*^B B 3E S 5E S ^
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ASSESSMENT OF THE EFFECT OF POLLEN GRAINS OF MAIZE
f
(ZEA MAYS L.) ON CERCOSPORA ARACHIDICOLA HORI. 
AND ON INFECTION OF LEAVES OF GROUNDNUT 
(ARACHIS HYPOGEA L.) CAUSED BY THE FUNGUS.
A Thesis presented by
JOHN LAWER TERLABIE
In part fu lfilm ent o f the requirements fo r the
M.PHIL. DEGREE
of the University of Ghana.
From: The Department of Botany, 
University of Ghana, 
LEGON.
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d e d i c a t i o n
Do ^ o r a  ^Jerlabie, m y louely a n d  d ep en d a lie  ivi^e a n d  to 
my m um  who d en ied  herdel^a  lo t o ^com forts  to mahe me w ha t  
^3  am  today a n d  to my tw in  iidter.
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DECLARATION
I hereby certify that this thesis is my own original work. All assistance and 
references to relevant literature have been duly acknowledged. This thesis has 
not been submitted, either in whole or in part for a degree or other qualification in 
any other university.
 ................
JOHN LAWER TERLABIE
EMERITUS PROFESSOR G. C. CLERK 
(SUPERVISOR)
II
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ACKNOWLEDGEMENT
I wish to express my sincere gratitude and appreciation to Emeritus 
Professor G. C. Clerk who suggested this problem, for his guidance and 
his invaluable assistance through and through which have made this work 
a success.
Special thanks also go to other lecturers of the Botany Department who 
showed interest in the progress of this work, and to technicians of the 
department especially Mr. Kofi Baako for their various technical assistance 
and direction.
My profound gratitude goes to all my friends and relatives particularly 
Lovelace, Gilbert, Leonard, Lawyer Fred, Ebenezer Owusu.Joycelyn, 
Roselyn ,Uncle Edwin , Nyakor , and a very special one to sister Kpobia 
and Buama who assisted me in diverse ways.
My sincere thanks go to the Narteys whose computer I used almost 
entirely for this work, to my loving parents and finally to my wife for her 
invaluable support.
in
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DEDICATION 1
DECLARATION 1
ACKNOWLEDGEMENT
TABLE OF CONTENTS iv
LIST OF FIGURES viii
LIST OF PLATES xii
ABSTRACT xiv
I. INTRODUCTION AND LITERATURE REVIEW.................................... 1
II. MATERIALS AND GENERAL METHODS............................................ 16
i. MATERIALS............................................................................................ 16
a. Cercospora arachidicola.....................................................................16
b. Groundnut seeds................................................................................. 16
c. S o il........................................................................................................ 16
d. Pollen grains of m a ize ....................................................................... 17
e. Chem ica ls............................................................................................ 17
f. Tubers of Irish Potato .......................................................................17
ii. GENERAL METHODS: .........................................................................18
a. Raising of maize p la n ts .....................................................................18
b. Collection of pollen g ra in s ................................................................ 18
c. Raising of groundnut p la n ts ..............................................................18
d. Collection of conidia of Cercospora arachidicola ..........................18
e. Culture media .................................................................................  19
Table of Content
IV
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f. Methods of sterilization....................................................................... 21
g. Spore Germination Chamber- Petri d ish.........................................22
h. Spore Germination Chamber -  Van Tieghem Cell 22
i. Humidity chamber for Longevity Test.............................................. 24
j. Preparation of Spore Suspension for Germination
Tests..................................................................................................... 24
k. Preparation of Spore-Pollen Grain Suspension for
Germination Test................................................................................24
I. Preparation of Spore Print for Germination of Storage
in Viability Test at different Relative Humidities......................... 25
m. Assessment of Conidial Germination on Glass Slides
and Lids of Solid Watch Glasses................................................... 25
n. Assessment of Viability of Conidia stored at different
Relative Humidities.......................................................................... 26
o. Conidium Germination on the Surface of Groundnut
Leaflets................................................................................................26
p. Assessment of Germination of Conidia on the surface of
leaflets.................................................................................................26
q. Incubation of Conidia in Germination and Survival
Tests.....................................................................................................27
r. Buffer solutions for Conidial Germination Tests............................28
s. Method for Testing Phytotoxicity of Hydrogen
Peroxide to Groundnut leaves.........................................................30
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t. Assessment of Efficacy of Hydrogen Peroxide
to Disinfect the surface of Groundnut Leaflets............................. 30
u- Infection Tests...................................................................................... 31
v Experimental Precautions..................................................................32
III EXPERIMENTAL DETAILS
A. Morphology and germination process of conidia of
Cercospora arachidicola.............................................................................33
B Influence of Sterile and Non-sterile maize pollen grains
on germination of conidia of Cercospora arachidicola ........................ 34
C. Influence of Exudates of maize pollen on pH of the
germination medium....................................................................................36
D. Studies of the influence of maize pollen grain on germination
of aged conidia of Cercospora arachidicola...........................................37
E. Germination of Cercospora arachidicola on groundnut leaflets
with normal and reduced phylloplane microbial population................38
F. Influence of pollen of maize on infection of groundnut
leaflets by conidia of Cercospora arach id ico la .................................... 40
IV. RESULTS:....................................................................................................41
A. Morphology and germination process of conidia of
Cercospora arachidicola.........................................................................41
B Influence of Sterile and Non-sterile maize pollen grains
on germination of conidia of Cercospora arachidicola.....................44
VI
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C. Influence of Exudates of maize pollen on pH of the
germination medium..................................................................................60
D. Studies of the influence of maize pollen grains on
germination of aged conidia of Cercospora arachidicola .................64
E Germination of Cercospora arachidicola on groundnut
leaflets with normal and reduced phylloplane microbial
population...................................................................................................92
F. Influence of pollen of maize on the infection of groundnut
leaflets by conidia of Cercospora arachidicola ...............................102
V. GENERAL DISCUSSION...............................................................................109
VI. SUMMARY....................................................................................................... 119
REFERENCES................................................................................................ 123
VII
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LIST OF FIGURES.
FIG 1. Camera Lucida drawing of conidia of C.arachidicola germinating
in sterile distilled water after 24 hours at 30°C..............................................43
FIG.2a. Percentage viability of conidia of C.arachidicola stored 
at 30°C in light intensity of 76 lux for varying periods at
different relative humidities................................................................................79
FIG.2b. Mean lengths of germ tubes produced by apical cells 
of germinating conidia of C.arachidicola stored at 30°C 
in light intensity of 76 lux for varying periods at different
relative humidities...............................................................................................80
FIG.2c. Mean length of germ tubes produced by Basal cells of
germinating conidia of C.arachidicola stored at 30°C in light
intensity of 76 lux for varying periods at different relative
humidities..............................................................................................................81
Fig 3a Graph showing percentage of germinated conidia of C arachidicola 
stored at different relative humidities for 10 days and later 
germinated in distilled water at 30°C with maize pollen 
(500-600 m l'1) showing different patterns of germ tube
development recorded after 24 hours of incubation.......................................82
Fig 3b Graph showing percentage of germinated conidia of C. arachidicola 
stored at different relative humidities for 10 days and later 
germinated in distilled water at 30°C without maize pollen
VII I
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showing different patterns of germ tube development recorded
after 24 hours of incubation..............................................................................S3
Fig 3c Percentage of germinated conidia of C. arachidicola
stored at different relative humidities for 20 days and later 
germinated in distilled water at 30°C with maize pollen 
(500-600 ml"1) showing different patterns of germ tube
development recorded after 24 hours of incubation..................................84
Fig.3d Percentage of germinated conidia of C. arachidicola stored at 
different relative humidities for 20 days and later germinated in 
distilled water at 30°C without maize pollen showing different 
patterns of germ tube development recorded after 24 hours of
incubation............................................................................................................. 85
Fig.3e Percentage of germinated conidia of C. arachidicola stored
at different relative humidities for 30 days and later germinated 
in distilled water at 30°C with maize pollen (500-600 m l'1) 
showing different patterns of germ tube development
recorded after 24 hours of incubation.............................................................. 86
Fig. 3f Percentage of germinated conidia of C arachidicola stored at 
different relative humidities for 30 days and later germinated in 
distilled water at 30°C without maize pollen showing different 
patterns of germ tube development recorded after 24 hours of 
incubation.................................................................................  qj
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Fig.3g Graph showing percentage of germinated conidia of C. arachidicola 
stored at different relative humidities for 40 days and later germinated 
in distilled water at 30°C with maize pollen (500-600 m l'1) 
showing different patterns of germ tube development recorded
after 24 hours of incubation...............................................................................88
Fig.3h Percentage of germinated conidia of C arachidicola stored at 
different relative humidities for 40 days and later germinated in 
distilled water at 30°C without maize pollen showing different 
patterns of germ tube development recorded after 24 hours of
incubation..............................................................................................................89
Fig.3i Percentage of germinated conidia of C. arachidicola stored at 
different relative humidities for 50 days and later germinated in 
distilled water at 30°C with maize pollen (500-600 m l'1) showing 
different patterns of germ tube development recorded after 24 hours
of incubation............................................................................................................90
Fig 3j Percentage of germinated conidia of C. arachidicola stored at 
different relative humidities for 50 days and later germinated in 
distilled water at 30°C without maize pollen showing different 
pattern of germ tube development recorded after 24 hours of 
incubation................................................................................................................g-j
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Fig 4a Percentage of leaflets of groundnut previously washed with 
sterile distilled water showing leaf spot symptoms after 
inoculation with drops of spore suspension of C. arachidicola
containing Zea mays pollen at different densities.....................................105
Fig. 4b Percentage of leaflets of groundnut previously washed with 
10 per cent H2O2 solution showing leaf spot symptoms after 
inoculation with drops of spore suspension of C. arachidicola
containing Zea mays pollen at different densities..................................106
Fig. 4c Percentage of leaflets of groundnut previously washed with 
20 per cent H2O2 solution showing leaf spot symptoms after 
inoculation with drops of spore suspension of C. arachidicola
containing Zea mays pollen at different densities................................... 107
Fig. 4d Percentage of leaflets of groundnut previously washed with 
30 per cent H20 2 solution showing leaf spot symptoms a after 
inoculation with drops of spore suspension of C. arachidicola 
containing Zea mays pollen at different densities....................................108
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PLATE 1
PLATE 2
PLATE 3
PLATE 4
LIST OF PLATES
Photograph showing phylloplane fungal Colony-Forming 
Units of washing of Non-sterile Groundnut leaflets on 
Potato Dextrose Agar after incubation for 5 days at
30°C (x 5/9)..........................................................................................100
Photograph showing residual phylloplane fungal Colony- 
Forming Units of washing of Groundnut leaflets previously 
sterilized with 10% H2O2 on Potato Dextrose Agar after
incubation for 5 days at 30°C (x5 /9 )............................................ 100
Photograph showing residual phylloplane fungal 
Colony-Forming Units of washing of groundnut leaflets 
Previously sterilized with 20% H20 2 on Potato Dextrose
Agar after incubation for 5 days at 30°C (x5/9)...............................101
Photograph showing residual phylloplane fungal 
Colony-Forming Units of washing of groundnut leaflets 
Previously sterilized with 30% H20 2 on Potato Dextrose 
Agar after incubation for 5 days at 30°C (x5/9)...............................101
XI I
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PLATE  5 Photographs of previously surfaced-sterilized (treated 
with 30% H20 2 for 1 minute) Groundnut leaves showing 
C.arachidicola leafspots on the 8th day after inoculation 
with C.arachidicola conidia-Maize pollen suspension 
and incubated under continuous fluorescent light in 
closed Petri dishes with humid internal atmosphere 
(x4/9).......................................................................................... 104
XII I
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ABSTRACT
Conidia of Cercospora arachidicola are straw-coloured or olivaceous, sub- 
fusiform, multicellular, 28.3-68.1(jm in lengths. They contain 3-7 cells. They 
germinated in distilled water and at 85-100% R.H. The optimum pH for 
germination was 5.0-6.0; any cell of the conidium germinating produced only one 
germ tube. The germ tube commonly comes from the end cells with occasional 
germ tube emerging from one or two median cells. Pollen of Zea mays, glucose 
(1.0-8.0%) and peptone (0.1-2.0%) failed to stimulate more median cells to 
germinate. Maize pollen, however, stimulated germination of the conidia at pH 3 
and 10.
During germination tests using conidia in water drops on sterile glass slides, the 
conidia germinated better and produced slightly longer germ tubes in the 
absence of pollen grains of Zea mays than in the presence of both non-sterile 
and autoclaved pollen grains. Particularly with the sterile pollen grains, 
percentage germination decreased with increasing pollen density. Maize pollen 
when added to solutions of glucose (1.0-8.0%) and peptone (0.1-2.0%) did not 
improve germination of the conidia. Conidia in pollen-free aqueous suspension 
drops on leaflets of groundnuts also germinated well than in suspension drops 
containing the pollen. Percentage germination in the presence of the pollen on 
leaflets with reduced fungal flora (a third of the original) and bacterial flora (a 
sixth of the original) was still lower than in the absence of pollen Maize pollen at 
moderate densities did not alter the infection rate of groundnut by C. arachidicola
x i v
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but high density of pollen on non-sterile groundnut leaves encouraged excessive 
phylloplane microfloral population which suppressed germ tube growth.
Conidia stored at 0, 20, 40, 60, and 80% R.H. survived better during the initial 30 
days at 20, 40 and 60% R.H. than at 0 and 80% R.H. The pattern of survival 
changed thereafter and conidia stored at 0% R.H. showed the highest survival on 
the 50th day; 35.1 percentage viability at 0% R.H. decreased with increasing 
relative humidity to 29.2% at 80% R.H. Maize pollen could not rejuvenate the 
aged conidia.
It was concluded that since maize pollen reduced percentage germination o f C. 
arachidicola conidia, and high pollen density suppressed germ tube growth on 
non-sterile groundnut leaves, a closer spacing of maize plants in mixed farms 
could be recommended to encourage greater pollen deposit on the groundnut 
plants. This would be an environmentally friendly control measure provided the 
conidia of the other Cercospora leaf spot fungus, Cercospora personata, are 
similarly affected by maize pollen.
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CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
Pollen grains vary considerably in composition and probably also in food value. 
The chief constituents of pollen are carbohydrates, fat, proteins and various 
inorganic mineral substances. Carbohydrate content is high. It can be as high as 
48.35 per cent as found in pollen of Pinus contorta The next in abundance are 
proteins. Date Palm (Phoenix dactylifera) pollen is richest in protein, reaching 
35.5 per cent. All have a very low fat content of only one-three per cent. Other 
important components are mineral elements (calcium, iron, magnesium, 
phosphorus and potassium) (Todd and Bretherick, 1942), plant growth 
substances (auxins,, ethylene, gibberellins hydroxypiridin cytokinins and steriods) 
(Redemann, 1949; ) and vitamins (nicotinic acid, panthetic acid, pyridoxine and 
riboflavin) ( Stanley and Liskens, 1974).
It is obvious that it is this outstanding array of compounds, shown inTable 1 
which makes pollen of certain plants including Crocus albiflorus, Papaver sp., 
Plantage sp., Pyrus sp. Triflolium sp. and Zea mays of particular biological value 
to honey bees (Maurizio, 1951). This stimulates the development of brood food 
glands, ovaries and fat bodies, and also prolongs the life span of honey bees 
(Maurizio, 1951).
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Table I.The  major components of maize pollen reported in the literature.
COMPONENT REFERENCES
Mineral element; Calcium, magnesium 
Nitrogen, phosphorus, sulphur Knight et. al (1972)
Carbohydrates (34.26%) Anderson and Kulp (1922)
Specific carbohydrates:
Reducing sugar, Non-reducing sugar 
Starch.
Galactose, Glucose, Glucosamine, 
Mannose, Raffinose, Stachyose
Todd and Bretherick (1942) 
Ueno (1954)
Protein (28.30%) Anderson and Kulp (1922)
Amino acids 
Essential amino acids 
Proline
Tseluiko (1968)
Anderson and Kulp (1922)
Lipid (1.55%) Knight et.al.1972
Enzymes (Catalase) Beckman,Scandalios and 
Brewbaker(1964)
Vitamins (Nicotinic acid, Pantothenic 
acid, Pyridoxine, Riboflavin) Nielson, Grommer and Lunden (1955)
Hormones
Hydroxypiridin Redemann (1949)
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Stanley and Liskens (1974) noted that upon wetting, viable pollen grains become 
leaky and the leachate of the pollen contains many of the compounds of the 
cytoplasm. A pertinent report of Hutchinson and Barron (1977) showed that the 
leachate could effect positive chemotropism in fungal hyphae. In successful 
positive chemotropism of tests carried out on Water Agar in Petri dishes, hyphae 
of the test fungi in most instances entered live pollen grains of Pinus nigra either 
by physical penetration or lysing of the wall of the pollen tubes. They coiled 
inside the pollen grain to form structures similar in morphology to pelotons of 
endomycorrhizal fungi. One hundred and sixty-two fungal species were tested, 
made up of 26 members of Ascomycota and Deuteromycota and 136 members 
of the Basidiomycota. In all, only 45 species were attracted by the leachate of the 
pollen grain of P nigra. Of these, 32 exhibited a consistent positive response 
while 13 species exhibited a weak or inconsistent positive response. The 45 
species, with the exception of 2 (Ambiyosporium botrytis, a member of the 
Deuteromycota and Chaetomium cochliodes, a member of the Ascomycota) 
were members of the Basidiomycota. This means that as many as 117 fungal 
species were not attracted by the leachates. It is therefore obvious that the 
relationship is not universal.
In nature, both viable and non-viable pollen grains could play a valuable role in 
the ecosystem. Stark (1972) observed that pollen grains falling in the temperate 
forest on the litter surface in the spring would move into the decomposition layer 
principally by water and would subsequently be attacked by filamentous fungi. 
Stark (op cit.) proposed that the nitrogen and phosphorus contained within the
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pollen grains were sufficient to allow these fungi to complete litter decomposition 
in environments where nitrogen and phosphorus were limiting for litter decay 
fungi. Lee Kenkel and Broth (1996) determined that 1.0 g of Pinus banksiana L. 
pollen yields 20 mg of nitrogen and that only 0.1 per cent of this was leached 
from P. banksiana pollen after 24 hours. Thus, 80 kgha'1y '1 of pollen recorded in 
a mixed pine forest in Wisconsin by Doskey and Ugosgwu (1989), for instance, 
would provide a potential 1.6 kgha1y '1 of nitrogen that could be utilised by 
lignicolous fungi. This amount is comparable to the estimated amount of nitrogen 
contributed by free-living nitrogen-fixing bacteria in wood, woody debris and litter 
(Jurgensen, et al. 1987; Roskosti, 1980; Weber, and Sundman, 1986).
Another general natural event is that the leaves of plants are showered with 
pollen. This pollen deposit plays a critical role in the life of a number of 
specialised phyllosphere fungi (Oliver, 1978; 1983) as well as supplementing the 
nutritional requirements of pathogenic and saprophytic fungi in the phyllosphere 
(Chu-Chou and Preece, 1968). During an investigation on a group of 
phyllosphere fungi of evergreen trees and shrubs in South Africa, Oliver (1978) 
observed that the fungi Retiarius bovicornutus and Retiarius superficiaris 
captured pollen grains deposited on leaves by wind. This was first noticed on 
Cassine peragua L. Pollen caught by the fungal traps was visible to the naked 
eye as a net-like deposit at the tips and downwardly directed edges of leaves and 
along the veins, and all regions where rain-water and dew accumulated. In 
microscopic preparations the fungi could be seen to form radiating anastamosing 
colonies on leaf surface to which they were firmly attached by the mucilaginous
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outer layer of the hyphal wall. From these net-like systems a number of short 
pointed branches protrude vertically. When pollen grains landed on or near these 
mycelial traps, the short hyphae grew towards them, penetrated the pollen wall 
and established a mycelium within the grain.
The directional growth of the elongating hyphae appears to be a chemotropic 
response to a stimulus from the pollen grain. The stimulating principle seemed to 
come from the germinal region as attachment and penetration always took place 
there. It was remarkable that if a pollen grain landed on the germinal region, the 
approaching short hypha encircled the grain to reach the germinal region. The 
outer layers of the pollen grain persisted throughout the dry summer and until the 
rainy season is well established, then the mycelium in the grains put out short 
hyphae, which broke through the exine and produced the characteristic spiked 
mycelium and branched conidia.
Studies by Knox and Heslop-Harrison (1970) also showed that fungal attack of 
pollen of Pinus banksiana invariably took place through the dorsal region 
between the wings where the intine bound protein is located. No appressorium is 
formed but there is a swelling of the hyphal tip before the penetration of the 
pollen wall, apparently by mechanical pressure (Oliver and William, 1975). When 
the penetrating-hypha reaches the lumen of the pollen it develops into a 
branched moniliform mycelium, which rapidly fills the grain. The hyphal walls 
gradually become thickened, followed by formation of chlamydospores. The 
pollen coat normally remains intact for along time.
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The rapidity of the parasitic reaction suggested that the attractant began to leach 
out of the pollen immediately on moistening. Indeed, several relevant reports 
indicate that when dry pollen is moistened, it releases enzymic protein within a 
few seconds. Thereafter, substantial amounts of other compounds will be 
released. For example, 10 per cent of the dry weight of Pinus elliottii pollen in 
water is lost after 60 minutes (Stanley and Search, 1971).
Studies by Chu-chou and Preece (1968) showed that pollen diffusate had been 
dialysed against distilled water for 24 hours, the non-dialysable portion (inside 
the tube) had no effect at all on Botrytis spore germination, whereas heat­
concentrated dialysate promoted spore germination to the same extent as freshly 
prepared pollen diffusate. The effective component of the diffusate was, 
therefore, water-soluble, dialysable and heat-stable. Many workers (Deverall and 
Wood, 1961;Kosnge and Hewitt, 1964; Last, 1960; Purkayastha and Deverall, 
1965) have demonstrated that sugars highly stimulated germination of Botrytis 
cinerea spores and infection of its host. But, it seems that the concentration of 
sugars in the diffusate was too low to influence germination of the spores to the 
extent recorded by Chu-chou and Preece (op cit). Neither glucose nor fructose 
detected in the diffusate by paper chromatography at 10 times the concentrations 
occurring in the diffusate stimulated spore germination. Evidently, other 
substances besides sugars must be involved in the stimulation of germination of 
the spores.
The percentage of B. cinerea spores, which germinated in distilled water, 
decreased sharply with the age of the culture providing the spores. Thirty-five per
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cent of spores from one-week old cultures germinated while only two per cent 
from two-week old cultures did so, and none of the spores from four-week old 
cultures germinated. Spores from one-, two-, three-, four- and five-week old 
cultures, on the other hand, showed 100, 96, 90, 85 and 55 per cent germination, 
respectively in the presence of strawberry pollen
Effects on lesion development on petals of strawberry paralleled the rejuvenation 
effects of pollen on the germination of old spores. For example, 88 per cent of 
successful inoculations were obtained by adding pollen to spores from five-week 
old cultures, whereas only 11 per cent success was recorded in corresponding 
tests using distilled water only. Germination of spores in suspension droplets of 
'moribund' spores of five-week old cultures in the absence of pollen on this 
occasion was due, most probably, to the presence of nutrients in the strawberry 
petal exudate.
Stimulated fungal growth has been observed near pollen grains on leaves, 
suggesting possible effects of the presence of pollen at the early stages of 
infection by plant pathogenic fungi. Ogawa and English (1960) reported pollen- 
stimulated Botrytis cinerea spore germination and germ tube growth on almond 
(Prunus amygdalus) petal. Similarly, Bachelder and Orton (1963) observed that 
a heavy deposit of pollen grains served as a locus of infection of flowers and 
other parts of American holly (Ilex opaca) by B. cinerea. Chu-chou and Preece 
(1968) carried out extensive relevant studies on the effect of pollen of strawberry 
(Fragaria moschata) on the germination of conidia of B. cinerea and the infection 
of petals and fruits of strawberry and leaves of broad bean by the germ tubes.
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Pollen grains were added to spore suspension droplets by placing in a droplet 
one whole anther, which immediately released its pollen grain, approximately 
2,500 in number into the droplet. Spore inoculum densities of four and eight 
spores per unamended aqueous droplet produced zero and 14 per cent infection 
respectively. Addition of pollen of to the spore suspensions resulted in improved 
successful infections of 63 and 93 per cent, respectively, by the two spore 
densities.
Grey mould attack of strawberry fruits caused by B. cinerea usually starts near 
the base where anthers still remain attached. (Jarvis, 1961; Moore, 1961; 
Powelson, 1960). On the premise that infection was encouraged by residual 
pollen, Chu-chou and Preece (op cit) compared the rate of infection of inoculated 
(sprayed with B. cinerea spores) intact fruits from which the anthers had been 
removed. They found out that presence of anthers markedly stimulated both the 
speed and severity of infection. For example, removal of the anthers of immature 
green inoculated fruits completely prevented infection while fruits with intact 
anthers showed 88 per cent infection, eight days after inoculation with B. cinerea. 
Pollen of strawberry also greatly promoted infection of leaves of broad bean by
B. cinerea resulting in increased number of spreading lesions sim ilar to lth e  
symptoms of aggressive infections described by Wilson (1937).
The effect of pollen on the inoculum threshold of B. cinerea on strawberry petals 
was studied by Gaumann (1950) using different densities of spores of 16-1600 
spores per suspension droplet. The results showed that the inoculum threshold 
was near 160 spores per droplet of unamended distilled water. Below this
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concentration, petals could not be infected by B. cinerea. At 160 spores per 
droplet the percentage inoculation that caused infection was only seven per cent. 
The figure increased with increasing inoculum density, reaching 78 per cent with 
800 spores per droplet. When pollen grains were added to droplets, the inoculum 
threshold was reduced to 16 spores per droplet, which caused 18 per cent 
infection. At 160 spores per droplet, 97 per cent of inoculations with pollen added 
were successful, and 100 per cent success was achieved with a density of 400 
spores per droplet. Mansfield and Deverall (1971) also showed later that pollen 
enabled Botrytis cinerea to overcome the inhibitory action of Wyerone acid, an 
antifungal product of infected bean leaves.
Besides Botrytis sp. pollen grains or entire anthers enhanced infections by 
Aiternaria (Channon, 1970J, Fusarium  (Fokkema, 1968; Strange and Smith, 
1971) Cladoasporium  and Heiminthosporium Bipolaris (Fokkema, 1971) and 
Phoma (Warren, 1972). Fokkema (1970) also observed a vastly improved growth 
of Heiminthosporium sativum  in the presence of pollen of rye.
Saprophytic fungi are also affected by pollen. Barnes (1969) showed that 
Scanning Electron Microscopy could make out the spots where pollen grains had 
been deposited on leaves after the grains had been removed. When the leaf 
surface of red clover (Trifolium pratense) was examined, 80 per cent of the 
germinated resident saprophytic fungus spores were found close to pollen grains 
or to spots where pollen had been.
The presence of pollen may not necessarily lead to greater infection. Warren 
(1972) observed differences in response to pollen by phylloplane microflora. He
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found that leaves of sugar beet (Beta vulgaris) showered with its own pollen 
supported large populations of 280-500 c.f.u.cm '2 of leaf surface of yeast, 
Cladosporium sp. and Aureobasidium pullans but spores of Phoma betae applied 
to those leaves gave only 3-5 per cent aggressive infection. On the other hand, 
plants that were not in bloom and, therefore, had no pollen deposits on their 
leaves and low phylloplane microflora population of 9,700 c.f.u.cm '2 of leaf 
surface, gave 88 per cent aggressive infection when they were inoculated with 
Phoma betae together with pollen of rye (Secale cercale). It was concluded that 
natural deposit of pollen apparently promotes growth of antagonistic microflora 
that inhibits pathogens. Apparently, the intensity of infection may be decided by 
the time of arrival of the pollen and the pathogen in the infection court, and the 
proximity of the propagule of the pathogen to the pollen grains.
Obomanu (1988) showed that, addition of maize (Zea mays) pollen grains at 
densities of 412-13,200 ml to aqueous conidial suspension of Curvularia lunata 
only slightly increased percentage germination but greatly improved germ tube 
growth, almost by a factor of two in some cases. The rate of infection of plants 
inoculated with conidial suspension without pollen grains was close (54.5 per 
cent) to rates of infection of 57.1-61.1 per cent of plants inoculated with conidial 
suspension with pollen grains of densities of 3.300-26400 grains m l'1. Application 
of pollen grains to the leaves four and eight hours before conidium inoculation did 
not significantly increase the infection rate. Obomanu (1988) also observed that 
the pollen had no effect on bacterial and yeast populations on maize leaves but
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caused greater development of Mucor sp. The pollen grains also had no 
significant effect on the rate of maize leaf infection by Curvularia lunata. 
Anyebuno (1990) defined the conditions, which make the pollen grains of maize 
stimulatory, ineffective or inhibitory. The percentage germination of conidia of 
Alternaria sp. and Cercospora arachidicola increased with increasing extract 
concentration from 1/8 dilution to the standard concentration (0.1g pollen grains 
in 10 ml distilled water). Germ tubes also grew longer with increasing extract 
concentration and more cells of the multicellular conidia in higher extract 
concentration, produced germ tubes.
Higher pollen extract concentration above the standard concentration became 
inhibitory and C. arachidicola were most severely depressed. With all three 
species, the higher concentration of the extract beyond the standard 
concentration, the fewer the number of germ tubes produced. The high pollen 
extract concentration may by itself be inhibitory to the conidia. At the same time 
this inhibition may be accompanied by inhibition by metabolites of the increased 
phyllosphere bacterial population.
The stimulation of the phyllosphere microflora by the pollen could interfere with 
growth of leaf pathogens and saprophytic species as well. This, however, may 
always not be so. For studies which have been carried out to deliberately alter 
the phyllosphere population level have not led to a change in the activity of the 
test organisms. Kinkel and Andrews (1988) reported that leaves of apple (Malus 
pumila) with the normal phyllosphere microflora, and those treated with 15 per 
cent hydrogen peroxide solution for 75-90 seconds to remove 99 per cent of the
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surface bacterial and fungi and inoculated with Aureobasidium pullulans or 
Cladosporium cladosprioides supported growth of these fungi to the same extent. 
In the final analysis, the influence of the phyllosphere population depends more 
on quality of the members of the population rather than on quantity.
The content of pollen of maize is very complex causing it, on one hand, to 
stimulate spore germination and growth of microorganisms and on the other, to 
inhibit, depending on circumstances.
The spreading tassel at the apex of the maize stem with hundreds of dangling 
anthers produces immense quantity of pollen. Pohl (1937) estimated that one 
maize plant produces 18,500,000 pollen grains. In addition, the maize pollen is 
so large (700,000 |jm3)) and heavy that unless a fair breeze is blowing, it falls 
almost vertically to the ground (Percival, 1965). Consequently, the over-towering 
maize plant showers any inter-planted crops below during mixed farming with 
abundant pollen.
Mixed farming is probably the most common farming practice in Ghana. It is a 
good system because it facilitates very effective use of the available soil nutrients 
during a growing season. The deep-rooted crops will be able to absorb nutrients 
that would be lost to shallow-rooted crops. Also, fibrous-rooted plants will be able 
to hold the soil and reduce soil erosion. If legumes are included, they improve the 
nitrogen status of the soil. Different crops have different plant nutrient 
requirement, and in this system of farming, the available plant nutrients are used 
more evenly.
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Maize is an important and common crop in mixed farms in West Africa since it is 
the staple food of most of the inhabitants of this region. It is planted in mixed 
farms with the legumes, bambara bean (Vigna subterranea (L) verde), cowpea 
(Vigna unguiculata L.) and groundnut (Arachis hypogea L .), and the root tuber 
crop -  cassava (Manihot escutenta Cranz.) There is bound to be an interaction 
between leaf fungal parasites of these crops and the pollen of maize deposited 
on the leaves.
Leaf spot is one of the commonest plant fungus diseases in Ghana, the greatest 
proportion being caused by Cercospora species ("Clerk, 1974). Leaf spots are 
patches of dry and brittle dead tissue or necrotic spots, some as tiny as 
pinheads, without extending any further and are sharply demarcated from the 
surrounding healthy tissue. The cells of the dead tissue or a necrosis maintain 
their form because death is predominantly or even exclusively caused by toxins 
rather than by cell wall disintegrating enzymes. The dead leaf spot seldom 
exceeds 10mm in diameter and would not extend any further even under most 
favourable environmental conditions The lesion is either sharply demarcated 
from the surrounding healthy tissues or separated from them by a band of yellow 
tissues of dying cells It is generally believed that the dead host cells become 
filled with substances that in turn either kill the parasite or prevent its further 
growth (Clerk, 1974). The spots may also occur on petiole and stems. 
Sometimes, a spot on the petiole may damage so much tissue that the entire leaf 
dies.
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The Cercosporas belong to the Deuteromycota. Their conidia are, generally, 
long, slender and multiseptate and borne on stiff erect, oddly crooked 
conidoiphores, which emerge in tufts through the stomata. Bambara bean, 
cassava, cowpea and groundnut all suffer from serious Cercospora leaf spot 
diseases. Those of Bambara bean, cassava, and cowpea are caused by C. 
canescens, C. Henningsii and C. cruenta, respectively. Groundnut has two leaf 
diseases caused by C. arachidicola and C. personata, respectively. These two 
Cercospora diseases have distinctive distinguishing symptoms. Cercospora 
arachidicola produces irregular circular spots, up to 10mm in diameter, and light 
tan or yellow in colour when freshly formed. With age the spots become reddish- 
brown to black on the lower surface of the leaf and light brown on the upper 
surface and surrounded by a bright yellow halo (Jenkins, 1938). Observations 
tend to show that the halo surrounding each lesion is perhaps in some way 
related to the carbohydrate content of the leaf cells, since they appear best 
developed on leaves supposedly high in carbohydrates. Spots caused by 
Cercospora personata are circular and smaller and not more than 7mm in 
diameter and with no noticeable yellow halo. The spots are dark-brown or black 
on both surfaces of the leaf. The structures of the fungi also show differences. 
Haustoria are formed by C. personata but not by C.arachidicola. C.arachidicola, 
besides, has very long slender conidia up to 100pm in length while conidia of C. 
personata are shorter broader up to 60pm long (Clerk, 1974). The work reported 
in this thesis focussed on C.arachidicola because its leaf spots were far more
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abundant during the course of this work and provided conidia in quantities 
needed for the work.
The work reported in this thesis was carried out to examine the effect of pollen of 
maize (Zea mays) on conidia of Cercospora arachidicola and on infection of 
groundnut caused by the pathogen. The thesis contains mainly results of studies
i. The germination of conidia, pattern of germ formation and growth germ 
tubes of Cercospora arachidicola as affected by leachate of maize pollen 
grains only or in combination with nutrients, groundnut leaf exudates and 
metaboltesof phylloplane microorganisms.
ii. Longativity of conidia of C.arachidicola stored at different relative 
humidities and
effect of maize pollen on germination and germ tube evelopment of aged 
conidia.
iii. Effect of maize pollen grains on infection of groundnut with varying 
phylloplane microfloral populations by C. arachidicola.
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CHAPTER TWO 
MATERIALS AND GENERAL METHODS
MATERIALS
Cercospora arachidicola Conidia
Conidia of C. arachidicola used were obtained from naturally infected 
leaves of groundnut (Arachis hypogea) plants growing in three localities:
1. Plants raised in the Teaching Garden of the Department of Botany, 
University of Ghana, Legon.
2. Plants in local private farms near the University of Ghana, Legon.
3. Plants in farms in the Dodowa area, about 20 kilometres from Legon. 
Groundnut (Arachis hypogea) Seeds
Seeds of the commonly cultivated ‘white’ variety were purchased from the 
store of the Ghana Seed Company in Accra.
Soil
Groundnut plants were raised in plastic buckets, with drainage holes at the 
bottom, for infection experiments. Garden loamy soil was obtained from 
plots previously used for cultivation of legumes for this purpose The soil 
was found to contain compatible Rhizobium  strain for nodulation of 
groundnut.
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d. Pollen Grains of Maize (Zea mays)
Pollen grains of maize used throughout this study were provided by 70-80 
days old ‘Composite Four’ variety plants raised specifically for this project 
in the Teaching Garden of the Department of Botany, University of Ghana, 
Legon. The grains were obtained from the store of the Ghana Seed 
Company, Accra.
e. Chemicals
Hydrogen peroxide and Glycerol were purchased from a local pharmacy. 
Chloral hydrate, ethanol, glucose, peptone, sulphuric acid and Tween 80 
used came from the chemical store of the Department of Botany, 
University of Ghana, Legon. They were originally purchased from British 
Drug House (BDH), Poole, England, United Kingdom.
f. Tubers of Irish Potato (Solanum tuberosum).
Tubers of Irish Potato were purchased from a local grocery shop 
whenever needed.
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ii. GENERAL METHODS
a. Raising of Maize Plants
Grains of the ‘composite four’ maize variety were soaked in distilled water 
for 48 hours and those showing no discolouration and looking apparently 
healthy were sown on plots prepared in the Teaching Garden. They were 
planted at a spacing of 100 x 100 cm, and were watered daily at the initial 
stages and once in three days from the age of 30 days until they tasselled,
b. Collection of Pollen Grains
Transparent cellophane bags were tied round the tassels just before the 
anthers dehisced. The pollen grains spilled out into the bags when the 
anthers broke open. The bags were carefully removed and taken into the 
laboratory where the collected pollen was transferred with a sterile micro­
spatula into sterile McCartney tubes and stored in a refrigerator at 4° C 
until needed.
c. Raising of Groundnut Plants.
Seeds of the ‘White’ groundnut variety were soaked overnight in distilled 
water and the swollen and wholesome ones among them sown on beds 
prepared in the Teaching Garden They were planted at a spacing of 30 x 
30cm so that they would be sufficiently crowded as they grew to 
discourage the breeding of the aphid, craccivora that transmits the 
Groundnut Rosette Virus Disease. The aphid is unable to thrive under
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conditions of high humidity around crowded groundnut plants (Clerk, 
1974) .The plants were watered daily.
d. Collection of Conidia of C.arachidicola
The growing plants were naturally infected, and infected leaflets with spots 
bearing mature conidia, were carefully detached and conveyed to the 
laboratory in transparent polythene bags. The conidia were harvested in 
different ways for different purposes and these would be described at 
appropriate places in the Experimental Details.
e. Culture Media
Different culture media were used for different purposes at various stages 
of the work. The composition and preparation of the media used are as 
follows:
1. Potato Dextrose Agar (PDA) (Ainsworth and Bisby, 1971).
Potato tuber (peeled)....................................................................... 200 g
Dextrose............................................................................................... 10 g
Agar-Agar..........................................................................................  15 g
Distilled water...................................................................................... 1L
The peeled potato tubers were cut into pieces and boiled in 400ml water 
until they started to break up. The suspension was strained with muslin 
cloth and the extract collected in 500ml beaker and allowed to cool. It was 
then poured into a 1L measuring cylinder and topped up with distilled 
water to the 1L mark. The extract was then transferred into a 2L 
Erlenmeyer flask and the glucose and agar-agar added. The mixture was
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stirred and heated in a water bath to melt the agar-agar before the 
medium was dispensed into 250ml Erlenmeyer flasks. The flasks were 
plugged with non-absorbent cotton wool and autoclaved. The plugs were 
covered with aluminium foil before autoclaving to prevent the entry of 
water vapour.
2. Nutrient Agar (oxoid)
Twenty-eight grams of Nutrient Agar were dissolved in one litre of distilled 
water. The suspension was then heated in a water bath to melt the agar 
and dispensed into 250ml Erlenmeyer flasks. The flasks were plugged 
with non-absorbent cotton wool and then autoclaved. The plugs were 
covered with aluminium foil before autoclaving to prevent the entry of 
water vapour.
3. Water Agar
Fifteen grams of Agar-agar were dissolved in one litre of distilled water in 
a 1L Erlenmeyer flask and heated in a water bath to dissolve the Agar. 
The preparation was dispensed into 250ml Erlenmeyer Flasks and the 
flasks plugged with non-absorbent cotton wool, and autoclaved The plugs 
were covered with aluminium foil before autoclaving to prevent the entry of 
water vapour.
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f. Methods of Sterilization
Nutrient and Culture media, distilled water, pollen grains,McCartney 
tubes,beakers and measuring cylinders, were sterilized by autoclaving at 
1.1 Kg cm'2 steam pressure at 121°C for 15 minutes. Petri dishes were 
sterilized by heating at 160 °C for 6 hours in an electric oven.
Tips of forceps, inoculating needles and loops and micro-spatula were 
flamed to red heat and air-cooled before use. Glass slides thoroughly 
cleaned, were stored in 90 per cent ethanol and flamed just before use. 
Glass lids of solid watch glasses were cleaned with 5% aqueous Dettol 
solution rinsed with sterile distilled water and stored in 90 per cent ethanol 
and then flamed just before use.
The inoculating room was sterilized by spraying heavily with 5.0 per cent 
aqueous Dettol solution and allowed to stand for 20 minutes before use. 
Top of the working bench in the inoculating room was wiped with 70 per 
cent ethanol. The interior of the incubator where inoculated media were 
incubated was sprayed with a mixture of 1.0 per cent potassium 
permanganate solution and 1.0 per cent aqueous Dettol solution and the 
door closed for 20 minutes before the incubator was used.
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g. Spore Germination Chamber- Petri Dish
Spore germination tests in suspension drops or at 100 per cent R.H. were 
carried out in Petri dishes. The bottom of the dishes was lined with sterile 
moistened filter paper. Slides carrying conidial suspension drops or dry 
conidia were placed on supporting V-shaped glass rods in the Petri dishes, 
with the ‘spore side’ facing upwards. The moist filter paper maintained an 
internal atmosphere of 100 per cent relative humidity. In prolonged tests, the 
filter paper was periodically re-moistened to ensure that the air in the 
chamber remained humid.
h. Spore Germination Chamber- Van Tieghem Cell
Solid watch glass, measuring 4x4x1.5 cm with a well 3.5 cm in diameter and 
1.0 cm deep, was used as Van Tieghem Cell for the conidial germination 
tests under different relative humidities. An amount of 2ml of the appropriate 
solution (5.0g in the case of solid reagents) was put into the well of the 
watch glass to maintain the desired relative humidity. The top edge of the 
watch glass was luted with petroleum jelly to provide an airtight seal when 
the lid was placed on it. The conidia were dusted onto the centre of the 
cover glass within a circumscribed area of about 1.0 cm in diameter. The lid 
was then placed on the watch glass with the conidium-bearing surface 
facing downwards. Aqueous sulphuric acid (Tetra-oxo-sulphate VI acid) 
solutions used in maintaining the different relative humidities are presented 
in Table 2.
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Table 2
Aqueous sulphuric acid (Tetra-oxo-sulphate VI) solutions for maintaining 
desired constant relative humidities at 25°C (Extracted from data of 
Solomon, 1952).
%Relative humidity at Weight in gm. of H2S 0 4 Weight of water in gm.
25°C per 100g of solution per 100g of solution
5 69.44 30.56
10 64.45 35.55
15 60.80 39.20
20 57.76 42.24
25 55.01 44.99
30 52.45 47.55
35 50.04 49.96
40 47.71 52.29
45 45.41 54.59
50 43.10 56.90
55 40.75 5925
60 38.35 61.65
65 35.80 64.20
70 33.09 66.91
75 30.14 69.86
80 26.79 73.21
85 22.88 77.12
90 17.91 82.09
95 11.02 88.98
Distilled water provided 100% R.H.
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I. Humidity Chamber for Spore Longevity Tests.
The method described in Section (h) was used in the study of survival o f
C. arachidicola conidia at different relative humidities. Conidia stored for 
different lengths of time were then germinated in nutrient broth to test their 
viability.
j. Preparation of Spore Suspension for Germination Tests.
A leaflet with a leafspot was folded across the spot with the sporulating 
surface outermost. The medium drop on a glass slide was touched with 
the lesion to transfer the conidia to the drop. The suspension drop was 
then quickly examined microscopically to assess the spore density. More 
conidia were added to suspensions of low density, while a drop of the fluid 
was added to dilute very dense suspension. Averagely, a spore density of 
20-30 conidia in the microscope field under High Power objective- X40 
was used in all the germination tests.
k. Preparation of Spore-Pollen Grain Suspension for Germination Tests
Pollen grains were transferred from the McCartney tubes with flamed 
inoculating pin into the spore suspension drop prepared as described in 
Section (j). The pin was dipped into the pollen to a marked depth and 
washed in the drop. The transfer was standardised so that pollen densities 
of 500-600, 1000-1200, 1500-1800, 2000-2400, 3000-3500,
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4000 -4500, 5000 -5500 and 6000 -7000 per ml of suspending medium 
were obtained by adding dips of 1, 2, 3, 4, 6, 8, 10 and 12 respectively, 
estimated by actual haemocytometer counts.
1. Preparation of Spore Print for Germination or Storage in Viability 
Tests, at Different Relative Humidities
The glass lid was gently touched with the outer spore-bearing surface of 
the leafspot to get a spore print. The lid was then placed on the solid watch 
glass with the spore-bearing surface facing downwards thereby exposing 
the conidia to the internal atmosphere, 
m. Assessment of Conidial Germination on Glass Slides and Lids of 
Solid Watch Glasses
At the end of the desired incubation period, 0.01 ml of 1.0 per cent 
Formaldehyde was added to each suspension drop or to each ‘dry’ spore 
print to arrest further spore germination and germ tube development. 
Percentage germination in each treatment was estimated based on not 
less than 300 randomly observed conidia under the High Power objective 
of the microscope. Any conidium with a discernible germ tube was 
considered as having germinated. The lengths of terminal and basal germ 
tubes of at least 25 germinated conidia of every treatment were measured 
using an eye piece graticule and the Mean Germ Tube Length calculated.
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n. Assessment of Viability of Conidia Stored at Different Relative 
Humidities
After the desired storage period, conidial prints on the glass lids were 
suspended in drops of Potato Dextrose Broth and incubated at 30°C for 24 
hours, after which the percentage germination (percentage viability) was 
determined and lengths of germ tubes measured with an eye-piece 
graticule.
o. Conidium Germination on the Surface of Groundnut Leaflets
This test was carried out on detached leaves placed on a sterile moist filter 
paper in a sterile Petri dish. A drop of sterile distilled water (about 0.01 ml) 
was placed on each leaflet and the conidia introduced into the drop as 
described for medium drops on glass slides. Pollen grains were added to 
the spore suspension drops of half of the preparation, while none was 
added to the drops of the remaining half. The preparations were then 
incubated at 30°C.
p. Assessment of Germination of Conidia on Surface of Leaflets
At the end of the incubation period, the leaflets were floated on a 
concentrated solution of Chloral hydrate for 24 hours to clear, with the 
conidium-bearing surface facing upwards in order to retain the conidia on 
the leaflets. The conidia were then stained in sjtu with lactophenol cotton 
blue. Percentage germination was then determined and the lengths of the 
germ tubes measured using a microscope.
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q- Incubation of Conidia in Germination and Survival Tests.
The conidia for the various tests were incubated at 30°C but the period of 
incubation varied as indicated in Chapter III- Experimental Details
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r. Buffer Solutions for Conidial Germination Tests
Buffer solutions were prepared according to the data in Tables 3 and 4 
and used in Conidial germination tests.
Table 3
MclLVaine’s Standard Buffer Solutions Stock Solution A : 0.1M Citric acid 
(C6H80 7) and Stock Solution B: 0.2M Disodium hydrogen orthophosphate 
(Na2HP04) (Hale, 1966)
pH Solution A (ml) Solution B (ml)
3 15.87 4.11
4 12.29 7.71
5 9.70 10.30
6 7.37 12.63
7 3.53 16.47
8 0.55 19.45
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Table 4
Boric acid: NaOH (Clark and Lubs) Buffer Solution. Stock Solution A : 0,2M 
Boric acid +0.2M Potassium Chloride. Stock Solution B: 0.2M NaOH 
(Clark and Lubs, 1958)
pH Solution A(ml) Solution B(ml) De-ionised Water
8 50.0 4.0 146.0
9 50.0 21.4 128.6
10 50.0 43.9 106.1
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s. Method for Testing Phytotoxicity of Hydrogen Peroxide (H20 2) to
Groundnut Leaves
Prior to Experiment that disinfected the surface of leaflets of groundnuts 
with H20 2, the ‘safe’ concentration of H20 2 to be applied was determined 
Samples of undetached leaves were immersed in H20 2 solutions of 
different concentrations for different lengths of time and rinsed with sterile 
distilled water. The samples were then left to stand for 24 hours and 
examined for bronzing. The highest concentrations of H20 2l which did not 
cause leaf bronzing, was adopted for disinfecting the leaf surface in the 
subsequent tests.
t. Assessment of Efficacy of Hydrogen Peroxide (H20 2) to Disinfect the
Surface of Groundnut Leaflets.
The residual microbial population after disinfecting the leaflet surfaces with 
H20 2 was quantified using a wash-dilution plating method. Fungal 
populations were determined with Potato Dextrose Agar containing 
Ampicillin solution to suppress bacterial growth, and bacterial populations 
with Nutrient Agar. For each test, four leaflets were immersed in 10ml 
phosphate buffer solution containing 0.01 per cent Tween 80 in a 
McCartney tube. The Tween 80 was added to disperse the bacterial cells 
and fungal spores. The tubes were kept in the refrigerator at 4°C for one 
hour. Each McCartney tube was vigorously shaken, and for the 
assessment of the fungal population, 1.0 ml of the undiluted washing and
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0.1ml of 1.0-% ampicillin was put in each of three Petri dishes and 20ml of 
melted PDA added. The dishes were gently agitated with a circular 
motion before the medium solidified. For bacterial population assessment, 
1.0ml of the undiluted washing and 20ml of Nutrient Agar were put in each 
Petri dish and mixed. Plates of PDA were incubated at 30°C for five days 
and those of NA were kept at 37°C for 48 hours. At the end of each 
incubation period the colony forming units (CFU) per ml on each plate 
were counted and the mean calculated for each treatment,
u. Infection Tests
Because the epidermal hairs on the leaflets made spore suspension drops 
to roll off leaves still attached to the stem, the leaflets were detached and 
placed on moist sterile filter paper lining the bottom of sterile Petri dishes, 
and inoculated with 0.2ml C. arachidicola conidial suspension droplets. 
The inoculated leaves were kept at room temperature under constant 
fluorescent light to keep the leaflets green for as long as possible. The 
preparations were examined each morning for signs of infection, 
v. Experimental Precautions
1 In setting up spore germination tests using Van Tieghem Cell, care was
always taken to prevent the solution in the well from coming into contact 
with the spores.
2 The density of conidia in the various spore suspensions was strictly
standardiized, giving 20-25 spores per High Power objective of the 
microscope field.
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3 Care was taken to obtain uniform distribution of the conidia on the slide 
and host leaflets for germination tests. Leaflets with unduly crowded 
conidia were discarded.
4 Glassware was scrupulously cleaned with detergents,washed well with tap 
water and finally rinsed with distilled water and allowed to air-dry before 
use.
5 Glycerol-water mixtures and Tetra-oxo-sulphate (vi) acid solutions used to 
maintain different relative humidities were thoroughly shaken during 
preparation to obtain homogenous mixtures. Fresh solutions were 
prepared for each experiment.
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CHAPTER THREE 
EXPERIMENTAL DETAILS
A. Morphology and Germination Process of Conidia of C.arachidicola
The first exercise carried out was to study thoroughly the morphology of 
the conidia and the mode of development of germ tubes on germinating 
conidia incubated in distilled water. A conidial suspension was prepared 
with distilled water and drops on slides were stained with lactophenol 
cotton blue to make the conidia clearly visible under the microscope. The 
drops were covered with cover slips and observed under high power of the 
microscope. One hundred conidia were selected randomly from five 
suspension drops and the number of cells in each conidium was recorded. 
For each of these conidia the length was measured with an eye piece 
graticule. The conidia contained three, four, five, six or seven cells. From 
the data of measurements obtained, the mean conidial length and the 
range of conidial length were obtained for each category of conidia. The 
percentage of 100 conidia measured belonging to each of the five 
categories was then calculated. The data obtained are shown in Table 5. 
Another set of conidial suspension drops on sterile slides, but on this 
occasion without cover slips, was incubated at 30°C for 24 hours so that 
the pattern of germ tube development could be studied. The different 
patterns of germ tube development by the germinated conidia after 24 
hours are illustrated by the Camera Lucida drawings in Fig. 1
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B. Influence of Sterile and Non-sterile Maize Pollen Grains on 
Germination of conidia of C. arachidicola
A series of experiments were next carried out to study the effect of pollen 
grains of maize at different densities on the germination of the conidia of 
C.arachidicola
(a.) In the first experiment different aqueous pollen suspension of either 
non-sterile pollen or autoclaved pollen grains were used. Each set 
employed pollen densities of 500-600, 1000-1200, 1500-1800, and 
2000-2400 pollen grains per ml estimated as described at Section k of 
Materials and General Methods. For each treatment of sterile and the 
non-sterile pollen grains, three well spaced pollen suspension drops 
were put on a sterile slide and sufficient conidia, about 20-30 per 
microscope field under X40 objective were added to each droplet to 
provide the desired conidial concentration as described in the Materials 
and Methods. The slides were placed in humid chambers and 
incubated at 30°C for 24 hours. Percentage germination of each 
treatment was determined after the incubation period and the lengths 
of germ tube emerging from the basal and apical cells of the conidia 
measured. The studies made in the preceeding experiment in A above 
showed five different modes of germ tube development. . Quantitative 
data of these details were therefore recorded to assess any possible 
influence of the pollen. Results obtained are presented in Tables 6 and 
7.
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(b.) The preceeding experiment showed a reduced percentage 
germination of conidia in suspension containing either sterile or non- 
sterile pollen at the highest densities of 2000-2400 pollen grains per 
ml. A subsequent test was therefore carried out to find the effect of 
higher pollen densities on germination of the conidia. The 
experiment was repeated using pollen densities of 3000-3500, 4000- 
4500, 5000-5500, and 6000-7000 pollen grains per ml of the 
suspension. The data obtained are presented in Tables8 and 9.
(c.) Indeed, these higher pollen densities depressed further the 
percentage germination of the conidia. An experiment was therefore 
carried out to find out whether externally supplied nutrient would 
offset the depressant effect of the higher concentrations of the 
pollen. The highest pollen density of 6000-7000 pollen grains per ml, 
which gave the lowest percentage germination, was used in this 
experiment. The nutrients used were glucose at concentrations of
1.0, 2.0, 4.0 and 8.0 per cent and peptone at concentrations of 
0.1,0.5, 1.0 and 2.0 per cent. Since in the earlier experiments spore 
germination in suspension containing non-sterile pollen and that in 
suspension with sterile pollen was quite close only non-sterile pollen 
was used in this experiment. The same preparations were made and 
the relevant data were collected after incubation at 30°C for 24 
hours. The results appear in Tables 10, 11, 12 and 13.
35
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(d) Because addition of either glucose or peptone to conidial drops 
containing 6000-7000 pollen grains per ml improved germination of 
the conidia, this experiment was carried out as a sequel to find the 
combined effect of glucose and peptone. Different combinations and 
permutations of glucose, of concentrations- 1.0, 2.0, 4.0 and 8.0 and 
peptone, of concentrations of 0.1, 0.5, 1.0 and 2.0 per cent, were used as 
the germination media containing pollen or without pollen. The results 
obtained are shown in Tables 14and 15.
C. Influence of Exudates of Maize Pollen on pH of the Germination 
Medium
(a) The depression of percentage germination of very high densities of 
maize pollen could probably be due to alteration of the pH of the 
suspension. An experiment was designed to find the extent to which pH of 
the suspension might have been altered by the pollen. Pollen suspensions 
were prepared with either sterile or non-sterile pollen in McCartney tubes 
at different densities of 500-600, 1000-1200, 1500-1800, 2000-2400, 
3000-3500, 4000-4500, 5000-5500, and 6000-7000 pollen grains per ml. 
Since the conidia normally began to germinate after 12 hours at 30°C, the 
McCartney tubes were kept at 30°C and samples of each treatment were 
withdrawn after 3, 6, 9 and 12 hours, respectively, and the pH measured. 
The results obtained are presented in Table 16.
36
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(b) Although the results obtained and tabulated in Table 16, showed 
very little difference among the various treatments, the leaf surface 
environment could possibly shift the pH values beyond the limits of the pH 
range provided by the different densities of the pollen. The germination of 
the conidia over a wide range of pH was, therefore, studied. Two buffer 
solutions were used for this investigation. MclLvaine’s Standard Buffer 
solutions provided pH 3, 4, 5, 6, 7 and 8 and Clark and Lubs Standard 
provided pH 8 and 9. The results obtained after incubation at 30°C for 24 
hours appear in Tables 17 and 18.
D. Studies on the influence of maize pollen grains on germination of aged 
conidia of C. arachidicola
(a) Before aged conidia could be used, it was necessary to establish the 
relative humidities where conidia would germinate. The conidia could then 
be stored below those humidities and the effect of pollen on the 
germination of such conidia could be tested at intervals. The conidia were 
dusted onto dry glass lids of solid watch glasses containing solutions of 
Tetra-oxo-sulphate (vi) acid of appropriate concentrations to provide 
humidities of 75, 80, 85, 90 and 95% R.H. Solid watch glass containing 
distilled water in their wells provided atmosphere of 100 per cent relative 
humidity. Percentage germination of the conidia was calculated after 24 
hours’ incubation at 30°C as showed in Tables 19 and 20.
37
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(b) Germination occurred at the upper humidities of 85, 90, 95 and 100 per 
cent relative humidity and the conidia as a consequence could be stored 
for the ageing experiment at humidities from 0 to 80 per cent R.H.. Five 
humidities from this range namely 0, 20, 40, 60 and 80 per cent R.H. were 
adopted and the conidial were stored at these humidities as described in 
the Materials and Methods over 50 days. Conidial samples were 
withdrawn at 10 days interval and germinated by suspending in sterile 
distilled water with or without pollen grains. The results are shown in 
Tables 21-30 and in Figs. 2 and 3.
E. Germination of C. arach id ico la  conidia on groundnut leaflets with 
normal and reduced phylloplane microflora population
In this investigation made up of four different experiments, the conidia 
were germinated in the presence of maize pollen grains on natural leaf 
surface and on leaf surface, which had been disinfected with hydrogen 
peroxide.
(a) Only non-sterile pollen grains were added to conidial suspension drops 
on the groundnut leaflets. There were five treatments: spore suspension 
without pollen grains, and spore suspension with pollen of four densities, 
500-600, 1000-1200, 1500-1800 and 2000-2400 pollen grains per ml of 
medium. The preparations were incubated at 30°C in humid atmosphere 
for 24 hours. The test was repeated five times and the results obtained for 
the five are presented together on Table 31.
38
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(b) Washing the leaves with hydrogen peroxide did the removal of the 
phylloplane microfloral. This, however, required the determination of the 
appropriate concentration of the disinfectant, which would not destroy the 
leaves. The leaf samples were washed with different concentrations of 
hydrogen peroxide (10, 20 and 30%) for different lengths of time and 
rinsed quickly thereafter with sterile distilled water as already described in 
the Materials and Methods. The observations made are shown in Table 
32.
(c) The ‘safe’ treatments were then repeated and the plate counting 
method as already described assessed the degree of disinfection of the 
leaflets. The results are shown in Tables 33 and 34 and Plates 1-4
(d) Similar to other relevant studies in literature, the microfloral population 
was reduced by hydrogen peroxide but not completely eliminated. The 
germination of the conidia on leaflets of normal microfloral population and 
that on leaflets treated with 30% H2O2 for one minute with reduced 
population were compared. The results of the germination test on these 
two types of leaflets in the presence and absence of pollen grains are 
presented in Tables 35 and 36.
39
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F. Influence of pollen of maize on the infection of groundnut leaflets by 
conidia of C. arachidicola.
Although the previous experiment showed that the germination of conidia 
was slightly better on leaves in the absence of pollen than in the presence 
of pollen subsequent infection may or may not follow the same trend. 
Infection tests were carried out with non-sterilized and surface-sterilized 
leaflets to establish the trend of infection. Unsterilized leaflets and the 
leaflets disinfected with 10, 20 and 30 per cent hydrogen peroxide were 
inoculated with conidial suspension containing different densities of pollen 
grains of maize. The inoculated plates were left under a continuous supply 
of white fluorescent light for 8 days at 30°C and the infection rate of the 
different treatments recorded. The results obtained are shown in Table 
37,Fig 4 and in Plate 5.
40
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CHAPTER FOUR 
RESULTS
The conidia of C.arachidicola were solitary, dry, septate (multicellular) and 
subfusiform in shape. They were straw-coloured or olivaceous and 
smooth. A conidium contained 3, 4, 5, 6 or 7 cells and they ranged from 
28.3 to 68.1 nm in length, and approximately 10.4 thick in the widest 
part. The data in Table 5 show that the 5-celled conidium was the most 
abundant (36.0 per cent) while the 3 and 7-celled conidia represented only
6.0 and 9.0 per cent of the population, respectively.
Five patterns of conidial germination were identified and illustrated in 
Fig.1. One germ tube emerged from the distal cell only or from the basal 
cell only (Figs 1a and 1b). A conidium may also produce one germ tube 
from the distal cell and one from the basal cell at the same time (Fig 1d.) 
In other instances, a conidium either produced germ tube from the medium 
cells only (Fig. 1c) or from an end cell and a medium cell (Fig. 1e). The 
commonest forms are those illustrated in Fig. 1a and Fig 1d.
A. Morphology and Germination Process of Conidia of C.arachidicoda
41
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TABLE 5
Details of Morphology of 100 Randomly Selected Multicellular Conidia of
C.arachidicola
No. of Cells 
in Conidium
Mean length of 
Conidia (|um)
Range of length 
of Conidia (^m)
Percentage of 
Spores in Group
3 35.8 28.4-48 .3 6.0
4 40.3 35.2 -52 .8 33.0
5 42.6 32.4-55.1 36.0
6 46.3 38.8 -65 .6 16.0
7 54.5 42.1 -68.1 9.0
42
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lb
Vig.l: Corners lucid a drawings ol: Don.i.h.i ol '• ..'.r.v::ii iiicoin
germinating in steril e distilled vatv - fir’ ?A liours. 
at 30°C.
Note the different points of emergence of the gunns 
tubes and the swelling of some of the cells of the 
multicellular conidia.
43
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B. Influence of Sterile and Non-sterile maize pollen grains 
on Germination of Conidia of C.arachidicola.
(a) The data in Table 6 indicate that the conidia germinated very well in 
distilled water achieving a percentage germination of 80.5 percent. 
Percentage germination decreased when pollen of maize was added, 
in the case of both sterile and non-sterile pollen grains. Percentage 
germination in pollen suspensions of densities of 500 -  1800 pollen 
grains per ml of suspending medium was practically the same; but 
germination was further depressed, especially with sterile pollen in 
media with 2000 -  2400 pollen grains per ml. of suspension. The germ 
tubes in most cases were also shorter in the pollen suspensions than in 
distilled water without pollen, while germ tubes produced in the 
presence of non-sterile pollen were generally longer than those 
produced in suspension of sterile pollen. The germination pattern was 
also somehow affected by the pollen grains. The data in Table 7 
revealed a number of features which can be summarised as follows:
i) The five different types of germination occurred in different 
proportions, with the greatest proportion being conidia producing 
germ tubes from both the distal and basal cells, followed closely by 
conidia with germ tube from the apical cell only. Very few conidia 
produced germ tubes from the median cells only.
ii) With both sterile and non-sterile pollen, the percentage of conidia 
producing germ tubes from both the distal and basal cells 
increased with increase in pollen density. This was more 
accentuated in media of non-sterile pollen.
44
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iii) A reverse trend was observed in conidia producing germ tubes 
from the apical cells only.
iv) The percentages of the three other patterns of germ tube 
development seemed to be independent of pollen density.
(b) In the subsequent experiment in which higher pollen densities of 2000 
-  7000 pollen grains ml'1 of suspension were used, percentage 
germination progressively decreased as shown in Table 8. Thus, 
whereas conidial germination in the pollen-free medium was 83.0 
percent, it was only 58.4 per cent and 48.2 per cent, respectively in the 
presence of non-sterile and sterile pollen of a density of 6000 -  7000 
pollen grains ml'1 of suspending medium. The higher pollen densities, 
on the other hand, uniformly supported greater production of germ 
tubes from both the apical and basal cells as indicated in Table 9.
(c) The results of germination tests carried out to find out whether addition 
of either glucose or peptone would improve conidial germination in 
aqueous suspension containing the highest pollen density of 6,000 -
7.000 non-sterile pollen grains ml"1 are presented in Tables 10, 11, 12 
and 13. The already high percentage germination of the conidia in 
distilled water was further increased by Glucose at concentrations of
4.0 and 8.0 per cent as shown in Table 10 On addition of Glucose at 
concentrations ranging from 1.0 to 8.0 per cent, germination in 
suspensions with pollen grains increased from 67.5 percent (without 
Glucose) to 74.7 -  80.6 per cent. The treatment also improved the 
growth of the germ tubes.
45
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It was noted that increase in Glucose concentration increased the 
percentage of conidia that produced germ tubes from both the apical 
and basal cells and from median cells and apical and/or basal cells, 
whereas the percentage of apical cells producing germ tubes 
decreased (Table 11).
Tests with peptone also showed that addition of peptone also improved 
the percentage germination of the conidia in the presence of maize 
pollen grains (Table 12) and increased the percentage of conidia 
producing germ tubes from both the apical cells and basal cells and 
those of conidia producing germ tubes from apical and/or basal cells 
and median cells while the percentage of conidia bearing germ tubes 
from the apical cells only declined (Table 13).
) Tables 14 and 15 contain data of results of tests, which used combined 
glucose and peptone as germination medium to which pollen grains 
had been added. The data in Table 14 showed that the lengths of 
germ tubes from both the apical and based cells showed very little 
variation. Percentage germination in the media on the other hand, 
showed clearly that, the percentage decreased when there was low 
concentration of both glucose (1.0 and 2.0%) and peptone (0.1 and 
0.5%) but was greater at higher peptone concentrations of 1.0 and 2.0 
per cent. At higher glucose concentrations of 4.0 and 8.0 per cent, 
very high percentage germination occurred at all concentrations of 
peptone.
46
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Data in Table 15 showed that the percentage of conidia bearing germ 
tubes from both apical and basal cells was directly related to concentration 
of the peptone rather than to glucose concentration.The percentage 
increased with increase in peptone concentration.The percentage of 
conidia bearing apical germ tubes only,on the other hand,showed an 
inverse relationship with peptone concentration.
47
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TABLE 6
Germ ination o f Conidia o f C.arachidicola in aqueous suspension o f sterile and
non-sterile pollen o f Zea mays at 30°C in light intensity o f 76 lux in 24 hours
Pollen
Treatment
Pollen density 
in conidial 
suspension 
(No ml"1)
Total
Number of
Conidia
observed
Percentage
Germination
Mean length of 
Germ tubes (urn) 
from
Basal Apical 
Cell Cell
Non-sterile 500 -  600 481 704 58.9 66.7
1000- 1200 491 73.6 61.7 60.4
1500-1800 537 71.5 54.7 61.1
2000 -  2400 456 69.7 58.2 59.3
Sterile 500 -  600 446 70.7 51.2 58.2
1000-1200 589 73.5 48.9 59.6
1500-1800 516 71.2 52.5 55.1
2000 -  2400 512 61.6 53.9 57.5
Control No pollen 563 80.5 60.5 65.5
48
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TABLE 7
Pattern of formation of germ tubes by 50 randomly selected germinated 
conidia of C.arachidicola incubated in aqueous suspension of pollen of 
Zea mays at 30°C in light intensity of 76 lux for 24 hours.
Percentage of Conidia with germ tubes emerging 
from indicated cells
Pollen
Treatment
Pollen 
density in 
Conidial 
Suspension 
(No. ml"1)
Apical 
and 
Basal 
Cell Co- 
jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical 
and/or 
Basal cell 
and 
Median 
cell
Non-
sterile 500 -  600 26 36 16 6 16
1000-1200 36 30 10 6 18
1500-1800 44 24 16 4 12
2000 -  2400 46 20 12 2 20
Sterile 500 -  600 24 44 10 2 20
1000- 1200 34 30 6 0 30
1500- 1800 30 30 14 6 20
2000 -  2400 36 24 14 4 22
Control No pollen 27 20 10 9 34
49
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Germination of Conidia of C.arachidicola in aqueous suspension of higher 
densities of sterile and non-sterile pollen of Zea mays at 30° C in light 
intensity of .76 lux in 24 hours.
TABLE 8
Pollen
Treatment
Pollen density 
in conidial 
suspension 
(No. ml'1)
Total 
Number of 
Conidia 
observed
Percentage
Germination
• Mean length of 
Germ tubes (^im) 
from 
Basal Distal 
Cell Cell
Non-sterile. 2000 -  2400 676 70.5 58.22 58 22
3000 -  3500 614 66.4 62 48 62 48
4000-4500 533 64.2 55.38 63.19
5000 -  5500 433 59.9 54.67 50.41
6000 -  7000 445 58.4 56.8 60.35
Sterile 2000 -  2400 484 65.5 53 96 47 57
3000 -  3500 465 63.3 59 64 61.54
4000 -  4500 503 53 4 61.06 62.51
5000 -  5500 540 53.2 61.06 62.24
6000 -  7000 546 48.2 53.96 58.93
Control No pollen 651 83.0 59 64 67 45
50
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TABLE 9
Pattern of formation of germ tubes by 50 randomly selected conidia of
C.arachidicola incubated in aqueous suspension of higher densities of pollen 
of Zea mays at 30°C in light intensity of 76 lux for 24 hours.
Percentage of Conidia with germ tubes emerging 
from indicated cells
Pollen
Treatment
Pollen 
density in 
Conidial 
Suspension 
(No. m l'1)
Apical and 
Basal Cell 
Co-jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical 
and/or 
Basal cell 
and 
Median 
cell
Non-sterile 3000 -  3500 36 32 12 4 16
4000 -  4500 40 20 8 8 24
5000 -  5500 44 24 8 4 20
6000 -  7000 48 20 8 4 20
Sterile 3000 -  3500 40 24 8 8 20
4000 -  4500 44 16 12 4 24
5000 -  5500 40 20 8 4 28
6000 -  7000 44 8 8 4 24
Control No pollen 33 22 12 8 25
51
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TABLE 10
Germ ination o f Conidia of C.arachidicola in aqueous suspension o f non-sterile
Zea mays pollen o f density o f 6000 -  7000 grains ml"1 amended with G lucose
at 30°C in light intensity o f 76 lux in 24 hours.
Concentration
Total 
Number of
Mean length of 
Germ tubes (|im) 
from
Treatment of Glucose
(%)
Conidia
observed
Percentage
Germination
Apical
Cell
Basal
Cell
Without pollen 0.0 1069 83.2 81.5 79.2
1.0 970 79.7 80.1 79.6
2.0 1005 81.7 80.4 80.2
4.0 996 86.9 77.3 73.8
8.0 1048 87.3 78.7 76.6
With pollen 0.0 1002 67.5 65.4 62.3
1.0 800 78.8 75.6 74.0
2.0 977 80.0 80.2 78.6
4.0 1146 74.7 77.5 76.0
8.0 1010 80.6 74.0 72.7
52
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Pattern of formation of germ tubes by 50 randomly selected germinated 
conidia of C.arachidicola incubated in aqueous suspension of non-sterile Zea 
mays pollen of density of 6000 -  7000 pollen grains ml'1 amended with 
Glucose at 30°C in light intensity of 76 lux in 24 hours.
TABLE 11
Percentage of Conidia with germ tubes 
emerging from indicated cells
Treatment
Concentration 
of Glucose
(%)
Apical and 
Basal Cell 
Co-jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical 
and/or 
Basal 
cell and 
Median 
cell
Without pollen 1.0 28 36 16 8 12
2.0 40 28 16 4 12
4.0 44 24 12 4 16
8.0 40 24 12 4 20
With pollen 1.0 32 40 12 8 8
2.0 32 32 16 4 16
4.0 40 32 8 4 16
8.0 44 16 12 4 24
53
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TABLE 12
Germ ination o f conidia o f C.arachidicola in aqueous suspension o f non-sterile
Zea mays pollen of density o f 6000 -  7000 pollen grains m l1 amended with
Peptone at 30°C in light intensity o f 76 lux in 24 hours.
Concentration
Total 
Number of
Mean length of 
Germ tubes 
(urn) from
T reatment
of Peptone
(%)
Conidia
observed
Percentage
Germination
Apical
Cell
Basal
Cell
Without pollen 0.0 1069 83.2 81.5 79.2
1.0 1019 73.5 71.4 68.8
5.0 949 78.8 69.2 71.6
1.0 1004 82.1 71.6 67.4
2.0 873 82.7 58.78 58.76
With pollen 0.0 1002 67.5 65.4 62.3
0.1 1092 77.5 80.1 79.6
0.5 1282 73.6 80.4 80.2
1.0 1081 72.9 77.3 73 8
2.0 879 75.4 78.7 76.7
54
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TABLE 13
Pattern of formation of germ tubes by 50 randomly selected germinated 
conidia of C.arachidicola incubated in aqueous suspension of non-sterile 
pollen of Zea mays pollen of density of 6000 -  7000 pollen grains ml'1 
amended with Peptone at 30°C in light intensity of 76 lux in 24 hours.
Percentage of Conidia with germ tubes emerging from 
indicated cells
Treatment
Concentration 
of Peptone
(%)
Apical 
And Basal 
Cell Co- 
jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical and/or 
Basal cell 
and Median 
cell
Without pollen 0.1 28 40 12 8 12
0.5 40 32 16 0 12
1.0 40 24 12 4 20
2.0 48 24 12 0 16
With pollen 0.1 32 36 16 8 8
0.5 40 28 16 4 12
1.0 44 20 12 4 20
2.0 44 24 8 4 20
55
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Germination of Conidia of C.arachidicola in aqueous suspension of non-sterile 
pollen of Zea mays, of pollen of density of 6000 -  7000 pollen grains m l1 
containing Glucose and Peptone in different ratios, at 30°C in light intensity of 
76 lux in 24 hours.
(Each value of Percentage Germination based on a total of 400 -  500 conidia)
TABLE 14
Percentage
Mean length of germ tubes (^im) 
from apical and basal cells of 
conidia in suspension
Concentration 
of Glucose
(%)
Concentration 
of Peptone
(%)
Germination 
In suspension 
With Without 
pollen pollen
With
Apical
Cell
pollen
Basal
Cell
Without pollen 
Apical Basal 
Cell Cell
1.0 0.1 68.0 76.3 63.1 59.7 67.6 65 5
0.5 74.3 75.1 65.6 66.9 67.8 65.4
1.0 77.0 78.6 66.6 61.6 64.8 64.5
2.0 78.2 84.8 66.2 64.6 64.2 65.3
2.0 0.1 79.2 83.4 71.4 65.4 75.6 72.6
0.5 75.5 79.0 71.5 69.3 69.9 69.3
1.0 85.7 78.5 71.5 69.6 65.3 64.2
2.0 86.4 83.5 72.2 70.3 73.6 70.9
56
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Table 14 Cont’d
Concentration 
of Glucose
(%)
Concentratio 
n of Peptone
(%)
Percentage 
Germination 
In suspension 
With Without 
pollen pollen
Mean length of germ tubes 
(|xm) from apical and basal 
cells of conidia in suspension
With pollen Without pollen 
Apical Basal Apical Basal 
Cell Cell Cell Cell
4.0 0.1 85.6 71.6 70.7 70.4 65.1 63.4
0.5 79.2 75.6 69.3 68.2 64.5 65.3
1.0 82.2 77.8 65.2 63.0 65.4 63.8
2.0 80.0 73.0 67.2 68.3 75.2 74.1
8.0 0.1 81.7 81.1 65.6 63.7 74.3 71.3
0.5 83.9 82.8 63.3 62.2 76.2 73.1
1.0 83.7 81.0 62.2 59.6 72.3 71.9
2.0 85.9 87.3 61.2 60.3 69.6 68 9
57
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TABLE 15
Pattern of formation of germ tubes by 50 randomly selected conidia of C.arachidicola
incubated in aqueous suspension of non-sterile pollen of Zea mays of pollen density 
of 6000 -  7000 pollen grains ml'1 containing glucose and peptone in different ratios at 
30°C in light of 76 lux for 24 hours.
Germina­
Percentage of Conidia with germ tubes emerging 
from indicated cells
tion 
medium 
(without 
pollen: -) 
(with 
pollen: +)
Concen­
tration of 
Glucose
(%)
Concen­
tration of 
Peptone
(%)
Apical 
and 
Basal 
Cell Co- 
jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical 
and/or 
Basal cell 
and 
Median 
cell
- 1.0 0.1 32 36 12 8 12
0.5 32 32 12 4 20
1.0 36 28 8 4 24
2.0 44 28 8 0 20
+ 1.0 0.1 36 40 12 0 12
0.5 32 36 12 0 20
1.0 40 24 16 4 16
2.0 40 20 12 8 20
- 2.0 0.1 32 32 12 8 16
0.5 36 28 12 4 20
1.0 40 24 16 0 20
2.0 40 20 8 4 28
+ 2.0 0.1 28 36 16 8 12
0.5 32 28 12 8 20
1.0 44 24 12 4 20
2.0 40 28 8 0 24
58
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Table 15Cont’d.
Germina­
Percentage of Conidia with germ tubes emerging 
from indicated cells
tion 
medium 
(without 
pollen: -) 
(with 
pollen: +)
Concen­
tration of 
Glucose
(%)
Concen­
tration of 
Peptone
(%)
Apical 
and 
Basal 
Cell Co- 
jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical 
and/or 
Basal 
cell and 
Median 
cell
- 4.0 0.1 24 40 16 8 12
0.5 32 32 12 8 16
1.0 36 28 12 4 20
2.0 40 28 8 0 24
+ 4.0 0.1 28 32 16 8 16
0.5 40 28 8 4 20
1.0 40 24 12 0 20
2.0 36 24 12 4 24
- 8.0 0.1 28 36 20 4 12
0.5 36 28 16 4 16
1.0 40 28 12 0 20
2.0 40 24 12 4 20
+ 8.0 0.1 28 36 16 8 12
0.5 36 32 12 4 16
1.0 40 24 12 0 24
2.0 40 28 8 4 20
59
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C. Influence of Exudates of maize pollen on pH of the 
germination medium
(a) Irrespective of the density of maize pollen grains added to the aqueous 
suspension, the pH of the media showed very slight changes (Table 
16). The pH of the different suspensions ranged from pH 6.0 to pH 7.0, 
3 hours after the introduction of the pollen grains, and from pH 6.0 to 
pH 6.5, 12 hours after introduction of the pollen grains. The effects of 
the exudates on the pH showed no relationship with density of the 
pollen grains.
(b) The data in Table 17 showed that the pollen of maize could affect the 
response of conidia of C.arachidicola to pH of the medium. In the 
absence of the pollen grains, the conidia germinated over the pH range 
of pH 4.0 to pH 9.0 with an optimum at pH 5.0 -  pH 6.0. On addition of 
the pollen, however, conidial germination also occurred at pH 3.0 and
10.0, thus extending the pH range which supported germination. 
Germination at pH 3.0 was, however, tardy (5.2 per cent). The 
optimum pH anyway remained still at pH 5.0 -  pH 6.0. The respective 
pH’s did not influence the pattern of germ tube formation (Table 18).
60
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Hydrogen ion concentration of aqueous suspension of pollen of Zea mays 
recorded 3, 6, 9 and 12 hours after introduction of the pollen into 
sterile distilled water of pH 6.5 and incubated at 30°C.
TABLE 16
Pollen Density Nature of pH of suspension after (h)
(No ml'1) Pollen 3 6 9 12
500 -  600 Sterile 6.5 6.5 6.5 6.5
Non-sterile 6.5 6.5 6.0 6.0
1200 - 1200 Sterile 7.0 6.5 6.5 6.5
Non-sterile 6.0 6.0 6.0 6.5
1500-1800 Sterile 6.5 6.5 6.5 6.5
Non-sterile 7.0 7.0 6.5 6.5
2000 -  2400 Sterile 6.5 6.5 6.5 6.5
Non-sterile 6.5 6.5 6.5 6.0
3000 -  3500 Sterile 6.5 6.5 6.5 6.0
Non-sterile 6.5 6.5 6.0 6.0
4000 -  4500 Sterile 6.5 6.5 6.5 6.5
Non-sterile 7.0 7.0 6.0 6.0
5000 -  5500 Sterile 6.0 7.0 6.0 6.0
Non-sterile 6.0 7.0 6.0 6.0
6000 -  7000 Sterile 6.5 7.0 7.0 6.0
Non-sterile 6.0 7.0 7.0 6.0
61
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Effect of pH on germination of Conidia of C.arachidicola 
at 30°C in light intensity of 76 luix in 24 hours.
TABLE 17
Treatment pH
Total 
Number of 
Spore 
observed
Percentage
Germination
Mean length of 
Germ tubes (nm) 
from 
Apical Basal 
Cell Cell
Without pollen 3.0 430 0.0 - -
4.0 350 68.3 55.3 51.3
5.0 411 80.2 68.2 73.6
6.0 418 76.6 68.6 63.5
7.0 398 51.8 49.4 51.2
8.0 403 42.4 37.7 33.6
9.0 379 39.7 32.5 29.8
10.0 382 0.0 - -
With pollen 3.0 460 5.2 28.3 25.7
(500 -  600) 4.0 410 56.5 51.6 48.5
5.0 380 60.7 61.2 58.2
6.0 418 59.1 60.4 57.4
7.0 365 56.4 58.7 54.1
8.0 340 50.2 52.3 53.3
9.0 392 39.4 48.5 43.6
10.0 403 28.7 36.2 30.2
62
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TABLE 18
Pattern of formation of germ tubes by 50 randomly selected germinated 
conidia of C.arachidicola incubated in buffer solution of various pH at 
30°C in light intensity of 76 lux in 24 hours.
Percentage of Conidia with germ tubes emerging 
from indicated cells
T reatment pH
Apical 
And 
Basal Cell 
Co-jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical 
and/or 
Basal cell 
and Median 
Cell
Without pollen 3 0 0 0 0 0
4 34 30 24 4 8
5 40 26 18 10 6
6 36 24 20 8 12
7 30 36 28 12 4
8 34 40 22 0 4
9 28 38 24 0 0
10 0 0 0 0 0
With pollen 3 * - - - -
(500-600 ml-l) 4 28 34 22 10 6
5 36 30 14 6 14
6 36 28 24 8 4
7 32 30 28 4 6
8 34 32 24 8 2
9 28 34 18 8 12
10 32 34 22 6 6
• Germinated conidia were too few to allow evaluation.
63
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D. Studies on the influence of maize pollen on germination of aged 
conidia of C.arachidicola
(a) The conidia were incubated at 75 -  100% R.H. to establish the threshold 
relative humidity above which germination would take place. The 
percentage germinations at the different relative humidities are shown in 
Table 19. The conidia did not germinate at 75%and 80% RH. Over the 
range of 85% -  100% R.H, which supported germination, percentage 
germination increased with increasing humidity. Thus, percentage 
germination at 85% RH was 11.4 per cent and 64.0 percent at 100% R.H. 
Relative humidity had marked effect on the germination pattern. Table 20 
indicated clear trends.
i) The percentage of conidia bearing apical and basal germ tubes
declined from 56 per cent, at 100% R.H with decreasing humidity to16
percent at 85% R.H
ii) The percentage of conidia bearing germ tubes from end cells and
median cells also increased with increasing relative humidity.
iii) A reverse trend was shown by conidia bearing one germ tube only
from one of the end cells and the percentage of conidia involved
decreased with increasing relative humidity.
64
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Germination of Conidia of C.arachidicola at different relative 
humidities at 30°C in light intensity intensity of 76 lux in 24 hours.
TABLE 19
Relative
Humidity
(%)
Total Number 
of Conidia 
observed
Percentage
Germination
Mean length of Germ 
tubes (|im) from
Apical Basal 
Cell Cell
100 501 64.0 69.2 64.0
95 676 57.3 61.5 61.6
90 470 46.3 62.8 57.1
85 604 11.4 52.4 51.2
80 505 0.0 - -
75 490 0.0 - -
65
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TABLE 20
Pattern of formation of germ tubes by 50 randomly selected germinated 
conidia of C.arachidicola incubated at different relative humidities at 30°C in 
light intensity of 76 lux in 24 hours.
Percentage of conidia with germ tubes emerging 
from indicated cells
Relative
Humidity
(%)
Apical 
And 
Basal Cell 
Co-jointly
Apical
Cell
Only
Basal
cell
only
Media
n
cell(s)
only
Apical 
and/or 
Basal cell 
and 
Median 
Cell
100 56 16 4 0 24
95 32 32 12 4 20
90 24 52 16 0 12
85 16 40 20 12 12
80 - - - - -
75 - - - - -
66
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(b) The data in Tables 21 -  30 provide sufficient information on the survival of 
the conidia of C.arachidicola and the effect of maize pollen on germination 
of conidia of different ages. The important details could be summarised as 
follows:
i) Between 17 and 35 per cent of the conidia at the different relative 
humidities survived 50 days storage.
ii) The conidia survived best at 20% R.H as clearly shown by data on 10. 
20 and 30 days old conidia which showed 76.0 and 80.2, 65.1 and 
71.3, and, 51.0 and 53.2 per cent viability, respectively.
iii) Longevity decreased appreciably at Zero and 80% R.H:
Conidia at 0% R.H. showed 46.9 and 51.8, 42.9 and 46.2, and 34 1 
and 35.5 per cent viability after 10, 20 and 30 days, respectively.
Conidia at 80% R.H showed 47.2 and 50.0, 30.5 and 35.7 and, 34.7 
and 36.1 per cent viability after 10, 20 and 30 days, respectively.
iv) The lengths of the germ tubes produced in 24 hours at 30°C 
decreased as the age of the conidia increased
v) Conidia stored at 20% R.H consistently produced the highest 
percentage of conidia producing germ tubes from both the apical 
and basal cells.
vi) Germination of conidia of different ages was not affected by pollen 
grains of maize, and conidia of the same age showed closely 
similar percentage germination in the presence and in the absence 
of the pollen grains of maize.
67
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Some of these details are depicted in Figs 2 & 3.
vii) Conidia stored at 0% R H for 40 and 50 days then showed the 
highest percentage viability (Tables 27 and 29) and viability 
decreased with increasing relative humidity of storage.
viii) The maize pollen did still not affect germinating 40 and 50 days old
conidia. In the numerous germination tests carried out in this 
investigation the germ tubes of C. arachidicola were never attracted by the 
pollen grains in the suspension droplets.
68
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TABLE 21
Percentage of conidia of C.arachidicola able to germinate in Distilled 
Water and Aqueous Suspension of non-sterile Pollen of Zea mays of density 
of 500 -  600 pollen grain ml'1 in 24 hours at 30°C in light intensity of 76 lux 
after storage at 0 -  80% R.H. at 30°C in light intensity of 76 lux for 10 days.
Mean length of 
Germ tubes (urn) 
from
Germination
medium
Storage 
Humidity 
(% R.H)
Total
Number of 
Conidia 
observed
Percentage
Germination
Apical
Cell
Basal
Cell
Without pollen 80 364 47.2 47.2- 53.8
60 342 68.3 55.3 51.3
40 331 80.2 68.2 73.6
20 600 76.6 68.6 63.5
0 488 51.8 49.4 51.2
With pollen 80 362 50.0 56.6 55.1
60 345 56.2 53.6 54.5
40 435 64.1 56.5 58.5
20 618 80.2 74.3 69.9
0 415 46.9 50.2 50.3
69
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TABLE 22
Pattern of formation of germ tubes by 50 randomly selected conidia of 
C.arachidicola germinated in distilled water and aqueous suspension of non- 
sterile pollen of Zea mays of density 500 -  600 pollen grains ml'1 in 24 hours 
at 30°C in light of 76 lux after storage at 0 -  80 % R H at 30°C in light of 76 lux
for 10 days.
Percentage of Conidia with germ tubes 
emerging 
from indicated cells
Germination
medium
Storage
Humidity
(%R.H)
Apical
and
Basal
Cell
Co-
jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical 
and/or 
Basal 
cell and 
Median 
Cell
Without 80 16 48 20 8 8
60 32 36 12 4 16
40 24 20 16 8 32
20 40 12 4 4 40
0 20 36 20 12 12
With pollen 80 12 40 24 12 12
60 20 28 20 16 16
40 32 24 12 8 24
20 52 16 12 4 16
0 16 44 16 8 16
70
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TABLE 23
Percentage of conidia of C.arachidicola able to germinate in Distilled 
Water and Aqueous Suspension of non-sterile Pollen of Zea mays of density 
of 500 -  600 pollen grains ml'1 in 24 hours at 30°C in light intensity of 76 lux 
after storage at 0 -  80% R.H. at 30°C in light intensity of 76 lux for 20 days.
Storage
Total 
Number of
Mean length of 
Germ tubes (urn) 
from
Germination
medium
Humidity 
(% R.H)
Conidia
observed
Percentage
Germination
Apical
Cell
Basal
Cell
Without pollen 80 397 30.5 46.8 41.9
60 398 58.2 53.8 54.5
40 432 63.6 59.9 56.5
20 384 71.3 70.5 71.0
0 396 46.2 40.0 51.3
With pollen 80 392 35.7 47.5 43.0
60 390 55.1 56.3 57.2
40 407 61.9 62.5 60.0
20 381 65.1 71.0 76.6
0 401 42.9 43.6 48.0
71
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TABLE 24
Pattern of formation of germ tubes by 50 randomly selected conidia of 
C.arachidicola germinated in distilled water and aqueous suspension of non- 
sterile pollen of Zea mays of density 500 -  600 pollen grains ml'1 in 24 hours 
at 30°C in light intensity of 76 lux after storage at 0 -  80% R.H at 30°C in light 
intensity of 76 lux for 20 days.
Percentage of Conidia with germ tubes 
emerging 
from indicated cells
Germination
medium
Storage
Humidity
(%R.H)
Apical
and
Basal
Cell
Co-
jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical 
and/or 
Basal 
cell and 
Median 
Cell
Without pollen 80 12 60 12 8 8
60 16 48 20 12 4
40 20 40 12 8 24
20 56 20 4 4 20
0 24 44 20 0 12
With pollen 80 16 60 12 8 4
60 24 32 16 4 20
40 20 40 24 4 12
20 44 20 4 0 32
0 24 28 16 4 28
72
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TABLE 25
Percentage of conidia of C.arachidicola able to germinate in Distilled 
Water and Aqueous Suspension of non-sterile pollen of Zea mays of density 
of 500 -  600 pollen grains m l1 in 24 hours at 30°C in light intensity of 76 lux 
after storage at 0 -  80% R.H. at 30°C in light intensity of 76 lux for 30 days.
Germination
medium
Storage 
Humidity 
(% R.H)
Total
Number of
Conidia
observed
Percentage
Germination
Mean length of 
Germ tubes (^m) 
from 
Apical Basal 
Cell Cell
Without pollen 80 415 34.7 45.2 43.1
60 432 37.3 50.3 51.4
40 442 45.0 52.8 51.2
20 370 53.2 62.0 60.5
0 419 34.1 37.6 33.7
With pollen 80 413 36.1 49.8 49.2
60 438 39.2 53.5 51.9
40 403 44.4 54.9 50.0
20 387 51.0 58.7 55.4
0 414 35.5 36.9 33.2
73
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TABLE 26
Pattern of formation of germ tubes by 50 randomly selected conidia of 
C.arachidicola germinated in distilled water and aqueous suspension of non- 
sterile pollen of Zea mays of density 500 -  600 pollen grains ml'1 in 24 hours 
at 30°C in light intensity of 76 lux after storage at 0 -  80 % R.H at 30°C in light 
intensity of 76 lux for 30 days.
Germination
medium
Storage
Humidity
(%R.H)
Percentage of Conidia with germ 
emerging 
from indicated cells
tubes
Apical
And
Basal
Cell
Co-
jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical 
and/or 
Basal 
cell and 
Median 
Cell
Without pollen 80 24 44 16 12 4
60 20 36 12 4 28
40 28 24 16 8 24
20 48 16 20 12 4
0 24 48 16 4 8
With pollen 80 12 52 28 0 8
60 20 32 24 4 20
40 24 40 20 0 16
20 40 24 16 8 12
0 16 56 24 4 0
74
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TABLE 27
Percentage of conidia of C.arachidicola able to germinate in Distilled 
Water and Aqueous Suspension of non-sterile Pollen of Zea mays of density 
of 500 -  600 pollen grains ml'1 in 24 hours at 30°C in light intensity of 76 lux 
after storage at 0 -  80% R.H. at 30°C in light intensity of 76 lux for 40 days.
Germination
medium
Storage 
Humidity 
(% R.H)
Total
Number of
Conidia
observed
Percentage
Germination
Mean length of 
Germ tubes (nm) 
from 
Apical Basal 
Cell Cell
Without pollen 80 408 15.0 41.3 39.8
60 450 17.1 44.5 41.7
40 417 18.4 45.8 44.4
20 405 20.9 53.0 50.8
0 410 21.2 48.0 47.0
With pollen 80 418 13.1 40.1 37.7
60 465 18.0 43.9 41.6
40 399 18.0 46.2 45.4
20 423 19.1 56.1 51.7
0 424 20.8 48.1 47.2
75
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TABLE 28
Pattern of formation of germ tubes by 50 randomly selected conidia of 
C.arachidicola germinated in distilled water and aqueous suspension of non- 
sterile pollen of Zea mays of density 500 -  600 pollen grains ml'1 in 24 hours 
at 30°C in light intensity of 76 lux after storage at 0 -  80 % R.H at 30°C in light 
intensity of 76 lux for 40 days.
Percentage of Conidia with germ tubes 
emerging 
from indicated cells
Germination
medium
Storage
Humidity
(%R.H)
Apical
And
Basal
Cell
Co-
jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical 
and/or 
Basal 
cell and 
Median 
Cell
Without pollen 80 20 48 8 4 20
60 28 36 16 4 16
40 36 40 8 0 16
20 48 28 12 0 12
0 24 56 4 0 16
With pollen 80 28 52 4 0 16
60 40 40 8 4 8
40 32 44 4 0 20
20 44 32 8 4 12
0 32 48 4 0 16
76
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TABLE 29
Percentage of conidia of C.arachidicola able to germinate in Distilled 
Water and Aqueous Suspension of non-sterile Pollen of Zea mays of density 
of 500 -  600 pollen grain m l1 in 24 hours at 30°C in light intensity of 76 lux 
after storage at 0 -  80% R.H. at 30°C in light intensity of 76 lux for 50 days.
Germination
medium
Storage 
Humidity 
(% R.H)
Total
Number of
Conidia
observed
Percentage
Germination
Mean length of Germ 
tube (urn) from 
Apical Basal 
Cell Cell
Without pollen 80 405 19.2 41.0 40.7
60 440 25.4 47.7 43.1
40 435 29.7 48.5 44.7
20 436 34.3 51.5 45.9
0 408 35.1 53.6 47.7
With pollen 80 430 17.3 42.2 39.0
60 423 23.8 46.4 43.7
40 435 26.4 48.0 43.3
20 420 32.3 53.8 46.6
0 434 33.7 52.4 48.6
77
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TABLE 30
Pattern of formation of germ tubes by 50 randomly selected conidia of
C.arachidicola germinated in distilled water and aqueous suspension of non- 
sterile pollen of Zea mays of density 500 -  600 pollen grains ml"1 in 24 hours 
at 30°C in light intensity of 76 lux after storage at 0 -  80 % R.H at 30°C in light 
intensity of 76 lux for 50 days.
Percentage of Conidia with germ tubes emerging 
from indicated cells
Germination
medium
Storage
Humidity
(%R.H)
Apical
And
Basal
Cell
Co-
jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical 
and/or 
Basal 
cell and 
Median 
Cell
Without 80 24 44 4 4 24
pollen
60 32 40 12 8 8
40 40 32 16 0 12
20 48 24 8 0 20
0 20 52 20 4 4
With pollen 80 28 48 4 0 20
60 36 40 8 4 12
40 36 28 20 8 8
20 56 20 12 0 12
0 28 60 8 0 4
78
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# 20
20 40 60
1% R.H. of Storage Chamber!
30 Days
i R.H. of Storage Chamber
Fig.26.: Percentage viability of conidia of Cercospora
arachidicola stored at 30°C in light of 76 lux for 
varying periods at different relative humidities.
79
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10 Days
20 Days
|  40 
|  30
I  20 
I  105 „
70 T 
60
50 f  
40 |  
30 I 
20 { 
10 
0 J- 
0
30 Days
40 Days
20 40 60
% R H of Storage Chamber
80
50 Days
20 40
%R  .H of Storage Chamber
—■—  pollen
60 80
• pollen
Fig.2b: Mean length of germ tubes produced by Apical cells
of germinating conidia of Cercospora arachidicola 
stored at 30°C in light of 76 lux for varying periods 
at different relative humidities
|o
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Fig.2c: Mean length of germ tubes produced by Basal cells
of germinating conidia of Cercospora arachidicola 
stored j»t 30°C in light of 76 lux for varying periods 
at different relative humidities
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Fig. 3a 
Percentage 
of conidia 
of C.arachidicola 
stored 
at different relative 
hum
idities 
for 10 
days 
and 
la 
germ
inated 
in 
distilled 
water at 30°C 
with 
maize 
pollen(500-600m
l'1) showing 
different patterns 
of germ 
1 
developm
ent recorded 
after 24hrs 
of 
U
cuW
Vic'o
% of Germinated Conidia
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germ
inated 
in 
distilled 
water at 30°C 
without maize 
pollen 
showing 
different patterns 
of germ 
tube 
developm
ent recorded 
after 24hrs 
of incubation.
University of Ghana                              http://ugspace.ug.edu.gh
□ 
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88
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□ 
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germ
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□ 
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E. Germination of C.arachidicola conidia on groundnut 
leaflets with normal and reduced phylloplane microflora 
population
(a) Germination on the surface of groundnut leaflets of the conidia of 
C.arachidicola in suspension drops of different pollen densities, 0, 500 
-  600, 1000 - 1200, 1500 -  1800 and 2000 -  2400 pollen grains ml'1 
in all five tests carried out showed germination ranging from 31.0 to 
52.9 per cent (Table 31). There was no recognised trend in relation to 
pollen density.
(b) Test of phytotoxicity of Hydrogen peroxide to the leaflets of 
groundnut identified the treatment, which could be tolerated by the 
leaflets as shown by the results tabulated in Table 32. The safe’ 
treatments were application of 20% H2O2 and 30% H2O2 for only one 
minute and 10% H20 2 for two minutes.
(c) When the ‘safe’ treatments were used to disinfect the surface of
groundnut leaflets, the population of the phylloplane bacteria (Table 33) 
and phylloplane fungi (Table 34) were reduced to different levels but 
were not totally removed (plates 1-4). The various treatments removed 
from 14.3 per cent (10% H2O2 for one minute) to 70.8 per cent (30%
H20 2 for one minute) of bacterial flora and from 46.1 per cent (10%
92
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H20 2 for one minute) to 87.5 per cent (10% H20 2 for two minutes and 
20% H20 2 for one minute) of the fungal population.
(d) The data in Table 35 showed that the partial removal of the 
phylloplane microflora slightly improved the germination of the 
conidia on the leaflets of groundnut. Without pollen, germination 
on the unsterilized and surface-sterilized leaflets was 50.7 and 
55.3 per cent, respectively and with pollen, the respective range of 
percentage germination was 37.4 -  48.7 and 42.5 -  51.8 percent. 
The pattern of germ tube development presented in Table 36 did 
not indicate any clear effect of surface sterilization of the leaflets.
93
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Germination of conidia of C.arachidicola in drops of aqueous suspension of 
non-sterile Zea mays pollen on the abaxial surface of freshly detached leaflets 
of groundnut lying on sterile moist filter paper at 30°C in light intensity of 76
lux in 24 hours.
TABLE 31
Test No.
Pollen density 
(No. ml"1)
Total No. of 
conidia observed
Percentage
Germination
1 0 492 52.4
500 -600 553 43.7
1000-1200 499 40.0
1500- 1800 576 50.5
2000 -  2400 413 49.2
2 0 486 50.2
500 -  600 462 38.1
1000- 1200 452 31.0
1500- 1800 402 42.3
2000 -  2400 438 44.7
3 0 390 41.5
500 -600 438 52.9
1000- 1200 422 30.3
1500-1800 488 44.2
2000 -  2400 408 33.3
4 0 418 36.2
500 -  600 392 34.2
1000-1200 488 37.0
1500- 1800 466 33.5
2000 -  2400 514 36.9
5 0 428 43.9
500 -  600 434 35.9
1000- 1200 470 37.2
1500- 1800 460 36.5
2000 -  2400 440 47.7
94
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TABLE 32
Reaction of leaflets of groundnut immersed for varying periods in 
Hydrogen peroxide (H20 2) solutions of different concentrations at 30°C.
Concentration of 
H20 2
(%)
Period of Immersion 
(minutes)
Bronzing of 
Leaves
10 1 -
2 -
4 +
8 +
20 1 -
2 +
4 +
8 +
30 1 -
2 +
4 +
8 +
95
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TABLE 33
Effectiveness of Hydrogen Peroxide (H202) solution as 
groundnut leaf surface sterilant against phylloplane bacteria
Test No. Concentration of 
H20 2 (%)
Period of 
immersion of 
leaflets in H20 2 
solution 
(minutes)
Mean No. of Bacterial colony 
-  forming Units (CFU) per me 
of leaf washing (to the nearest 
whole number)
1 10 1 148
10 2 125
20 1 100
30 1 70
Untreated leaflet - 240
2 10 1 120
10 2 80
20 1 73
30 1 53
Untreated leaflet - 140
3 10 1 187
10 2 124
20 1 123
30 1 95
Untreated leaflet - 226
96
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TABLE 34
Effectiveness of Hydrogen Peroxide (H202) solution as 
groundnut leaf surface sterilant against phylloplane fungi
Test No.
Concentration of 
H20 2 (%)
Period of 
immersion of 
leaflets in H20 2 
solution 
(minutes)
Mean No. of Bacterial colony 
-  forming Units (CFU) per me 
of leaf washing (to the nearest 
whole number)
1 10 1 11
10 2 6
20 1 8
30 1 6
Untreated leaflet - 48
2 10 1 16
10 2 8
20 1 5
30 1 4
Untreated leaflet - 29
3 10 1 7
10 2 4
20 1 3
30 1 3
Untreated leaflet - 13
97
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Germination of Conidia of C.arachidicola in aqueous suspension of non-sterile 
Zea mays pollen placed on abaxial surface of unsterilized and 30% H2O2 -  
treated surface-sterilized leaflets of groundnut at 30°C in light intensity of 76
lux in 24 hours.
TABLE 35
Pollen
Total
Number
of
Mean length of 
Germ tubes (^m) 
from
Leaf
Treatment
Density 
(No. ml"1)
Conidia
observed
Percentage
Germination
Apical
Cell
Basal
Cell
Unsterilized 0 350 50.7 56.3 53.7
500 -  600 420 48.3 54.6 55.3
1000-1200 330 37.5 48.7 45.2
1500- 1800 310 38.2 50.4 47.0
2000 -  2400 380 44.4 49.0 48 0
Surface-Sterilized 0 420 55.6 60.6 58.3
500 -  600 402 51.3 54.6 53.6
1000- 1200 380 49.5 55.0 50.4
1500- 1800 350 46.2 48.2 46.2
2000 -  2400 450 42.4 48.0 47.7
98
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TABLE 36
Pattern of formation of germ tubes by 50 randomly selected germinated 
conidia of C.arachidicola incubated in aqueous suspension of non-sterile Zea 
mays pollen placed on abaxial surface of unsterilized and H20 2 treated 
surface-sterilized leaflets of groundnut at 30°C in light intensity of 76 lux for 24
hours
Percentage of Conidia with germ tubes emerging 
from indicated cells
Leaf
Treatment
Pollen
Density
in
conidial 
suspension 
(No. ml"1)
Apical
and
Basal
Cell
Co-jointly
Apical
Cell
only
Basal
cell
only
Median
cell(s)
only
Apical 
and/or 
Basal cell 
and 
Median 
Cells
Unsterilized 0 24 30 16 14 16
500 -  600 30 30 20 6 14
1000- 1200 40 20 12 0 28
1500 -1800 36 24 10 14 16
2000 -  2400 44 28 8 8 12
Surface
Sterilized 0 34 26 16 10 14
500 -  600 40 24 18 6 12
1000- 1200 32 28 22 8 20
1500 -1800 48 36 10 0 6
2000 -  2400 36 30 14 10 10
99
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PLATE 1
PLATE 2:
Photograph showing residual phylloplane fungal Colony - Forming Units of 
washing of Non-sterile Groundnut leaflets on Potato Dextrose Agar after 
incubation for 5 days at 30°C (x 5/9)
Photograph showing residual phylloplane fungal Colony - Forming Units of | 
washing of Groundut leaflets previously sterilized with 10% H2Q2on 
Potato Dextrose Agar after incubation for 5 days at 30°C (x5/9)
loo
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PLATE 3:
Photograph showing residual phylloplane fungal Colony - Forming Units of 
washing of Groundut leaflets previously sterilized with 20% H20 2 on 
Potato Dextrose Agar after incubation for 5 days at 30°C (x5/9)
PLATE 4:
I Photograph showing residual phylloplane fungal Colony - Forming Units ol 
washing of Groundut leaflets previously sterilized with 30% H20 2 on 
Potato Dextrose Agar after incubation for 5 days at 30°C (x5/9)
I 1 0 1
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F. Influence of Non-sterile Pollen of Maize on the infection of 
groundnut leaflets by conidia of C.arachidicola.
The results of inoculation tests using non-sterilized and surface-sterilized 
groundnut leaflets and pollen grains at different densities are shown in Table
37. It was noteworthy that at the higher pollen densities of 4000 -  4500 and 
6000 -  7000 pollen grains ml'1 suspending medium, C.arachidicola did not 
infect the unsterilized leaflets. The first leaf spots appeared in 4 -  6 days after 
inoculation, and with one exception the rate of infection after 8 days ranged 
from 2/8, to 5/8. Fig. 4 shows the progress of infection and 
Plates 5 shows photographs of the inoculated leaves on the last days of 
incubation.
102
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TABLE 37
Infection of eight groundnut leaflets in each set previously surface-disinfected 
with different concentrations of H20 2 before inoculation with suspension of 
conidia of C.arachidicola containing pollen of Zea mays and incubated under 
continuous white fluorescent light of 76 lux at 30°C
Density of 
Pollen grains 
(ml'1)
Leaf disinfected 
with indicated 
H20 2
concentration
(%)
Time of appearance of 
first leaf spots after 
inoculation (Days) and 
number of leaflets 
infected in parenthesis
No of leaflets 
infected after 8 
days
0 0 4(1) 4
10 5(2) 4
20 5(2) 5
30 5(1) 6
1000-1200 0 5(1) 2
10 4(1) 4
20 4(2) 3
30 4(1) 5
2000 -  2400 0 6(4) 4
10 5(2) 4
20 4(1) 3
30 5(1) 5
4000 -  4500 0 - 0
10 6(3) 3
20 4(2) 4
30 4(3) 6
0
2
4
4
6000 -  7000 0
10
20
30
6 (2 ) 
6 (4) 
4(1)
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QJIO 13 
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Percentage infection
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CHAPTER FIVE 
GENERAL DISCUSSION
Garrett (1956) defined inoculum potential as ‘the energy o f growth of a 
parasite available for infection of a host at the surface of the host organs to be 
infected. This definition embraces both the infective capacities of the inoculum, 
that is, its total nutrient content and especially to its content of energy substrates, 
and the environmental conditions under which it is operating. The inoculum 
potential may, therefore, vary from a maximum value to zero, as environmental 
conditions vary from optimal to completely inhibitory. Given environmental 
conditions optimal for activity of the inoculum, the most important would be the 
carbon compounds available to the fungus as energy substrates, whether they 
were carbohydrate reserves in the fungus itself or supplied externally by the 
immediate environment of the inoculum.
It was speculated as far back as 1908 (Brooks, 1908) that the externally 
supplied energy substrate play critical role in infection by fungal parasites. 
Precision was lent to this speculation by the observation of Last (1960) on 
infection of leaves of Vicia fuba by spores of Botrytis fabae. Maximum 
percentage germination of spores in water was maintained as cultures of B. fabae 
from which the spores were taken, aged to 40 days, whereas Infectivity to bean 
leaves declined to 1/10th at 25 days and 1/100th at 35 days of the parent culture, 
Infectivity of the ageing spores could be restored by suspending them in orange 
juice which contained, in addition to proteins, minerals, vitam ins about 1.5% citric
109
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acid and 4.5% sucrose. Last (1960) had commented, ‘Ageing conidia o f B. fabra 
seem to contain adequate reserves for germ ination but insufficient to meet the 
demands for infection'.
It was presumed to begin with, that pollen of maize in mixed farms would provide 
external substrate for conidia o f Cercospora arachidicola  landing on leaves of 
groundnut for, carbohydrate content of pollen grains is usually high. It was found 
for example to be as high as 48.35 percent in pollen of Pinus con to rta jTodd and 
Brethrick, 1942), and that would have been the basis for the high stimulation of 
infection of plants by fungi in the presence of pollen. For instance, there was an 
increased infection o f strawberry by Botrytis clneria_on addition of strawberry 
pollen to suspension droplets from 14 percent to 93 percent (Chu-chou and 
Preece, 1961).The object o f the present study was to determ ine the effect of the 
pollen of maize on germ ination o f conidia of Cercospora arachid icola^ the major 
infectionjjn its and on infection o f groundnut.leaves by the conidia.
Teyegaga (1970) reported that the conidia of a local isolate of Cercospora 
canescens causing leafspot o f bambara bean (Vigna subterranea) germinated in 
water best at 25, 30 and 35° C attaining 100% germination in three hours at all 
three temperature. Because o f the absence o f sufficient numbers of incubators to 
provide a range o f temperatures, all experiments were carried out at 30°C 
Furthermore, the earliest germ inating conidia in distilled water produced germ 
tubes after 12 hours o f incubation and percentage germ ination was. therefore, 
assessed for all tests after 24 hours’ incubation.
no
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The high percentage germination o f 80.5 per cent in sterile distilled water 
might be due to the presence of a large amount of endogenous substrate in the 
conidia (See Table 6). The considerable growth of the germ tubes would 
contribute to successful infection of groundnut leaves. The conidia in the 
presence of sufficient moisture would not only successfully germ inate in the 
absence of nutrients in the infection court but would also produce extensive germ 
tubes that would also produce extensive germ tubes that would effect penetration 
of the host tissues.
In the absence of liquid water, the conidia would also germinate in air 
provided the relative humidity is sufficiently high. Percentage of the conidia 
germinated at 90, 95 and 100% RH was 64,0, 57.3 and 46.3% respectively (See 
Table 19). The germ tubes produced after 24 hours were almost as long as those 
formed by conidia germinating in distilled water. Even if the atmospheric humidity 
falls below 80% RH, it is very likely that a high enough level of humidity will be 
maintained at the transpiring leaf surface to support germination of the conidia. 
The pattern of germ tube formation in distilled water has been illustrated in fig 1. 
The conidia rarely produced germ tubes from the median cells only, while 
appreciable percentage of the conidia produced germ tubes either from apical 
cells only or from apical or basal cells together or from a median cell in addition 
to germ tube from the term inal ends. The difference between the capacities of 
the terminal cells and the median cell was indicative of a difference in the amount 
of endogenous nutrients in these cells. The behaviour of the conidia during 
germination shows that the cells of conidia of C. arachidicola  were independent
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of each other and adjacent cells were completely separated from each other. 
This feature is consistent with the nature of multicellular spores as established in 
observations with the electron microscope. Campbell (1970) showed that the 
pores in the separating walls of the multicellular conidia of Alternaria brassicola 
were effectively plugged, isolating one cell from the other. Thus every cell of the 
spore behaved independently of the others.
The pattern of germ tubes developments observed here is sim ilar to the 
observation of Woodroof (1933). He, however, stated that 'often several tubes 
emerged from a single conidium o f C arachidicola. That was, however, not so in 
the isolate studied during this work, because no spore formed more than four 
germ tubes. Woodroof (op cit) also reported that, with insufficient moisture the 
cells might swell w ithout developing germ tubes’. Cells of many conidia incubated 
in drops of sterile distilled water during this work did swell visibly without 
developing germ tubes. From the evidence of the present work, swelling of cells 
of conidia without the production of germ tube was not caused by insufficient 
moisture.
The greater capacity o f the terminal cells than median cells for 
germination seems to be a common feature of the conidia of Cercospora 
species. Berger and Hanson (1963) reported that germ tubes may arise from any 
cell of the conidia of Cercospora zebrina, but the basal cell usually germinated 
first, while Baxter (1956) had earlier observed that the basal cell o f conidia of C. 
zebrina germinated first followed by the apical cell. Teyegaga (1970) also found 
that the germ tubes of conidia of Cercospora canascens commonly emerged first
112
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from the basal cell, followed within a short time by production from the apical cell, 
and lastly from the median cells. But not more than three of the numerous 
median cells (up to 12 cells) produced germ tubes in distilled water Anyway, the 
terminal cells, as a general rule, germinate so readily and produced abundant 
apical infective growing apices that the poor development of germ tubes by the 
median cells would not limit infection of the host.
More median cells of conidia of C.canascens were stimulated to germinate 
by external nutrients by Teyegaga and Clerk (1972) thereby augmenting the 
infective capacity of the conidia. As many as eight median cells of the conidia 
germinating in aqueous suspension droplets on the leaflets of the host plant, 
Vigna subterranea produced germ tubes compared to three cells that did so in 
droplets on glass slides. An even greater stimulation of the median cells was 
produced in solutions of casein hydrolysate, fructose, glucose, maltose, peptone 
sucrose and yeast extract. Yeast extract proved to be the most stimulating of all. 
Such stimulation could not be obtained when the conidia of C. arachidicola were 
incubated in solutions of glucose and peptone, (See Tables 7,11 and 13), which 
could be ascribed to inherent characteristics of C.arachidicola. Conidia of C. 
arachidicola belong to the same category as the 4-celled conidia of Curvularia 
lunata. Asomaning (1975) reported that only the two outer cells produced germ 
tubes in distilled water and solutions of various nutrients, and the two median 
cells could not be induced to produce germ tubes by fructose, glucose, 
haemoglobin, peptone, sucrose and yeast extract.
113
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Many reports in the relevant literature (eg. Chu-chou and Preece, 1961), 
have indicated that pollen grains provide nutrients to fungal spores and pollen 
induces chemotropism in hyphae (eg. Oliver, 1978).It was expected that if pollen 
of maize would similarly affect the conidia of C. arachidicola  it would do so in two 
ways, by first improving percentage germination of the conidia, and secondly, 
encourage production of more germ tubes by the median cells. Neither of these 
occurred in the presence of both autoclaved and non-sterile pollen. To 
summarise the events:
a. Percentage germ ination was not improved
b. The median cells were not induced to produce more germ tubes.
c. Pollen did not rejuvenate aged conidia (See Tables 21,23 25,27 and 29) and
d. High pollen densities of 4,000-7,000 pollen grains per ml. of suspending 
medium markedly reduced percentage germination of the conidia (See Table 
8)
e. Germ tubes were not attracted by pollen grains in the suspension droplets
However, the conidia in pollen suspension germinated at pH3 - pH 10, 
whereas those in pollen -  free suspension germinated at pH 4.0 -  pH 9 (see 
Table 17) indicating stimulation by the pollen at the extreme pH's and yet, 
germination at optimum pH of pH 5.0 and 6.0 in the absence of pollen was far 
higher, 80.2 and 70.6 percent, respectively, than the corresponding 60.7 and 
59.1 percent germination in the presence maize pollen (see table 17). Be as it 
may, the extreme pH of 3.0 and 10.0 are unlikely to occur in the groundnut leave 
surface microhabitat and the stimulation by pollen of maize at these pH’s would
114
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be of no practical value. It could, therefore, be concluded that, generally, pollen 
of maize will not increase infection of groundnuts by C. arachidicola and could 
even reduced infection if there are heavy deposits of the pollen on the leaves.
This supposition is supported by the results of the germination tests on the 
surfaces of the leaflets of groundnuts tabulated in Table 31.In a series of 
experiments, the mean percentage germination of the conidia in pollen-free 
aqueous suspension drops was 44.8 per cent compared to the mean percentage 
germination of 40.9, 35.1,43.2 and 43.1 percent, respectively in suspension 
drops containing 500 - 600, 1000 -  2000, 1500 -  1800 and 2000-2400 pollen 
grains per ml.of suspension.
Subsequent growth of the germ tubes and penetration into the host tissue 
will depend on the interaction among the pollen, germ tubes and the phylloplane 
microflora. Table 37 shows that abundant pollen grains (4000 -  7000 per ml. of 
the suspending medium) on non- sterile leaflets apparently stimulated prolific 
growth of phyloplane of bacteria and fungi, which smothered the germ tubes and 
prevented infection. On partially surface-sterilized leaflets with consequent 
microfloral population (See Tables 33 and 34) the germinating conidia caused 
infection. The pollen per se did not prevent infection.
Infection of new plants raised in a new cropping season depends on the 
capacity of the spores to survive the preceeding non-cropping period. Very great 
differences have been found in the survival potential of different kinds of fungi 
spores. The viability of all spores decreases with time and the rate of loss of 
vigour is dependent on inherent characteristics of the spores and upon
115
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environmental conditions, especially, temperature, humidity and light (Gottlieb, 
1950;Cochrane, 1958). At any given relative humidity, increasing the temperature 
generally decreases the viability of the fungal spores, and lower temperatures 
above freezing favour longevity. The relationship between relative humidity and 
viability does not appear to be so simple.
Four major types have been reported. Several investigators (e.g. 
Anderson, Henry and Morgan, 1948; Maclaughlin and True, 1952) have found 
that lower relative humidity favours retention of viability because of reduced 
accumulation of toxic metabolites of the low rate of metabolism in the dehydrated 
spore. Goos and Tschirch (1962), on the other hand, reported that spores of 
Gloeosporium musarum  could not withstand desiccation and survived longest at 
higher humidities (60-80%R.H.) than at lower humidities (0-20%R.H). Rosen and 
Wartman (1940) and Naqvi and Good (1957), respectively, found that 
uredospores of crown rust of oats and conidia of Monilinia fructicola retained their 
viability for a longer time at mid-humidities. Teyegaga and Clerk (1972) also 
found mid-humidities to be more favourable for survival of Cercospora canascens 
conidia than lower (0%R.H.) and higher (80%R.H) humidities. A reverse 
response occurs in Aspergillus flavus, Metarrrhizium anisopliae and Rhizopus 
oryzae. Teitell (1958) reported that the viability of conidia of A. flavus was 
preserved longest at zero and 85%R.H. and lost quickest at 75%R.H.while Clerk 
and Madelin (1965) found that conidia of Metarrhizium anisopliae died quickest at 
45-55%R.H. and survived longest at 0-35%R H. and 65- 
95%R.H..Sporangiospores of Rhizopus oryzae also belong to this category
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(Akushie and Clerk, 1981). The true causes for the survival patterns of the third 
and fourth categories are yet to be determined.
In this investigation the longevity of C.arachidicola conidia at different 
relative humidities was studied but at a temperature of 30°C and under light of 76 
lux only. These temperature and light condition were adopted because local 
atmospheric temperatures throughout the year are close to 30°C and because 
the dispersing spores are normally wind-borne for long periods exposed to light, 
and it is under such conditions that the longevity of the conidia becomes relevant 
to disease initiation. For initial 30 days of storage the conidia of C. arachidicola 
survived at 20,40 and 60% R.H than at zero and 80% (See Tables 21, 23 and 
25). The high moisture content of the conidia at 80% probably supported faster 
metabolism with attendant accumulation of toxic metabolites leading to quicker 
death while desiccation at 0%R.H disrupted metabolic processes. As the storage 
period extended beyond 30 days, viability at the median humidities fell to levels 
close to that at 0% R.H. while conidia at 80% R.H. showed the least viability (See 
Tables 27 and 29). Probably, as storage time lengthened, the internal 
accumulated toxic compounds generated at 20, 40 and 60% R.H. caused rapid 
death reducing longevity to the level of those at 0% R.H. The storage period 
should be extended beyond 50 days in future investigations to establish further 
behaviour of the conidia at these humidities.
The results, however, indicate that after 50 days, a fair proportion of the 
conidia remained viable at the humidities, which the conidia were likely to 
encounter in the field. Since groundnut is primarily a savanna crop and cultivated
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almost throughout the year the conidia would readily survive the normal brief 
intervals between successive cropping periods.
The influence of pollen of maize on the germination of conidia of C. 
arachidicola and on rejuvenation of the aged conidia has been studied. 
Fortunately, pollen of maize is not likely to increase infection rate of groundnut by 
C. arachidicola. Closer spacing of the maize plants will encourage thicker pollen 
deposits on the groundnut plants and consequently depress germination of the 
conidia. This proposal can be adopted if future investigation shows that the 
maize pollen affects the conidia of Cercospora personata, the other groundnut 
leafspot fungus in the same way.
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CHAPTER SIX
SUMMARY
1 The conidia of Cercospora arachidicola  incubated at 30°C germinated in
water and at 85-100%R.H. in 24 hours.
2 Germination was better in liquid water than in saturated atmosphere,and
percentage germ ination fell with decrease in atmospheric humidity.
3 The earliest germinating conidia in distilled water produced germ tubes in
10-12 hours.
4 The germinating multicellular conidia produced germ tubes first from the
end cells and later, occasionally, from one or two median cells.
5 In distilled water a maximum of only two median cells in conidia of seven
cells produced germ tubes.
6 The majority of the conidia (68%) contained 4-5 cells each.
7 There were five types of germination:
i. Germ tubes from the apical cell only.
ii. germ tube from basal cell only.
iii A germ tube each from the apical cell and basal cell together
iv. Germ tube from a term inal cell or both terminal cells and a median cell
v. Germ tube from a median cell only
8 The types of germination occurred in different proportions, with the 
greatest proportion of conidia producing germ tubes from both the distal 
and basal cell, whereas very few conidia produced germ tubes from a 
median cell only.
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9 Germ tubes produced by the apical cell are slightly longer (measured after 
24 hours’ incubation) than those from the basal cell in germinating conidia 
producing germ tubes from both end cells.
10 The conidia germinated better in distilled water on glass slides than on 
Surface of groundnut leaflets.
11 Pollen grain of Zea mays decreased germination of the conidia.
12 Non-sterile pollen caused less reduction in percentage than sterile pollen.
13 The germ tubes of conidia germinating in suspension with non-sterile 
maize pollen were slightly shorter than those of conidia in suspension with 
sterile pollen.
14 Percentage germination in suspension with both non-sterile and sterile 
pollen decreased with increase in density of pollen from 500 to 7000 pollen 
grains per ml. of suspending medium.
15 The proportions of the different germination types in distilled water were 
Apical and Basal cells co-jointly> Apical cell only> Apical and / or Basal cell 
and Median cell> Basal cell only> Median cell(s) only.
16 This order was not altered by the presence of pollen of maize and by 
germinating the conidia on the surface of groundnut leaflets.
17 Buffer solutions altered this order, perhaps due to the constituents of the 
solutions,to: Apical and Basal cells co-jointly > Apical cell only > Basal cell 
only > Apical and/or Basal cell and Median = Median cell(s) only.
18 Maize pollen stimulated germination of the conidia at pH 3.0and pH 10.0.
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19 Exudates of maize pollen hardly altered the pH of the suspending distilled 
water. The pH’s of all suspensions containing 500-7000 pollen grains per 
ml. were between pH 6.0 and pH 7.0
20 Conidia stored for 50 days at 0, 20, 40, 60 and 80% R.H., survived to 
different degrees. The percentage viability after 50 days at the different
humidities when germinated in the absence of maize pollen was :
0% R.H.: 35.1per-cent germination.
20% R.H.: 34.3 per cent germination 
40% R.H.: 29 per cent germination 
60% R.H.: 25 per cent germination 
80% R.H.: 19.2 per cent germination 
and in the presence of maize pollen was :
0% R.H. 33.7 per cent germination 
20% R.H.: 32.3 per cent germination 
40% R.H.: 26.4 per cent germination 
60% R.H.: 23.8 per cent germination 
80% R.H.: 17.3 per cent germination
21 Maize pollen could not re-juvinate the aged conidia
22 Leaflets of groundnut could be safely partially disinfected with 30% H20 2
for 1 minute, 20% H20 2 for 1 minute and 10% H20 2 for 2 minutes.
23 Treatment with :
I. 30% H20 2for 1 minute removed 64.1 per cent of the bacterial flora 
20% H20 2for 1 minute removed 51.3 per cent of the bacterial flora
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