.5                  98.5±2.9 a 
         100                            75.4                105.3±4.2 a                        71.3                101.4±1.9 a 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths in 
each horizontal row bearing the same letters are not significantly different at 5% level of 
probability. 
 
282 
 
APPENDIX T: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 120 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia). 
                                                  Pale-brown cultivar                               Pink cultivar 
       Storage                      Percen-              Mean           Percen-           Mean 
       Percentage                 tage                   germ-tube           tage                    germ-tube 
       Relative                     Germ-                length            Germ-                 length 
       Humidity                   ination               ±S.E (µm)                  ination          ±S.E (µm) 
              0                           30.8                    65.5±3.2 a                  28.2                  60.2±4.3 a 
            10                           33.2                    80.3±2.7 a                  31.4                  64.3±2.7 a 
            20                           37.4                    85.4±1.8 a                  34.7                  80.5±5.3 a 
            30                           40.3                    87.2±4.3 a                  35.3                  71.4±5.1 a 
            40                           44.5                    90.3±5.5 a                  40.8                   80.7±4.0 a 
            50                           47.4                    95.3±2.3 a                  42.3                   82.5±1.5 b 
            60                           48.5                     98.4±4.5 a                 45.4                   87.4±2.5 b 
            70                           50.8                   101.2±2.3 a                 47.3                   90.3±2.7 b 
            80                           53.4                   100.4±1.9 a                 52.5                   95.1±2.3 a 
            90                           61.3                   103.8±4.3 a                 56.5                   99.1±3.4 a 
          100                           65.4                   110.4±3.2 a                 60.3                  102.4±3.0 a 
 
        By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube 
Lengths in each horizontal row bearing the same letters are not significantly different at 
5% level of probability   
283 
 
APPENDIX U: Percentage of conidia of Aspergillus niger Isolate 5 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 160 days at different 
relative humidities at 32±2oC under 12-hour day-night cycle. (Percentage germination based 
on 400-500 observed conidia). 
                                                 Pale-brown cultivar                               Pink cultivar 
       Storage                      Percen-              Mean           Percen-           Mean 
       Percentage                 tage                   germ-tube           tage                    germ-tube 
       Relative                     Germ-                length            Germ-                length 
       Humidity                   ination               ±S.E (µm)                  ination          ±S.E (µm)
  
               0                          0.0                           -                                0.0                         - 
             10                          0.0                           -                                0.0                         - 
             20                          0.0                           -                                0.0                         - 
             30                          0.0                           -                                0.0                         - 
             40                         18.3                   60.5±1.3 a                       15.3                  58.5±3.2 a 
             50        20.2                    72.4±5.2 a                       19.7                  63.4±2.6 a 
             60                        23.8                     85.5±2.3 a                      20.3                  82.8±1.7 a 
             70                        30.4                     90.4±1.5 a                      33.2                  83.4±2.8 a 
             80                        32.1                     93.2±3.2 a                      38.3                  90.8±3.0 a 
             90                        43.2                   100.1±1.8 a                      40.2                  93.2±2.5 a 
           100                        50.1                   103.4±2.4 a                      43.9                 108.3±1.3 a 
 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths in 
each horizontal row bearing the same letters are not significantly different at 5% level of 
probability. 
284 
 
APPENDIX V: Percentage of conidia of Aspergillus niger isolate 5 formed on macerated 
bulbs of the two cultivars of Allium ascalonicum viable after storage for 200 days at different 
relative humidities at 32±2oC under normal day/ night light regime. (Percentage germination 
based on 400-500 observed conidia). 
                                                   Pale-brown cultivar                               Pink cultivar 
       Storage                      Percen-              Mean           Percen-              Mean 
       Percentage                 tage                   germ-tube           tage                       germ-tube 
       Relative                     Germ-                length            Germ-                    length 
       Humidity                   ination               ±S.E (µm)                  ination              ±S.E (µm) 
               0                             0.0                            -                            0.0                          - 
             10                             0.0                            -                            0.0                          - 
             20                             0.0                            -                            0.0                          - 
             30                             0.0                            -                            0.0                          - 
             40                             0.0                            -                            0.0                          - 
             50                             0.0                            -                            0.0                          - 
             60                             0.0                            -                            0.0                          - 
             70                             0.0                            -                            0.0                          - 
             80                           15.3                    53.4±1.2 a                     20.4                  58.4±3.1 a 
            90                            23.2                    60.2±1.3 a                     21.2                  61.3±2.8 a 
           100                           35.8                    78.4±3.8 a                     32.3                  73.4±5.5 a 
 
By the calculated Scheffe’s Confidence Limits, Values of pairs of Mean Germ-tube Lengths in 
each horizontal row bearing the same letters are not significantly different at 5% level of 
probability