ISOLATION, IDENTIFICATION AND PATHOGENICITY 
OF FUNGAL PATHOGEN (S) CAUSING BLACK 
SIGATOKA DISEASE OF LOCAL PLANTAIN AND 
BANANA IN GHANA
BY
FRANCIS KODJO TSIGBEY BSC. (HONS) AGRIC
A THESIS SUBMITTED TO THE BOARD OF GRADUATE 
STUDIES, UNIVERSITY OF GHANA, LEGON IN 
PARTIAL FULFILMENT FOR THE DEGREE OF MASTER 
OF PHILOSOPHY IN CROP SCIENCE
MAJOR - PLANT PATHOLOGY
JULY 1996
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(x 352731
S 6 ~ 7 S
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DECLARATION
I hereby declare that, except for references to 
other people's work which have been duly cited, this work 
is the result of my own original research and that this 
Thesis has neither in whole nor part been presented for 
another degree elsewhere.
FRANCIS KODJO TSIGBEY (STUDENT)
DR. K.A. ODURO (MAJOR SUPERVISOR)
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DEDICATIONS
Dedicated to my mother and to my father (Post humous).
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ACKNOWLEDGEMENT S
I am grateful to Dr K.A. Oduro, my major supervisor, 
for his patience, confidence, encouragement and very- 
useful criticisms when things were not going through. In 
addition for all his provisions throughout this research. 
To him I owe much.
The Lecturers and technical staff of the Crop 
Science Department, University of Ghana, Legon for their 
immense help.
The Manager, Volta River Estates Limited for 
providing some funds for completing this research. To my 
parents, Bertha Afare and Tefe Tsigbey, I owe my life for 
their absolute devotion to bring me thus far. To my 
brothers Emmanuel and David, my sisters Amy, Dora and
others, I thank and love for their absolute devotion to 
me. To my beloved wife Peace Amoako, my love always 
belong for her encouragement and the peace she brought
And finally to the many friends both past and
present, especially Ralph Bam, whose friendships have 
enriched my life, each one playing a part in God's
overall plan and purpose.
To God Belongs all the Glory.
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A study was conducted at the Crop Science 
Department, University of Ghana, Legon, to isolate, 
identify and test pathogenecity of fungal isolates 
causing the reported black sigatoka disease of plantain 
in Ghana and compare the isolate with that isolated from 
local tall banana having leaf spots.
Field symptoms of plantain and banana were compared 
and found to be similar. Initial specks indicating 
infection were minute, reddish-brown and were conspicuous 
only on the abaxial leaf surfaces on both plants. Specks 
turned dark-brown and later dark-black with adjoining 
yellow margins. The dark black areas dried out and 
turned grey on both plants.
The fungi species were isolated from necrosed banana 
and plantain leaves by Ascospore Discharge Technique. 
Ascospores from necrosed banana and plantain leaves were 
hyaline, two-celled, with one cell larger than the other 
and measured 13.9 by 3-2um and 13.8um by 3.2um 
respectively. Ascospores of both isolates germinated in 
Sterile Distilled Water (SDW) after 1^/2 hours and on PDA 
and Carrot Leaf Decoction Agar (CDA) between 12-18 hours 
and in all cases by bipolar germ tubes. No sporulation 
was observed for both isolates in SDW. Colony growth of 
both isolates on PDA and CDA were extremely slow and 
mycelia compact, erumpent, appearing dome-shaped from 
side view, and with a hard rind on the surface.
ABSTRACT
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Sporulation was not observed on PDA for both
isolates,but profuse sporulation was observed on CDA six 
days after inoculation. Conidia in culture was
Cercospora-type, with thickened hilum. Conidia of the 
plantain and banana isolates on the average measured 
18 .lum by 3.6um and 78.4um by 3.'Sum, respectively.
Greenhouse inoculations revealed that the plantain 
isolate on plantain had an incubation period of 18 days,
whilst the banana isolate pathogenic on the local tall
banana also had an incubation period of 23 days. Symptoms 
in both cases were typical for black sigatoka and 
progressed to the mature-spot stage with grey centres.
Cross-infection studies in the greenhouse revealed 
that plantain isolate of M. fijiensis was pathogenic on 
local tall banana with incubation periods of 29 days, and 
which produced typical black Sigatoka symptoms until the 
mature spot stage. Plantain ascospore inoculation on 
local tall banana also produced black sigatoka symptoms 
with 21 days of incubation period, after which symptoms 
progressed to the mature spot stage.
On plantain, the banana isolate of M. fijiensis took 
26 days for specks to appear and symptoms which were 
typical for black sigatoka progressed to the mature-spot 
stage. Control plants in the greenhouse did not develop 
symptoms. Neither ascospores nor conidia were produced 
in the greenhouse on inoculated plants due to
unfavourable environmental factors thus rendering
reisolation of the fungus from diseased leaves
impossible.
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The study therefore established that black sigatoka 
disease in Ghana is found on both plantain and the local 
tall banana, with the causal agent being Mycosphaerella 
fijiensis Morelet.
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TABLE OF CONTENTS
TITLE PAGE ................................................i
DECLARATION ............................................ ii
DEDICATION ..............................................ii;i-
ACKNOWLEDGEMENTS ........................................ iv
ABSTRACT ................................................ v
TABLE OF CONTENTS ..................................... viii
LIST OF PLATE FIGURES ..................................xii
LIST OF TABLES .......................................... xv
LIST OF APPENDICES .....................................xvi
LIST OF ABBREVIATIONS ............................... xvii
CHAPTER ONE ...............................................1
INTRODUCTION...............................  1
CHAPRTER TWO ............................................. 5
REVIEW OF THE LITERATURE ................................. 5
2.1. OVERVIEW OF PLANTAIN AND BANANA DISEASES ........... 5
2.2. MYCOSPHAERELLA DISEASE OF PLANTAIN AND BANANA...... 6
2.3. ECONOMIC IMPORTANCE OF YELLOW AND BLACK SIGATOKA
DISEASES ............................................6
2.4. DIFFERENCES AND SIMILARITIES BETWEEN BLACK
SIGATOKA (BS) AND YELLOW SIGATOKA (YS)DISEASES ....8
2.5. SOME CHARACTERISTICS OF THE CERCOSPORA s p p ........ 15
2.6. SOME FEATURES OF MYCOSPHAERELLA FIJIENSIS CULTURES 17
2.7. EPIDEMIOLOGY OF BLACK SIGATOKA DISEASE ........... 19
PAGE
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2 8 HOST-PARASITE INTERACTION OF BLACK SIGATOKA
DISEASE .......................................... 21
2.9. CONTROL OF YELLOW AND BLACK SIGATOKA DISEASE ....24
CHAPTER THREE
MATERIALS AND METHODS ............................ 29
3.1. FIELD SYMPTOMATOLOGY AND COLLECTION OF
DISEASED LEAVES OF PLANTAIN AND BANANA ......... 2 9
3.2. ISOLATION TECHNIQUES TO OBTAIN THE CAUSAL AGENTS .29
3.2.1. SINGLE CONIDIA ISOLATION ........................30
3.2.2. INDUCED MYCELIAL GROWTH .........................31
3.2.3. ISOLATION FROM PLANTAIN (BRODI YUO) BY ASCOSPORE
DISCHARGE TECHNIQUE (ADT) ....................... 33
3.2.4. ASCOSPORE DISCHARGE TECHNIQUE FOR ISOLATION
FROM LOCAL TALL BANANA (LTB) ..................... 34
3.3. MICROMETRY ....................................... 36
3.4. PHOTOMICROGRAPHY ................................. 36
3.5. INOCULATIONS AND INCUBATION TECHNIQUES
IN THE GREENHOUSE ................................ 36
CHAPTER FOUR
RESULTS ..................................................43
4.1. FIELD SYMPTOMS
4.1.1. FIELD SYMPTOMS ON PLANTAIN .......................43
4.1.2. FIELD SYMPTOMS ON LOCAL TALL BANANA.............. 43
4.2. ISOLATION AND CULTURAL CHARACTERISTICS OF THE
PLANTAIN ISOLATE ............................... 43
4.2.1. SINGLE CONIDIA ISOLATION..........................43
4.2.2. INDUCED MYCELIAL GROWTH ON MEDIA ............... 45
PAGE
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4.2.3. ISOLATION BY ASCOSPORE DISCHARGE .................45
4.2.4. GERMINATION OF PLANTAIN ISOLATE FROM SPORE
DISCHARGE IN STERILE DISTILLED WATER ............46
4.2.5. GERMINATION OF PLANTAIN ISOLATE
FROM ASCOSPORE DISCHARGE ON (PDA) ..............58
4.2.6. CULTURAL CHARACTERISTICS OF PLANTAIN
ISOLATE ON (CDA) .............................. 58
4.3. ISOLATION AND CULTURAL CHARACTERISTICS OF THE
BANANA ISOLATE .................................. 66
4.3.1. SINGLE CONIDIA ISOLATION .......................66
4.3.2. INDUCED MYCELIAL GROWTH ..........................66
4.3.3. ISOLATION BY ASCOSPORE DISCHARGE ................ 71
4.3.4. GERMINATION OF THE BANANA ISOLATE IN SDW ....... 71
4.3.5. GERMINATION OF THE BANANA ISOLATE ON PDA ....... 71
4.3.6. CULTURAL CHARACTERISTICS OF THE BANANA
ISOLATE ON C D A ................................... 71
4.4. PATHOGENECITY STUDIES ............................79
4.4.1. PLANTAIN ISOLATE INOCULATED ON PLANTAIN ........ 79
4.4.2. BANANA ISOLATE INOCULATED ON LOCAL TALL BANANA ..87
4.5. CROSS - INFECTION STUDIES ....................... 94
4.5.1. PLANTAIN ISOLATE ON LTB ..........................94
4.5.2. ASCOSPORES FROM PLANTAIN INOCULATED ON LTB ...... 94
4.5.3. BANANA ISOLATE INOCULATED ON PLANTAIN .......... 104
4.6. EFFECT OF ENVIRONMENTAL FACTORS ON DISEASE
EXPRESSION ...................................... 110
4.7. CONTROLS ........................................110
PAGE
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4.8. DISCUSSION:
4.8.1. FIELD SYMPTOMS .................................. Ill
4.8.2. SINGLE CONIDIUM ISOLATION ...................... Ill
4.8.3. INDUCED MYCELIAL GROWTH ....................... 112
4.8.4. ISOLATIONS BY ASCOSPORE DISCHARGE FROM
INFECTED BANANA AND PLANTAIN LEAVES ............113
4.8.5. CULTURAL CHARACTERISTICS OF BANANA AND
PLANTAIN ISOLATES IN SDW AND ON PDA ............114
4.8.6. CULTURAL CHARACTERISTICS OF PLANTAIN AND
BANANA ISOLATES ON CDA .............  116
4.8.7. PATHOGENECITY STUDIES .......................... 118
4.8.8. CROSS-INFECTION STUDIES ........................ 119
4.8.9. CONTROL TREATMENTS .............................. 120
4.8.9B EFFECT OF ENVIRONMENT (R.H.) ON SYMPTOM
EXPRESSION ...................................... 121
RECOMMENDATIONS ........................................ 125
LITERATURE CITED ....................................... 126
APPENDICES ..............................................133
PAGE
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LIST OF FIGURES
f i g.1: Incubation of diseased plantain (A) and banana
(B) leaf discs under bell-jar to induce conidia 
production.......................................... 30
FIG.2: Infected leaves plated on media, to induce
mycelial growth..................................... 32
FIG.3: Infected leaves stuck on the lid of Petri dish
for ascospore ejection onto media................... 35
FIG. 4: Method used in taking photomicrographs............... 37
FIG. 5. L: Method used for the incubation of inoculated
plants in the greenhouse ........................... 39
FIG.6: Inoculated plantain left in the open greenhouse to 
investigate the effect of humidity on disease 
expression. Note the absence of disease symptoms 
on leaves........................................... 42
FIGS.7 - FIG.11: Field symptoms of black sigatoka
disease on plantain ............................. 46-50
FIGS.12-14: Field symptoms of black sigatoka disease on
local tall banana ............................... 51-53
FIG. 15: Conidiophore (Cd) and detached conidia (c) of
Cordana musae on plantain and banana leaves
infected with black Sigatoka in the field........... 54
FIG. 16 (A,B): Ascospores of_M. fijiensis from
plantain ........................................ 55-56
FIG. 17: Ascospores of Leptosphaeria spp (arrowed),
ejected together with ascospores of_M. fijiensis
both germinating in SDW after two hours of
ejection............................................ 57
FIG. 18: 14 Days old colonies (non-sporulating) of
plantain isolate of M. fijiensis on media .......... 59
FIG. 19: Sporulating (s) and non-sporulating (NS)
12-days old Colonies of plantain isolate of M. 
fijiensis........................................... 59
FIG. 20: Hyphae of M. fijiensis from plantain on CDA
after 48 hours. Culture from ascospores (x 500). ...60
FIG. 21: Hyphae of M. fijiensis from plantain showing
dumb-bell-shaped cells (DBSC), (x 500)............... 60
FIG. 22: Sporulating colonies of M. fijiensis on CDA. (A)
Plantain isolate and (B) Banana isolate ............ 62
PAGE
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FIG.
FIG.
FIG.
FIG.
FIG.
FIG.
FIG.
FIG.
FIG.
FIG.
FIG.
FIG.
FIG.
FIG.
FIG.
FIG.
23 (A,B): Conidiophore (cd) of M. fijiensis in
culture intermingled in hyphae (x 500)........... 63-64
24 (A,B): Conidia of plantain M. fijiensis in
culture. Note thickened hilum (H) arrowed.......... 67
25. Sections on conidia of M. fijiensis from which 
measurements were taken............................. 68
26. Conidia of M. fij iensis from plantain showing 
bulbous cell (arrowed). Similar observations in 
banana (x 500)...................................... 69
27 (A,B): 14 days old colonies of plantain isolate
of M. fij iensis on CDA. Note the very whitish 
nature of colonies due to conidial germination in 
culture (arrowed)................................... 70
28. Ascospores of M. fij iensis from local tall
banana (x 500) ..................................... 72
29 and 30: Non-sporulating 7 days old colonies 
(arrowed) of banana isolate of M. fij iensis 
(A) CDA and (B) P D A ................................ 73
31 and 32: Hyphae of M. fij iensis from LTB, on CDA
after 48 hours. Culture from ascospores..............76
33. Conidiophore (cd) of banana M. fij iensis in 
culture. Note conidial scar (CS) .................77
34. Conidiophore (cd) of banana M. fijiensis
bearing a young conidium (c) in cultue.............. 77
35. Conidia of banana M. fijiensis in culture.
Showing thickened hilum (H) .....................78
36: Different stages of black sigatoka symptoms 
on greenhouse-grown plantain leaves inoculated 
with conidia of M. fij iensis isolated from 
plantain......................................... 81-83
37: (A,B) Some stages of black sigatoka on
greenhouse-grown plantain inoculated with M. 
fijiensis conidia isolated from plantain.........84-85
38. Array of inoculated plantains showing different
stages of black sigatoka symptoms .................. 86
39 (A,B,C) Symptoms of black sigatoka on greenhouse- 
grown Local Tall Banana, inoculated with banana 
isolate of M. fijiensis ....................  88, 89, 90
40: (A,B,C,) Symptoms on green house-grown LTB,
inoculated with M. fij iensis isolate from Local 
Tall Banana.................................. 91,92,93
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FIG. 41 (A,B,): Black sigatoka symptoms on greenhouse
grown Local Tall Banana inoculated with plantain 
isolate of M. fijiensis......................... 96-97
FIG.
FIG.
FIG.
42 (A,B,C): Black sigatoka symptoms on greenhouse
grown Local Tall Banana inoculated with plantain 
isolate of M. fijiensis.....................  98, 99, 100
43 (A,B,C) Black sigatoka symptoms on greenhouse 
grown Local Tall Banana inoculated with 
ascospores of M. fijiensis ejected from
plantain...................................101,102,103
44 (A,B,C): Black sigatoka symptoms on greenhouse 
grown plantain inoculated with banana isolate of 
M. fijiensis..............................  105, 106, 107
FIG. 45. Mature-spot (Ms) of black sigatoka symptoms on 
greenhouse grown plantain inoculated with banana 
isolate of M. fijiensis............................ 108
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LIST OF TABLES
PAGE
TABLE 1: Time course of disease progression on 
inoculated plants in the greenhouse ..............  95
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1. Ascospore dimensions of plantain M. fijiensis
on C D A ............................................ 133
2. Ascopore dimensions of banana isolate of M. 
fijiensis on C D A .................................. 134
3. Conidia lengths for plantain isolate of M. fijiensis 
on C D A .............................................135
4. Conidia lengths of banana isolate of M. fijiensis
on C D A ............................................ 136
5. Conidia breadths (diameters) for the plantain
isolate of M. fijiensis on C D A .................... 137
6. Conidia breadths (diameters) for the banana isolate
of M. fijiensis on CDA............................ 138
7. Conidiophore dimensions of plantain isolate of M.
fijiensis on C D A .................................. 139
8. Conidiophore dimensions of banana isolate of M.
fij iensis on C D A .................................. 140
LIST OF APPENDICES
Appendix PAGE
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LIST OF ABBREVIATION
LTB LOCAL TALL BANANA
ADT ASCOSPORE DISCHARGE
TECHNIQUE
r .H. RELATIVE HUMIDITY
BS BLACK SIGATOKA
YS YELLOW SIGATOKA
SDW STERILE DISTILLED WATER
PDA POTATO DEXTROSE AGAR
CDA CARROT LEAF DECOCTION AGAR
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CHAPTER ONE
INTRODUCTION
Banana (Musa sapientum L.) and Plantain (Musa 
paradisiaca L.) are staple foods for millions of people 
throughout the developing world, with banana especially 
eaten in the developed world. For others also, they 
comprise a significant portion of their diet.
In Ghana, plantain is a major source of 
carbohydrate. Cooked unripe plantain is pounded into 
"fufu", a local dish, and eaten with soup, whilst both 
the ripe and unripe forms are cooked and eaten with stew. 
Ripe plantain is also fried and eaten with cooked bambara 
beans and cowpea with palm oil as a major delicacy. 
Other use of plantain in Ghana is shredding unripe 
plantain and frying into chips for consumption. On the 
other hand,a greater proportion of banana produced in the 
country is eaten ripe with roasted groundnut as a meal.
It has been estimated by the Ministry of Agriculture 
that, national plantain production between 1986 and 1990 
was about 5,206,615 metric tonnes (mt) (Ministry of 
Agriculture, PPMED 1990,) There is however no production 
estimates of banana in the country.
Besides serving as food for domestic consumption, 
the two crops can be developed with proper agronomic and 
disease management practices, to serve as foreign 
exchange earners for the country. In light of this,
attention has been drawn to the crops for their 
improvement to increase yields. Areas of improvement
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include selection and breeding for high yielding 
varieties, guick and efficient methods of propagation, 
selection for dwarf varieties that can resist lodging 
during strong winds and the efficient control of 
economically important diseases.
Banana and plantain, like any other biological 
entities, have several diseases and pests that attack 
them. Important among these are Bunchy Top, Mosaic, Moko 
(Bacterial wilt) and Panama diseases (Vascular wilt). 
Banana and plantain leaf spot disease (Sigatoka) caused 
by Mycosphaerella spp is one of the most important 
diseases of banana and plantain and and causes serious 
losses in yields in many commercial plantations (Wardlaw, 
1961).
The commonest form of the disease (yellow Sigatoka) 
is caused by Mycosphaerella musicola Leach with the 
conidial state Pseudocercospora musae Zimm (Deighton),
while the other form and the more devastating, black 
Sigatoka, is caused by Mycosphaerella fijiensis, Morelet
with the conidial state Paracercospora fijiensis, Morelet
(Deighton).
In Ghana, yellow Sigatoka disease on improved 
commercial banana was reported in 1958 (Wardlaw, 1972) 
and had since been of epidemic proportions. This resulted 
in Sigatoka epiphytotics on the Subin banana plantation 
that led to its total collapse. Until the 1970s, plantain 
in the West-African sub-region was regarded to be
virtually free from economically important diseases 
including Sigatoka. Later, black sigatoka disease was
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reported to have reached Ghana in the early nineteen 
eighties (Wilson and Buddenhagen, 1986) .
An outbreak of a plantain disease in Ghana 
necessitated a scientific enquiry. Subsequently, Oduro et 
al. (1991) in a survey reported a 62.5% national
incidence and observed that the new plantain disease in 
Ghana was fairly similar to some aspects of the detailed 
symptoms of black sigatoka as reported by other workers 
like Meredith and Lawrence (1970) .
On basis of the symptoms alone, Oduro et al. (1991) 
considered the plantain disease in Ghana to be black 
sigatoka and therefore recommended further studies to 
identify the causative organism.
Oduro et al. (1991) also noted and supported by 
pictures that the leaf spot disease on the local tall 
banana in Ghana was symptomatologically similar to that 
of the new plantain disease.
Cultivation of the Local Tall Banana • (LTB) in Ghana 
is not done on any commercial basis, but normally planted 
to serve as shade crop for newly established cocoa 
plantations, and the produce eaten ripe as dessert or the 
unripe fruit eaten cooked. The local tall banana is very 
hardy and even long after the abandonment of the cocoa 
farms, they still exist as isolated grooves along roads 
and in secondary forests.
These grooves of local tall banana severely diseased 
by sigatoka, could serve as inoculum reservoir for the 
sigatoka pathogen. In addition, dried diseased leaves 
could be efficient means of transporting infective
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propagules to disease-free areas since such materials are 
used for packaging and as head pad. With this information 
about the local tall banana, any attempt in controlling 
Black Sigatoka of plantain without any scientific study 
on the relationship between black sigatoka of plantain 
and the leaf disease of local tall banana could be 
negated.
The purpose of this study therefore was firstly to 
isolate, identify and test the pathogenicity of fungal 
pathogen(s) on plantain in Ghana and compare it or them 
with the isolate from the local tall banana in the 
country.
Secondarily to establish the true relationship 
between the new plantain disease and the neglected 
disease of local tall banana.
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CHAPTER TWO
REVIEW OF THE LITERATURE
2.1. OVERVIEW OF PLANTAIN AND BANANA DISEASES
Banana and plantain (Musa spp), like any other 
plants, have several diseases that attack them. Almost 
all the pathogens that infect one species also attack the 
other. The pattern of growth, especially leaf
arrangement, which creates dense canopies when the crop 
is planted over large tracts of land in association with 
the high rainfall required for growth provide a conducive 
environment for pathogenic fungus infection.
Banana and plantain diseases can be grouped into 
five major categories thus: leaf, pseudostem, root,
rhizome and fruit diseases. All these diseases are of 
economic importance depending on the causative organism, 
environmental conditions and adopted control measures.
Some economically important diseases, include viral 
diseases such as Bunchy Top disease, Roxana disease, 
infectious chlorosis,and mosaic disease. Bacterial 
diseases include Moko (Bacterial wilt), Bacterial Head 
rot, Nematode diseases include the Burrowing nematode 
infection while Fungal diseases include Panama Disease 
(Vascular wilt), Cordana Leaf spot, Leaf Speckle, Yellow 
and Black Sigatoka Disease (Wardlaw, 1972).
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2.2. MYCOSPHAERELLA DISEASES OF PLANTAIN AND BANANA
Mycosphaerella diseases of plantain and banana are
two of the most economically important leaf spot 
diseases. They are popularly called Yellow Sigatoka and 
Black leaf streak or black sigatoka depending on which 
particular Mycosphaerella sp is causing the disease. The 
commonest form in Ghana, Yellow sigatoka, is caused by 
Mycosphaerella musicola Leach, with the conidial state 
Pseudocercospora musae Zimm. (Deighton) (Cercospora 
musae Zimm).
The current predominant form of the disease in 
Ghana, Black Sigatoka/Black Leaf streak, is caused by 
Mycosphaerella fijiensis (Morelet), with the conidial 
state Paracercospora fijiensis Morelet (Deighton). So
far, the two species only attack members of the Musa 
genus.
2.3. ECONOMIC IMPORTANCE OF YELLOW AND BLACK SIGATOKA 
DISEASE
Combined attack of Yellow and Black Sigatoka 
diseases have caused great economic losses wherever they 
had appeared. They have been reported that within a few 
years, a flourishing banana industry may be destroyed and 
this led Wardlaw (1939) to describe it as a disease of 
first class importance. Apart from monetary expenditures, 
by virtue of the nature of the Sigatoka disease, if 
judicious control measures are not employed,
environmental pollution with sprayed chemicals can
result.
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The onset of Sigatoka disease has been a tragedy to 
both individual planters and whole agricultural regions. 
Motz and Mallory (1944), gave an account of how sigatoka 
disease led to the collapse of the banana industry in 
Mexico. They indicated that, the disease was observed 
first in 1937; by 1941 production had fallen from 525,000 
metric tonnes to 240,000 metric tonnes. Similarly, 
sigatoka outbreaks led to the callapse of the banana 
plantation at Subri in Ghana (Oduro personal 
communication). Further, Guyot and Cuille (1958) remarked 
that, of the 62 million bunches produced in 1954 in 
Ecuador, only 19 million were exported. This was due to 
failure to provide protective measures against Sigatoka 
on the small farms and holdings.
Wardlaw (1972) reported that after the earlier 
incubation period, the disease could destroy a farm in 
two years in the absence of adequate control. In Ghana, 
the outbreak of the black sigatoka disease on plantain 
had catastrophic effects on plantain production. The 
major cause is due to the fact that plantain production 
is done on small scale by peasant farmers, hence control 
measures are not adequately administered. Consequently 
output fell with its attendant high cost of the produce 
on the Ghanaian market. Mbwaga (1990) reported that black 
sigatoka was first reported in 1989 in Tanzania and 
between 1989-90, caused almost 100% infection.
Black sigatoka disease inflict both physical and 
physiological damages on banana and plantain. 
Physically, the disease is responsible for the
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destruction of the photosynthetic tissue of the plant,
culminating in the loss of the leaf due to leaf necrosis. 
Stover (1974) reported that heavy spotting give rise to 
early leaf senescence and that it is one of the first 
effects of sigatoka on the plant and can shorten leaf 
life by as much as 25 days. Heavy spotting resulting in
4-5 viable leaves at bunch initiation give rise to
lighter bunches, reduced fruit weight and increase field 
ripening (Stover 1974, Ramsey et al. 1990). The average 
age of the youngest leaf spotted is the most sensitive
indicator of danger from .premature ripening (Stover, 
1974). Merchan V (1989) observed that under severe attack 
of black Sigatoka, bunches mature prematurely with low 
weights and fingers do not fill out.
2.4. DIFFERENCES AND SIMILARITIES BETWEEN BLACK SIGATOKA 
(BS) AND YELLOW SIGATOKA (YS) DISEASES
Yellow sigatoka (YS) is caused by Mycosphaerella 
musicola Zimm and is characterised by a general necrosis 
of the leaves of both plantain and banana. The evident 
feature of the disease is the presence of a profussion of 
small discrete spots on the lamina of older leaves, with 
areas of scorched or brown leaf tissue where the spots 
are closely grouped together. The first indication of
infection of YS is the appearance of light yellow or 
brownish-green, indistinct linear marking l-2mm or more 
in length lying parallel to the veins. These streaks are 
normally scattered on the upper surface of the leaf. 
These streaks increase slightly in size, forming dark
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muddy-brown to black, linear to oblong, or elliptic areas 
up to 1cm in length and about one-third in breadth. These 
spots are usually seen on the fourth to sixth leaves. On 
younger plants, similar but longer and broader spots are 
formed which appear dark-brown, oval to almost about 1cm 
in diameter. The centre of the elliptical brown spots 
dries out to light greyish-buff colour, with a narrow, 
well-defined dark-brown margin. Several workers including 
Stahel (1937) reported that between the dark-brown margin 
and the normal green areas of the leaf, there is 
frequently a bright-yellow transitional zone.
Stahel (1937) gave a precise description on the 
nature and sequence of symptom development. He observed 
that the first symptoms, 15-17 days after inoculation 
with conidia are minute yellow-green speckles or streaks. 
Some 22-24 days after inoculation, these speckles become 
streaks 8-10mm long with the leaf having a slightly rusty 
appearance- From here the spots show some enlargement and 
development of brown necrotic colour. After the brown 
spot stage, the spots collapse and yield the 
characteristics pale grey buff sigatoka spot.
Stover and Fulton (1966) reported that the pattern 
of spots over the surface of the lamina is influenced by 
the type of spore causing the infection. Conidial 
infections show linear distributions whereas ascospore 
infections do not, but the spots are generally 
distributed over the leaf. Furthermore, typical ascospore 
symptoms consist of heavy concentration of infection 
towards the drooping leaf-tip.
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The asexual fruiting body of M musicola was 
described by Leach (1946) as sporodochium which was 
defined by Ainsworth (1961) as a mass of conidiophores 
tightly placed together upon a stroma or mass of hyphae. 
Other terms used include acervulus (Stahel 1937), others 
are fascicles (for the mass of conidiophores) and 
Stromata (for the underlying mass of hyphae).
Meredith and Lawrence (1970) observed that 
sporodochium of M. musicola was amphigenous on leaf
materials which they examined, with the proportion being
higher on the lower than on the upper surface.
Meredith and Lawrence (1970) described the conidia 
of M. musicola as being pale to very pale olivaceous,
smooth, straight or variously curved, occasionally 
undulate and almost perfectly cylindric to obclavato- 
cylindric. The tip of conidia was obtuse or subobtuse 
and the basal cell shortly attenuate to the unthickened 
truncate hilum. They further observed that conidia were 
1-9 septate, with septa being indistinct and only fairly 
visible in unstained mounts. There were no constrictions 
at the points of septation. Conidia in culture were
longer (up to 102 um, average 72um) and narrower (2-3.5um 
average 3.0um) than those found on leaves (Meredith and 
Lawrence 1970).
Despite the widespread presence of YS disease, 
Trujillo and Goto (1963) published an account of the 
occurence of a new banana leaf spot disease in the 
Hawaiian Islands. The symptoms were similar to those of 
YS except that the individual lesions first appeared as
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elongated light brown streaks and not as yellow streaks. 
Rhodes (1964) first described black sigatoka and 
considered it similar to Yellow Sigatoka, but stated that 
it was more severe, as symptoms appear on younger leaves 
and, subsequently, more damage is done to photosynthetic 
tissue. Leach (1964 a,b) stated that, the main difference 
in symptoms between Yellow Sigatoka and black sigatoka 
occurs in the earlier stages of spot development.
For yellow sigatoka, the streaks that characterise 
the initial stages of the disease are yellow and clearly 
visible on the upper leaf surface but not conspicuous on 
the underside, while the surface of leaves infected with 
black sigatoka or black leaf streak are clearly 
distinguishable on the lower leaf surface (Leach 1964). 
The streaks are light brown to reddish-brown, often with 
a purplish hue when large numbers of streaks occur close 
together. The streaks remain inconspicuous on the upper 
surface until the time when they become water soaked and 
turn black rapidly (Meredith and Lawrence 1969). The most 
striking feature of black leaf streak or black sigatoka 
disease is the ability to form an enormous mass of narrow 
streaks on banana leaves as soon as infection becomes 
severe.
For so many years the two pathogens cannot be 
distinguished in culture if conidia are not produced. 
However, recently Johanson (1992) developed a technique 
capable of differentiating the two pathogens in culture 
by a Polymerase Chain Reaction technique.
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Meredith and Lawrence (1969), identified six stages 
of black sigatoka on mature banana plants as follows:
1. Initial speck stage (2) First streak stage
3. Second streak stage (4) First spot stage
5. Second spot stage (6) Third or mature spot stage 
Similar trends were observed for yellow sigatoka on 
banana and plantain.
According to Meredith and Lawrence (1969) both 
yellow sigatoka and black sigatoka in certain territories 
are sometimes present together in the same plantation. 
However, in the Pacific and the lowland areas of Latin 
America and Africa, yellow sigatoka could no longer be 
found and may have been completely replaced by Black 
Sigatoka (Jones and Mourichon, 1993).
Although there are no marked morphological 
differences between the perithecia and ascospores of M. 
musicola and M. fij iensis (Natural, 1988) there are 
notable differences between the Cercospora-type conidial 
state. Ascospores of M. fij iensis and M. musicola can be 
distinguished on V-8 juice agar. Germ tubes of ascospores 
of M. fij iensis are characteristically curved whereas 
those of M. musicola are straight (Meredith and Lawrence 
1969) . The number of perithecia per unit area on black 
sigatoka lesions was considerably greater than those on 
Yellow Sigatoka (Leach 1964). The conidiophores of M. 
fij iensis emerge singly or in diverging fascicles of 28 
stalks from many stomata on the lower surface of the 
leaf. Few conidiophores emerge from stomata on the upper 
surface of the lesion. Conidiophores are pale to medium
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olivaceous brown, becoming slightly paler towards the
tip. They are straight to variously bent, often with 
several geniculations, very rarely branched, basal 
swelling up to 8um diameter. One or more medium sized
scars are present near the tip of the conidiophore,
either lying flat against the apex or side of the 
conidiophore, or on a slightly sloping shoulder (Meredith 
and Lawrence, 1969). Apart from the colour of the
conidiophores, none of the above characteristics are
applicable to those of M. musicola. Conidiophores of M. 
musicola are straight to mildly curved, without septa, 
only very rarely branched, smooth, not shouldered or
geniculate and without conidial scars. Conidiophores are 
mostly bottle-shaped with rounded or nearly truncate
apices (Meredith and Lawrence 1969).
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The major comparisons of the conidial states of 
M.musicola and M. fijiensis after Meredith and Lawrence
(1969) are given
Conidiophores
First appearance
Habit
Distribution
Morphology
Dimensions (UM) 
CONIDIA
Morphology
below:
M. musicola
First spot stage
In dense fascicles 
(sporodochia) on 
a dark brown or 
black stroma
Abundant on both
surfaces of lesion 
sometimes more 
freguent on the 
upper surface.
Almost straight, 
hyaline, mainly 
without septation 
geniculation or 
branching, spore 
scars absent.
5-25 x 2-3.5
Cylindric to 
obclavato- 
cylindric, 
straight or 
curved, pale 
to very pale 
olivaceous, 
0-6 septate, 
indistinct 
basal hilum
M. fijiensis
Initial specks or 
first-streak stage
Emerge singly or 
in small groups 
(2-8stalks), 
sporodochia and 
stromata absent.
Largely confined 
to the lower leaf 
surface of lesion.
Straight or 
sly bent, pale to 
medium brown, 0-5 
septate, often 
geniculate, rarely 
branched near base 
well-definded, 
slightly thickened 
spore-scars.
.5-62.5 x 4-7
Obclavate to 
cylindro - 
obclavate, 
straight or 
curved, hyaline 
to very pale 
olivaceous 1-10 
septate, distinct 
slightly thickened 
basal hilum.
Dimensions (UM) 10-81 x 2-6 
(mean 59 x 3)
30-132 x 2.5-5 
(mean 72.5 x 4)
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2 .5 SOME CHARACTERISTICS OF THE CERCOSPORA SPP
Cercospora Fres. is reported to be one of the 
largest and most heterogeneous genera of Hyphomycetes 
(Subdivision Deuteromycotina) , and nearly 3, 000 specific 
names of Cercospora have been mentioned in the world 
literature (Pons 1989). Sporulation of several Cercospora 
spp in culture is difficult and depends mainly on the 
type of media used. Nagel (1934) reported that 
considerable difficulty is encountered in obtaining 
conidial production in pure culture of Cercospora 
beticola Sacc., and intimated that several workers 
presented conflicting evidence regarding conidial 
production of other Cercospora spp. Nagel (1939) 
reported that Cercospora brunkii Ell. and Galiv., grew 
well on several media but that it did not readily produce 
conidia. Aderhold (1900), dealing with C. beticola,
observed that conidia was produced somewhat sparingly
over the entire mycelial mat but in greater numbers at 
the borders of the colonies when the organism was grown 
on sugar-beet-leaf medium. Typical conidia of C. 
cerasella were produced in pure cultures from conidia and 
ascospores of the fungus, however when conidia were
plated on potato-dextrose agar, corn-meal agar, bean- 
dextrose agar, synthetic nutrient agar or cherry-leaf- 
decoction agar to which were added fragments of diseased 
leaves as additional source of nutrients, no fruiting was 
observed although the fungus made a good vegetative
growth (Jenkins, 1930) . However, if tubes of these media 
on which the organism had been growing for a time were
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again sterilised and plated with conidia, conidia was
produced in culture.
Nagel (1934) stated that certain of the Cercospora 
spp he studied demonstrated some specificity of medium as 
determined by the amount of sporulation. There was 
greater vegetative growth and less sporulation in the 
case of Cercospora beticola on potato-dextrose agar, 
while the same organism on beet-leaf agar produced little 
vegetative growth and greater numbers of conidia, whereas 
C. cruenta, C. davisi, C. dubia, C. physalidis and C. 
setariae fruited abundantly on ordinary potato-dextrose 
agar (Nagel 1934). Kilpatrick and Johnson (1956) reported 
that previous attempts by some other workers to obtain 
sporulation of Cercospora kikuchii (T. Matsu and 
Tomoyasu) Gardner in artificial culture have been 
unsuccessful and Murakishi (1951) tried 15 different 
types of media but failed to obtain sporulation. Nagel 
(1934), as well as Nagel and Dietz (1932), C. beticola 
reported that mycelial transfer of C. beticola resulted 
in sterile hyphae, whereas spore transfer yielded 
colonies that produced abundant sporulation. Sporulation 
of C. nicotianae Ell and EV. was obtained on steamed 
tobacco leaf decotion mixed with 1.2 percent agar 
(Diachum and Valleau, 1941).
Kilpatrick and Johnson (1956) obtained abundant 
sporulation of twelve Cercospora spp including C. 
kikuchii grown on agar medium prepared from carrot leaves 
steamed without pressure for one hour. They observed that 
sporulation did not occur on the medium prepared from
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carrot leaves sterilized under pressure, and cultures 
exposed to daylight also sporulated more abundantly than 
those in dishes incubated in darkness. Darkness however 
did not suppress sporulation completely. Calpouzos (1954) 
reported that Meredith and Butler (1939), were the first 
to observe sporulation of C. musae in pure culture. 
These observations were repeated by Leach (1941), Dantas 
(1948), and Fernandez (1953). However in all these cases 
the production of conidia was either not abundant or not 
consistent enough to offer a reliable supply of spores.
The critical factors controlling sporulation in 
culture by C. musae are genetic and not environmental 
(Calpouzos 1954). Calpouzos (1954) further reported that 
the slow growing colonies of C. musae show a heterogenous 
morphology, as they consist of patchwork of sectors 
differing in colour and texture, with some sectors 
sporulating.
2.6. SOME FEATURES OF M. FIJIENSIS CULTURES
Natural (1988) reported that the size of M. fijiensis 
conidia isolated varied with the cultivar from which they 
were obtained. Colonies derived from single spores 
became visible 4-6 days after incubation on PDA under 
continuous light, with the colonies of M. fij iensis 
compact, raised, hemispherical with irregular zonation, 
non-circular margin and a velvety surface (Natural,
1988). Natural (1988) also reported that ascospores were 
hyaline, 2-celled with one cell larger than the other, 
with a slight constriction at the septum and that
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ascospore germination which was bipolar occured 24 hours 
after discharge on PDA.
Meredith and Lawrence (1969) attributed the dark 
centres of M. fij iensis colonies to the presence of dumb­
bell-shaped hyphal cells. While Stover (1976) indicated 
that the presence of these cells is considered as an 
indication of pathogenic Cercospora. Several workers have 
reported conidial production of M. fij iensis on PDA 
(Meredith and Lawrence 1969, Natural 1988). Natural
(1988) reported that sporulation of M. fij iensis occured 
on PDA under continuous light after 14-21 days of 
incubation at 22-25°C, though some isolates failed 
produce conidia.
Pigment production was reported on 
such as PDA, Coconut Glucose Yeast Extract agar (CGYEa), 
Czapek's Yeast Extract Agar (CzYEA) and Synthetic medium 
Glucose Yeast Extract agar (SymGYEA), but this 
disappeared with age (Natural 1988). Subsequently, 
Natural (1988) reported that pigment production was 
highly correlated with toxin production in vitro. Though 
Asante et al (1977) and Fajola (1978) reported non­
pigment producing isolates of Cercospora spp., Upadhyay 
et al. (1991) isolated Fijiensin, a phytotoxic metabolite 
from a culture of M. fij iensis and found it to be 
phytotoxic to various banana cultivars. However it was 
observed that fijiensin production could only be detected 
in liquid cultures after 21-24 days though symptoms 
appeared after 10-15 days, on field plants.
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2.7. EPIDEMIOLOGY OF BLACK SIGATOKA DISEASE
The infecting propagules for black sigatoka disease 
are conidia and ascospores. Conidia production is at its 
maximum when the brown lesions begin to turn into a dark- 
brown or black spot and decreased when the center of the 
spot begin to turn light gray (Stover 1970) . Sporulation 
occured on successive nights in the absence of rain 
provided dew was present (Stover, 1970), whilst conidia 
production was abundant as a result of long hours of leaf 
wetness. Stover (1970) reported no sporulation on leaves 
during nights when dew was not formed. Conidia are 
released by rain and dew and then disseminated by water 
(Stover, 1970), and in the absence of spray during the 
dry months, spotting can gradually build up as a result 
of conidial inoculum. The emergence of streaks at these 
times is associated with longer incubation periods.
Ascospores produced by perithecia are rain-dependent 
and are disseminated by wind (Stover, 1970) and rapidly 
move disease outward from primary foci of infection, 
whereas conidial infection is localised. Frequent rains 
produce both conidia and ascospores which contribute to 
a rapidly increasing epidemic disease that is difficult 
to control (Stover, 1970) . Conidia survive 3-4 weeks on 
leaf surface whereas ascospores take 8-14 weeks in 
perithecia.
Stover (1968) reported a seasonal trend in the 
production of perithecia and sporodochia in different 
countries and climates. Perithecia and sporodochia 
production is high during wet seasons and declines in the
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dry seasons. Preponderance of perithecia or sporodochia 
varied from country to country and depended on the 
rainfall pattern (Stover, 1968). Consequently, when the 
rainfall is high throughout the year, perithecia 
production predominates and when the rainfall is lower, 
sporodochium production is high. Stover (1964) indicated 
that since perithecia produces only one major crop of 
ascospores, as opposed to sporodochium which produces 
conidia intermittently, the contribution of conidia to 
total inoculum is greater than that of ascospores during 
most time of the year. However perithecia are five to 
ten times more numerous in spots developed from mass 
infections than in scattered single spots (Stover, 1964).
Merchan V (1989) reported that in Columbia the 
highest concentration of ascospores was registered in the 
rainy week, with 62,700 ascospores/m^ of air. Stover and 
Fulton (1966) confirmed the findings of Brun (1963) that 
ascospores were dispersed long distance in horizontally 
moving air currents and deposited on the furled heart 
leaf giving rise to a typical left laminal infection. The 
unfurling heart leaf, is an ideal target for ascospores 
and conidia and this make adequate protection by 
fungicide spray very difficult (Stover and Fulton, 1966). 
Inadequate protection is due to the almost perpendicular 
heart leaf, coupled with a constantly expanding cylinder 
exposing new susceptible tissue.
The critical time for banana to suffer repeated 
attacks of sigatoka was during the three to four weeks 
prior to shooting (Stover, 1974) and a physiologically
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active substance is believed to be produced and 
translocated from the diseased leaf tissue (Stover 1974). 
Stover (1970) reported the production of ethylene by 
sigatoka pathogen in diseased tissue, which might be 
responsible for the premature fruit ripening associated 
with sigatoka attacks.
Artificial infection of banana and plantain by 
Mycosphererella spp was achieved by spraying conidial 
suspension in water on plant leaves (Stahel, 1937; Goos 
and Tschirch, 1963) and typical sigatoka disease symptoms 
were produced.
The presence of other fungus fructifications 
including Leptosphaeria musarum Sacc., Hendersonia spp. 
at the dry centres of cercospora spots on banana was also 
reported (Simmonds 1933, Parham 1934, Stahel 1937).
2.8 HOST-PARASITE INTERACTION OF BLACK SIGATOKA DISEASE
The infecting propagules of M. fij iensis are 
ascospores and conidia, and the courts of infection on 
plantain and banana are the adaxial and abaxial leaf 
surfaces. By microscopic examination, it was observed 
that the abaxial leaf surface might be more susceptible 
to infection than the adaxial leaf surface due to the 
abundance of stomata and the lesser amount of wax on this 
surface. Conidia and ascospores germinate by germ tubes 
in a film of water on the leaf surface and penetration 
into the leaf tissue is by way of the stomata.
Goos and Tschirch (1963) reported that conidia 
germinated within 24-48 hours on inoculated leaves
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enclosed in plastic bags in the greenhouse. They further 
reported that germ tube development was more extensive 
after 3 further 24 hours exposure to mist, whilst no 
conidia germinated on inoculated leaves left in the open 
green house. Stomatal penetration was reported to begin 
4-6 days after inoculation on leaves covered with plastic 
bags and it was suggested that fluctuating humidities 
were more favourable for this process (Goos and Tscirch, 
1963) . Goos and Tschirch (1963) reported that in the 
greenhouse spot developed within 28-35 days after 
inoculation on banana, and speculated that, the rate of 
disease development appeared to be related to the 
moisture relations of the plant and was affected by 
watering regimes.
Salle' et al. (1989) reported that after penetration 
via stomata, hyphae run parallel to the leaf surface in 
the large open areas of the spongy mesophyll and branches 
form which make contact with host cell walls without the 
development of haustoria. Twenty-eight days after 
inoculating a susceptible cultivar Grande Naine (AAA 
genotype), there were intravacuslar accumulations of tiny 
spherical globules in parenchyma cells which stained 
heavily black with paragon (Salle' et al. 1989).
Vasquez et al. (1988) reported that ascospores 
germinated on water-agar with three and four germ tubes 
and that ascospores germinated on both adaxial and 
abaxial surfaces of leaves. Under greenhouse conditions, 
Vasquez et al (1988) reported that the first symptoms 
appeared 22 days after inoculation. Scanning Electron
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microscope (SEM) studies by Vasquez et al. (1988) 
revealed bacteria in association with ascospores coupled 
with an apparent maceration of ascospores and speculated 
a possible biological control mechanism for M. fij iensis.
The pattern of symptom development in greenhouse- 
grown plants differ somewhat from that in the field, in 
that, infection occurs on all leaves in the greenhouse as 
greenhouse grown plants develop thinner wax deposits than 
field grown plants (Freeman and Turner, 1985).
Differences exist in the susceptibility of 
different Musa spp to both M. fij iensis and M. musicola. 
Plant characteristics which give them resistance to 
diseases may be morphological or biochemical, acting 
directly or indirectly on the parasite (Goodman et al. 
1986). The components of resistance may be present in the 
host before it comes into contact with the pathogenic 
agent (toxic compounds present before infection). In many 
cases however resistance mechanisms are induced in the 
host cell at contact with the pathogen (elicitation of 
phytoalexins) (Goodman et al. 1986).
Mourichon et al. (1989) reported that no correlation 
existed between duration of incubation and cultivar 
susceptibility to black sigatoka, but that the duration 
of the period of development of the symptoms is a perfect 
reflection of the degree of susceptibility. The ratio of 
necrotic guard cells to the number of penetration sites 
is a good indicator of the rate at which the host cells 
react in response to direct contact with the parasite 
(Mourichon et al. 1989). Mourichon et al (1989) further
23
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reported that preformed toxic substances exist in the 
leaves of banana plants to M. fijiensis. Salle et al
(1989) speculated that resistance in some highly 
resistant Musa might be due to a hypersensitive reaction 
resulting in the death of the fungus and reported that in 
a highly resistant cultivar "Yangambi" (AAA genotype), 
very small necrotic zones were formed with hyphae being 
very scarce in the tissues. Salle et al. (1989) published 
the findings of Jayapal and Mahadevan (1968) that a 
correlation exist between the polyphenolic content of 
healthy tissues and their resistance level against M. 
musicola and Helminthosporium gibberosporium on Musa.
Vasquez et al. (1988) suggested a relationship
between the stomatic density of a particular cultivar and 
its degree of resistance to M. fij iensis. The number of 
stomata per unit area decrease with an increase degree of 
ploidy and the decrease seems to be a function of the B- 
genome. (Vasquez et al. 1988). Laville (1983) observed 
that the presence of a single B genome rarely gives 
varieties higher resistance to M. fij iensis and that 
resistance seems to intensify only with genome ABB.
2.9 CONTROL OF YELLOW AND BLACK SIGATOKA DISEASE
In general, disease control is of utmost importance 
if crop productivity is to be increased and this can be 
achieved by any of the following methods: Cultural,
chemical, genetic or resistance control, or the 
combination of any of these methods depending on the 
crop, the environment and the availability of other
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logistics. For black and yellow sigatoka diseases, 
control attempts make use of any of the methods above and 
their combinations.
Chemical control by use of fungicides seems more 
effective. However, it is associated with certain 
potential environmental hazards due to spray chemicals. 
By the nature of the crop growth pattern and disease 
cycle, sigatoka disease control calls for frequent 
spraying of fungicides and most often on large 
plantations, by the use of helicopters which will 
subsequently increase spray drift. By any means, the 
early detection of the initial infection symptom of the 
disease is of utmost importance in disease control. 
Wieldmaker (1988) suggested the use of four strategies in 
control as follows:
1. monitoring the epidemiology of the disease through
adapted early warning disease survey,
2. the use of the systemic fungicide propiconazole
integrated with other mode-of-action fungicides,
3. low volume application techniques, and
4. change from overhead to under-canopy irrigation.
Stover (1989) gave a good account of how petroleum 
oil controls banana/plantain leaf spots. He stated that 
oil improves the spreading and sticking properties of all 
fungicides as well as enhancing the penetration of 
systemic fungicides and it has a fungistatic effect on
25
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the pathogen inside the leaf. Oil is effective whether 
applied as an oil in water emulsion or alone. However, a 
problem associated with oil sprays is their phytotoxicity 
effect which is characterised by extensive leaf bronzing.
Fungicidal control of banana and plantain leaf spot 
disease is currently the most widespread form of 
control.Protectant fungicides, such as Bordeaux mixture, 
were mostly used between 1936-1962 (Stover 1989), and 
they were found to be incapable of providing effective 
coverage for the ever increasing leaf surface against 
ascospores and conidia impingement. Control was basically 
by reducing conidial production in the leaf spot. Other 
protectant fungicides used include Dithane M-22 (maneb, 
Bravo 500, and Dithane flowable (Stover 1989). The 
systemic fungicides that were used first in Black 
Sigatoka control included Benomyl (Benlate) and 
Tridemorph (Calixin). The systemics were found to be 
superior because they penetrated the leaf and they acted 
on the pathogen long before it could sporulate (Stover
1989) . Other systemic fungicides in use include the 
triazoles - Propiconazole (Tilt), and Flusilazole 
(Punch), Stover (1989).
Eswaramurthy et al. (1989) reported that insertion 
of Bavistin (Carbendazim) 60-70mg, or aureofungin (90mg) 
capsules into the rhizome of banana gave good control of 
both Fusarium oxysporum f.sp. cubense and Mycosphaerella 
musicola. However, one major setback in the use of 
systemic fungicides is the appearance of resistant 
strains of the pathogen. Perez-Vicente et al. (1985)
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reported the appearance of benomyl-resistant race of IVL_ 
musicola in Cuba. Furthermore, there was cross-tolerance 
to thiophanate-methyl and carbendazim.
Fungicide resistance was manifest by normal 
ascospore discharge from leaves treated with benomyl and 
normal germination of conidia from benomyl treated areas, 
with subsequent colonies sporulating.
Genetic/resistance control of Black and Yellow 
Sigatoka diseases include the selection of resistant 
varieties through tissue culture and subsequent in vitro 
testing, using toxins or culture filtrates of M. 
fij iensis and breeding bananas and plantains resistant to 
black sigatoka. Maize tissues were found to be resistant 
to culture filtrate of Helminthosporium maydis race T. 
(Gengenback et al. (1977) quoted by Leopoivre and Acuna 
(1988). In a similar vein, Leopoivre and Acuna (1988) 
found a correlation between in vivo and in vitro 
phytoalexin production for M. fijiensis.
One major setback in banana/plantain breeding is the 
production of infertile seeds. Rowe and Rosales (1989) 
reported that several sub-species of wild Musa acuminata 
have high levels of resistance to black sigatoka and have 
been used in crosses. They found that the diploid 
hybrid, SH-3437 has an outstanding resistance to black 
sigatoka and can ,therefore, be used as a parental line 
in various phases of breeding.
Cultural methods of sigatoka disease control can be 
achieved by crop hygiene. Basically, it involves
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removal and burning of infected leaves before they show 
mature spot symptoms.
Emebiri and Obiefuna (1992) reported that leaf removal 
and/or intercropping with a fast growing multi-branched 
cassava cultivar reduced disease incidence by 16% and 
severity by 10%. Total crop destruction and strict 
quarantine restrictions can also be used to control black 
sigatoka disease (Vidal, 1992).
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CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Field Symtomatology and Collection of Diseased Leaves 
of Plantain and Banana
Symtoins of black sigatoka on both plantain and 
banana in the field were observed anytime samples were 
being taken. Both adaxial (upper surface) and abaxial 
(under surface) leaf surfaces were observed and disease 
stages noted. Photographs were then taken of infected 
leaves of both plantain and banana.
Diseased leaves collected for fungus isolation were 
those showing specks, streaks and scorching with grey 
centres, which were found at the leaf tip and margins. 
The scorched margin was cut-off from the other infected 
portions and placed in damp polythene bags to hasten 
perithecia maturation for ascospore discharge.
3.2 Isolation Techniques to Obtain the Causal Agents
Three methods were attempted in order to obtain the 
suspected pathogen Myscosphaerella fij iensis Morelet 
(Deighton), from diseased leaves of both banana and 
plantain:
(i) Single conidium isolation by Spore Pick Technique 
from diseased leaf surface.
(ii) Induced mycelial growth on media from first-streak 
stage diseased leaf discs.
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(iii) Ascospore Discharge Technique (ADT) from diseased 
leaves.
3.2.1 Single Conidium Isolation
Plantain and banana leaves showing various stages of 
black Sigatoka disease symptoms collected from the field 
were mounted under stereoscopic microscope and observed. 
With the aid of sterile mounting pins dipped in Sterile 
Distilled Water (SDW), spore picking was done.
In another instance, diseased leaves were washed in 
SDW to rid off surface contaminants and cut into small 
discs, which were then mounted on the inside of bell-jars 
containing a beaker of water to induce humid conditions 
(Fig. 1) . Microscopic examinations were made on the leaf 
discs after four days to scan for conidia.
Fig.l Incubation of diseased plantain (A)
and banana (B) leaf discs under bell- 
jar to induce conidia production.
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3.2.2 Induced Mycelial Growth
Fresh leaves of plantain and Local Tall Banana 
(LTB), showing first streak symptoms (reddish-brown 
streaks appearing on abaxial leaf surface) of black 
sigatoka were cut into small discs (5mm2), surface 
sterilised in 1% sodium hypochlorite (NAOCL) solution for 
one minute and then rinsed in SDW. The sterile leaf discs 
were then blotted dry on sterile Whatman No. 1 filter
paper. The leaf discs were then plated on the surface of 
Carrot Leaf Decoction Agar (CDA) and Potato Dextrose Agar 
(PDA).
The CDA was prepared from 300g of finely chopped 
fresh carrot leaves, 12g agar and 1 litre distilled 
water. 300g of fresh carrot leaves were washed under tap 
water and then finely blended in a good chopper. The
blended leaves was then mixed with 500ml distilled water 
in a conical flask. The material was then steamed for one 
hour without pressure and then strained through two
layers of calico and cotton wool. The strained decotion 
was then sterilised by filtering through sterile sintered 
glass filter (Gallen Kamp Sintered Glass 3,24/29). The 
sterile decoction was then added to 500ml distilled water 
containing 12g of disslolved agar antoclaved at 121°c at 
151b/in pressure for 15 minutes. The medium was
thoroughly mixed and poured into sterile petri dishes 
under aseptic conditions. The pH of the medium was found 
to be 7.3.This method of medium preparation was adopted 
after Kilpatrick and Howard (1956) .
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The Potato Dextrose Agar was prepared by weighing 
39g of dehydrated PDA (Manufactured by MERCK, Germany) 
and dissolving it in 1 litre of distilled water by gently 
warming and constantly stirring with a magnetic rod. When 
the media was fully dissolved, the Stirrer was removed 
and the media autoclaved for 15 minutes at 121°C at 15 
lb/in pressure, under aseptic conditions. The pH of the 
media was found to be 6.5.
The plated leaves on media (Fig.2) were incubated 
under room temperature. Plates were observed under the 
microscope for the growth of intercellular hyphae from 
the cut edges of the plated leaf discs.
Fig.2. Infected leaves plated on media, to 
induce mycelial growth.
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3.2.3 Isolation From Plantain (Brodi Yuo) By Ascospore 
Discharge Technique (APT)
Diseased .plantain leaves showing stage six or the 
mature spot stage of black Sigatoka disease, which was 
evident as necrosed leaf sections showing grey centres, 
were taken from "Brodi Yuo" in the field (A.R.S. Kade). 
The diseased leaves were incubated in polythene bags 
under moist conditions for three (3) days to hasten the 
maturation of perithecia. The diseased leaves were cut 
into 4cm^ discs and dried under the sun for two (2) days, 
and later taken to the laboratory to be washed under 
several changes of tap water in order to get rid of 
surface contaminants. The washed leaves were then further 
sterilised in 10% NaOCI solution for 3 minutes and rinsed 
in Sterile Distilled Water (SDW). The disinfected leaves 
were then immersed in SDW for 20 minutes, and then 
stapled onto a Whatman No.l filter paper.
The stapled leaf discs were placed in the lids of 
Petri dishes with the base dishes containing SDW, PDA and 
CDA. Fig. 3. The Petri dishes were then covered with the 
upper surface of the diseased leaf facing the media in 
the dish. This isolation method was adopted after 
Meredith and Lawrence (1969).
The plates were observed after 15 minutes under the 
microscope for the discharge of ascospores. It was found 
that a lot of ascospores were ejected onto the media. 
Ascospore discharge increased with time and after an hour 
the filter paper with the stapled leaf disc was removed 
and the plate incubated at room temparature (2 6°c) under
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continuous light. The light source was provided by 
40watts incandescent bulb, placed 32.5cm above the 
plates.
3.2.4. Ascospore Discharge Technique For Isolation From 
Local Tall Banana (LTB)
Since the Ascopore Discharge technique was found to 
be more reliable than the other methods described 
earlier, it was adopted for the isolation from the Local 
Tall Banana. Diseased banana leaves used were those 
showing stage six or the mature spot stage of black 
Sigatoka, which is evident as necrosed leaf portions with 
grey centres. The method of isolation and subsequent 
treatments were the same as those described for plantain 
above Fig. 3.
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3  &
A 6
Fig.3. Infected leaves stuck on the lid 
Petri dish for ascospore ejection 
onto media.
A - Infected plantain leaves 
B - Infected banana leaves 
Note: Viewing from above.
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3.3. Micrometry
Length and breadth of ascospores, conidia, and 
conidiophores were measured by means of an ocular 
micrometer mounted on the eyepiece of a binocular 
compound microscope which was standardized by using a 
substage micrometer. Assistance was received from Dr. 
K.A. Oduro during the process of standardization.
3.4 Photomicrography
Photomicrographs of fungal spores and other 
structures were taken by means of a camera mounted above 
a compound microscope (Fig.4). Help was sought from the 
technical staff during the loading of the film and the 
initial stages of the photography.
3.5 Inoculations and Incubation Techniques in The 
Greenhouse
Plantain and banana suckers were thoroughly washed 
in water and the old roots peeled off, in order to get 
rid of other root pathogens and surface contaminants. The 
suckers were planted in garden soils in plastic buckets. 
Watering was immediately done and subsequently three (3) 
times per week. However, during the dry season, watering 
was done on every other day.
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Fig.4. Method used in taking 
photomicrographs.
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Except in one instance where ascospores ejected from 
diseased plantain leaves were sprayed on leaves of LTB 
plants, all other inoculations were done with conidial 
inoculum produced from cultures of Mycosphaerella 
fij iensis grown on CDA produced from ADT as described 
earlier.
Inoculum was produced by macerating sporulating 
cultures of M. fij iensis grown on CDA, in SDW on a 
microscope slide.
Inoculation onto plantain and banana leaves was done 
by first rubbing the abaxial surface of the first fully 
expanded leaf of the plant at three-leaf stage with a dry 
sterile cotton wool to remove wax from the leaf surface.
Subsequently, the conidial suspension with mycelial 
fragments on the slide was gently pressed on the abaxial 
right-half of the leaf surface.
The inoculum was further evenly spread on the leaf 
with the fore-finger. The whole plant was then 
immediately enclosed in a transparent polythene bag as 
shown in Fig.5, containing a beaker of water to induce 
humid conditions. The humid condition was maintained 
continuously for the first eight (8) days and then 
removed in order to ensure conidia germination and 
penetration into leaf. Subsequently the polythene bag was 
replaced every other day until initial specks (first 
disease symptoms) were observed on the inoculated leaves.
Inoculations of the plants were done on leaf number 
one when the plants produced three (3) leaves on the
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average. This incubation method was adopted after Goos 
and Tschirch (1963).
Fig. 5. L: Method used for the incubation 
of inoculated plants in the 
greenhouse.
R: Uninoculated plants to detect 
presence of air spora.
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The inoculations were done to determine the 
following:
(a) pathogenecity of the plantain isolate from 
"Brodi Yuo" cultivar on plantain,
(b) pathogenecity of Local Tall Banana isolate on 
banana of the same variety,
(c) the effect of the plantain isolate on LTB.
(d) the effect of the banana isolate on plantain.
In one case, ascospores (instead of conidia) from 
diseased plantain were used to inoculate local tall 
banana. Diseased plantain leaves were wetted and plated 
over SDW for ascospores ejection into the liquid. When 
the presence of ascospores in SDW was confirmed under the 
microscope, the ascospore suspension was then sprayed an 
the banana leaves and incubated as described above.
Other experiments were set up to determine the 
effect of humidity on inoculation and disease expression, 
and they consisted inoculated but unincubated plantains 
and bananas that were not incubated under polythene bags 
(Fig.6).
In addition, uninoculated plantain and bananas were 
left in the open greenhouse in order to investigate the 
presence or absence of inoculum in the open greenhouse 
(Fig.5,R) .
Control treatments consisted of all uninoculated but 
incubated leaves. These were the other halves of all 
inoculated leaves that were incubated.
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Data collected during the greenhouse studies were as 
follows:
(i) Disease progression on plants:
I
(a) Time taken for initial specks to appear 
(incubation period).
(b) Time take for specks to reach the mature spot
stage (stage six), from the date of appearance 
of initial specks.
(c) Disease cycle (i.e. time taken from the day of
inoculation until mature spots developed with 
grey centres).
(ii) Visual recording of disease symptoms on plants by 
means of photographs.
Environmental conditions in the greenhouse were as 
follows:
Temperature, 24°C - 36.5°c.
R.H. in open greenhouse, 35%-87%
R.H in incubation chamber 94%-100%.
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Fig.6. Inoculated plantain left in the open 
greenhouse to investigate the effect 
of humidity on disease expression.
Note the absence of disease symptoms on 
leaves.
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CHAPTER FOUR 
RESULTS
4.1 FIELD SYMPTOMS
4.1.1 Field Symptoms on Plantain
From a distance, leaves o f  plantain appeared scorched as i f  fire had razed 
through the field (Fig. 7). On close observation it was found that symptoms were 
mostly concentrated along leaf margins and were first found on the mature leaves 
(Figs. 7 and 8). Other leaves showed reddish-brown streaks that were longer and 
broader with some water-soaked early in the mornings. Mature lesions which were 
constantly found at leaf margins appeared dark black with yellow halo-margins (Fig. 8 
and 9). Similar symptoms were found on young suckers (Fig. 7). Heavily spotted 
plants with very few or no green leaf tissues showed very poor bunch development 
(Fig. 10), whereas those w ith some green leaf tissues had some appreciable bunch 
development (Fig. 11).
4.1.2 Field Symptoms on Local Tall Banana (LTB).
Patterns o f  field symptoms followed essentially those on plantain. Infected 
grooves o f  LTB appeared blighted mainly at the leaf margins (Fig. 12) from afar. 
Closer observation revealed several stages o f  disease development on infected leaf 
strips (Fig. 13). The spots are broad especially at the leaf margins due to coalescence 
and appeared grey, surrounded by yellow halo-margins (Fig. 12). Heavily spotted 
plants with fewer green leaf tissues produced very poor bunches (fig. 14). Young 
suckers were similarly diseased as was found on plantain.
4.2 Isolation and Cultural Characteristics of the Plantain isolate
4.2.1 Single Conidium Isolation
Microscopic examination o f plantain leaves from the field showing typical 
black Sigatoka symptoms also revealed abundant conidiophores and conidia o f other 
fungi such as Cordana musae (Fig. 15,) and Chloridium  spp on the leaf surface.
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Typical conidiophores and conidia o f the suspected pathogen Mycosphaerella 
fijiensis  were observed. Conidia o f M. fijiensis  were intermingled among the abundant 
spores o f  Cordana and Chloridium, thus making spore picking very impossible.
Whereas Cordana was mostly found at the centres and margins o f  necrosed 
leaf tissues, Chloridium  was commonly found on portions o f  healthy green tissues 
showing reddish-brown speckles.
Isolation from leaf discs incubated under bell jars was not possible due to the 
slow growth o f  the pathogen from the leaf, which was outgrown by other 
contaminating fungi.
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4.2.2 Induced Mycelial Growth on Media
No growth o f mycelia o f M. fijiensis  was recorded on PDA and CDA from the 
cut edges o f leaf discs. Though growth o f  other contaminants believed to be present 
on the leaf surface were observed. Attempt to increase the period o f disinfection of 
leaf discs in NaOcl resulted in the scorching o f  the cut edges o f  the leaf discs.
4.2.3 Isolation by Ascospore Discharge
Ascospores were discharged into SDW, onto CDA and PDA fifteen (15) 
minutes after plating infected leaf.
Ascospores appeared hyaline to olivaceous green, two-celled, one cell o f 
which is larger than the other with a Septum dividing both cells, but not well-defined 
and only clearly visible in stain (Fig. 16a,b). Ascospores stained deep blue in 
lactophenol cotton blue and measured 10.7-16.6 um (Avg. 13.8um) by 2.4-3.8um 
diam. (Avg. 3.2um).
In addition to the ascospores o f M. fijiensis, spores o f  another fungus believed 
to be those o f  Leptosphaeria  spp were also ejected (Fig. 17). These spores germinated 
faster, after 30 minutes, with multiple hyphae than those o f  M. fijiensis.
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4.2.4 Germination of Plantain Isolate from Spore 
Discharge in SDW
Ascospore germination began after 1V2 hours in SDW and after 2 hours, germ 
tubes measured 0.7-9.0 um (avg. 4.3um). Germination was bipolar but frequently 
arising first from the top o f the smaller cell o f the ascospore.
No germ tubes arose from the sides o f  the cells o f  the ascospore and not more 
than two germ tubes were produced by an ascospore. Branching from the germ tubes 
were very seldom even after a week o f growth. Septation o f  germ tubes began over a 
period o f 12 hours. Germ tubes were very slender, less than 3.0um in diameter (Fig. 
16b) as compared to those on CDA measuring 3.45um.
Fig. 7: Heavily scorched leaf margins showing mature spots, 
with a young sucker(s) showing similar symptoms.
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Fig. 8: Black spots w ith halo-margins at leaf tips arrowed.
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A closer view o f black sigatoka symptoms on infected 
leaf strips. Note the very dark black spot (B) and grey- 
centred spot (G) and streaks (s).
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Fig. 10: Heavily spotted plant (arrowed s) with poorly developed 
bunch (arrowed b). Note the absence o f  healthy green 
leaves.
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Fig. 11: Infected plantain with scorched leaves showing
appreciable bunch development. Note the presence o f 
healthy green leaves.
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Fig. 12: Diseased banana grooves with heavily spotted leaf 
margins arrowed.
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Fig. 13: Closer view  o f disease on infected leaf strips.
Note the streak(s), black spots (b) and mature spots with grey 
centres (g).
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Fig. 14: Heavily spotted banana without viable leaves with a 
poorly developed bunch (arrows).
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\ SL
Fig. 15. Conidiophore (Cd) and detached conidia (c) o f  Cordana 
musae on plantain and banana leaves infected with 
black Sigatoka in the field, (x 1000).
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V ,
%
(x ioeo )
FIG. 16A: Ascospores o f  M. fijiensis  from plantain (x 500, x 
1000)
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FIG. 16B: Germinating ascospores o f  M  fijiensis  from Plantain 
in SDW after 12 hours, (x 500)
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J.
FIG. 17: Ascospores o f  Leptosphaeria spp (arrowed L), ejected 
together with ascospores o f  M. fijiensis  (arrowed_M) 
both germinating in SDW after two hours o f  ejection.
Note the longer and multiple germ tubes arising from 
Leptosphaeria  spores untypical o f  M. fijiensis  (x 500)
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4.2.5. GERMINATION OF PLANTAIN ISOLATE FROM ASCOSPORE 
DISCHARGE ON PDA, PH 6.5
Ascospore germination occured after 15 hours on media and was bipolar. 
Whitish fluffy colonies were rarely visible on the fourth day. After six days, colonies 
appeard whitish on the surface with the undersurface dark. Immediately below the 
superficial white hyphae was a hard rind. Colonies measured 1.5mm in diameter after 
ten days and still appeared whitish (Fig. 18a). Sporulation was not observed on PDA 
after four weeks and thereafter on cultures incubated under continuous light at 26°C. 
However, all other structures found on CDA were present on PDA. NOn-sporulating 
cultures transfered from CDA onto PDA did not sporulate but transfer o f  colonies 
from PDA to CDA sporulated.
4.2.6. CULTURAL CHARACTERISTICS OF PLANTAIN ISOLATE ON 
CDA, PH 7.3, TEMPERATURE OF INCUBATION 26<>C.
Ascospore germination began after 12 hours on CDA and became pronounced 
after 18 hours, with germ tubes measuring 8.6-38.Oum (avg. 25.6 um) by 3.5 um diam. 
Germ tubes were robust and the tips began swelling after 23 hours, a sign o f dumb­
bell-shaped cell development. Ocassionally, additional germ tubes arose from either or 
both cells o f  the ascospore, frequently from the tips o f  the cells after 18 hours. A 
maximum o f four germ tubes as opposed to two in SDW, emerged from an ascospore 
on CDA. After 48 hours, the germinated ascospores developed dumb-bell-shaped 
cells with numerous hyphal branches (Fig. 20).
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FIG. 18: 14 Days old colonies (non-sporulating) o f  plantain 
isolate o f  M. fijiensis on
A - PDA
B - CDA
Note whitish nature o f  colonies (arrowed)
FIG. 19: Sporulating (s) and non-sporulating (NS) 12-days old 
Colonies o f plantain isolate o f  M. fijiensis.
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FIG. 20: Hyphae o f M. fijiensis  from plantain on CDA after 48 
hours. Culture from ascospores (x 500).
Note the beginning o f dumb-bell-shaped cells 
development (arrows).
P«Sc
s .  
/'*
#» f
f*r
FIG. 21: Hyphae o f M. fijiensis from plantain showing dumb­
bell-shaped cells (DBSC), (x 500).
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Colonies resulting from ascospores germination o f the plantain isolate on CDA 
were first rarely visible to the naked eye on media only after four days o f  incubation 
under continuous light. Colonies appeared whitish and fluffy on the surface o f the 
media. The superficial fluffy hyphae appeared hyaline under the microscope and 
stained deep blue in lactophenol cotton blue and were highly branched (Fig. 21). 
After five days o f  incubation, the undersurface o f  the colonies became black, and 
hyphae in this portion appeared dark brown, with the cells dumb-bell-shaped, (Fig. 
21). Dumb-bell-shaped cells measured 3.45 um diam. Both hyaline and dumb-bell­
shaped cells are septate and frequently branched (Fig. 20, 21). The initially hyaline 
hyphae gradually changed to olivaceous brown with time and it was such light brown 
hyphae that gave rise to the conidiophores. Conidiophores generally arose from the 
very end o f a hyphal cell segement as i f  from the septum separating the adjoining 
hyphal cells (Fig. 23a,b). Conidiophores were light brown but became paler towards 
the tip and showing conidial scars, (Fig. 23b). Conidiophores are septate (1-13) but 
frequently 3 and measured 24.2-163.9um (avg. 54.7um) by 3.1-5.2 um (avg. 3.8um).
Conidial production was observed on the seventh day o f  incubation and 
became profuse on the ninth day. Consequently, the colonies appeared light grey 
(Figs. 19 and 22).
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t f f
A &
 J
FIG. 22: Sporulating colonies o f M. fijiensis  on CDA.
A - Plantain isolate 
B - Banana isolate
Note: Dark colour of colonies due to reflections from the 
undersurface of the colonies which were originally 
black
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9  %**
^  c<  V  ■'
v - \  V  X
-  Ajy
V>»-*T‘|
FIG. 23 A: Conidiophore (cd) o f  M. fijiensis  in culture intermingled in hyphae 
(x 1000).
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FIG. 23B Conidiophore (cd) o f plantain M. fijiensis  in culture (x 
500, x 1000)
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Entire colonies sporulated unlike in other reported cases that sporulation was 
only at the fringes o f  the colony. Cercospora-type conidia were formed (Fig. 24a, b). 
Conidia were very pale greenish or olivaceous, obclavate to cylindro-obclavate, 1-12- 
septate (commonly 3-8-septate), straight or slightly curved (Fig. 24a,b) w ith a short 
obconically truncate base having a visible and slightly thickened hilum 1.7-3.5um 
diameter (mean 2.5um), ocassionally bulbous tip but commonly obtuse tip 1.7-3.5um 
diameter (mean 2.4um), 31.1-158.7um long (mean 78.1um), 3.5-4.5um diameter 
(mean 3.6um) at the broadest point near the base, (Fig. 25). In older cultures, some 
cells o f  the conidia became bulbous (Fig. 26). The above conidial description and 
observation in culture agreed well with that o f  Meredith and Lawrence (1969) for 
Mycosphaerella fijiensis, the causative organism o f black sigatoka disease on banana 
in Hawaii.
Thus, based on these morphological characteristics, Mycosphaerella fijiensis  
was suspected. W ith time, sporulating colonies developed very whitish superficial 
hyphae as a result o f  conidial germination in culture (Fig. 27a,b). Below these white 
hyphae was found a very tough rind within which were embedded the dumb-bell­
shaped cells which were responsible for the dark appearance o f  the undersurface o f  the 
colonies. Microscopic examination o f  structures within this portion o f  the culture 
revealed round perithecia-like bodies, the walls o f  which were dark-brown with 
pseudoparenchymatous cells.
Crushing o f  these bodies revealed hyaline, rectangular-shaped cells believed to 
be spermatia which stained deep-blue in lactophenol cotton-blue. In general, growth o f 
the fungus in culture was very slow, colonies measured only 4.5mm on CDA after 
nine days o f  incubation, and 1.9cm in diameter after six weeks. Colonies were 
erumpent after one week and appeared dome-shaped when viewed from the side, with 
irregular zonation.
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4.3. ISOLATION AND CULTURAL CHARACTERISTICS OF THE 
BANANA ISOLATE
4.3.1. SINGLE CONIDIUM ISOLATION
Microscopic examination o f  diseased banana leaves revealed pathogens 
similar to those found on infected plantain (Fig. 15). Isolation was not possible due to 
the same difficulties encountered in the case o f  the plantain isolate.
4.3.2. INDUCED MYCELIAL GROWTH
Isolation was impossible due to the slow growth o f the fungus and other 
difficulties similar to those encountered in the case o f  isolation from plantain.
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FIG. 24A. Conidia o f  plantain M. fijiensis  in culture. Note 
thickened hilum (H) arrowed, (x 500).
FIG. 24B. Conidia o f plantain M. fijiensis  in culture. Note 
thickened hilum (H), arrowed, (x 1000).
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<b(Ko^£.i\ Poi*»7
FIG. 25. Sections on conidia o f  M. fijiensis  from which 
measurement were taken.
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(xt 9 0  o )
FIG. 26. Conidia o f  M. fijiensis  from plantain showing bulbous 
cell (arrowed). Similar observations in banana (x 500, x 
1000).
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FIG. 27A. 14 days old colonies o f  plantain isolate o f  M. fijiensis 
on CDA. Note the very whitish nature o f  colonies due 
to conidial germination in culture (arrowed).
FIG. 27B. 24 days old colonies (arrowed) o f  plantain isolate o f 
M. fijiensis  on CDA whitish nature o f colonies as a 
result o f germinated conidia in culture.
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4.3.3. ISOLATION BY ASCOSPORE DISCHARGE
Ascospores were ejected into SDW, onto PDA and CDA 15-20 minutes after 
plating o f infected leaf. Ascospores appeared hyaline to olivaceous green, two-celled, 
one o f  which was larger than the other, with a septum which was not well-marked. 
Septum was however clearly visible in stain. Ascospores stained deep-blue in 
lactophenol cotton blue and measured 10.8-16.7um (avg. 13.9um) by 2.5-3.9um (avg. 
3.2) (Fig. 28).
4.3.4. GERMINATION OF BANANA ISOLATE IN SDW
Ascospore germination, growth, hyphal septation and other characteristics 
were similar to those reported for the plantain isolate (Fig. 16b).
4.3.5. GERMINATION OF THE BANANA ISOLATE ON PDA
Ascospore germination and other cultural characteristics such as growth o f 
colony, colour and other structures were similar to those observed for the plantain 
isolate (Fig. 29 and 30). However, no sporulation occured on media, neither were 
conidiophores formed.
4.3.6. CULTURAL CHARACTERISTICS OF THE BANANA ISOLATE 
ON CDA, PH 7.3, TEMPERATURE OF INCUBATION 26°C.
Ascospore germination began after 12 hours on CDA and became pronounced 
after 18 hours, with germ tubes measuring 8.6-38.2um (avg. 25.6um) by 3.45um. 
Germ tubes in culture were robust and the tips began swelling after 24 hours as a sign 
o f  dumb-bell-shaped cell development.
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FIG. 29. Non-sporulating 7 days old colonies (arrowed) of 
banana isolate o f M. fijiensis on 
A  -CDA  
B -PDA
FIG. 30. 14 days old colonies o f banana isolate o f M. fijiensis  on 
PDA. Note whitish nature o f  colonies (arrowed)
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Additional germ tubes emerged from either or both cells o f the germinating 
ascospores after 18 hours and frequently from the tip o f  the cells. Maximum o f four 
germ tubes emerged from an ascospore on CDA.
Colonies resulting from ascospores on CDA media were first rarely visible to 
the unaided eye as fluffy white colonies only after four days o f  incubation under 
continuous light. Hyphae from the fluffy colonies were hyaline under the microscope, 
stained deep blue in lactophenol cotton blue, septate and were highly branched (Fig. 
32).
After 5 days o f  incubation, the under surface o f  the colonies appeared very 
dark black and hyphae in this portion were dark-brown, with the cells dumb-bell­
shaped (Fig. 32) measuring 3.45um in diameter. Both hyaline and dumb-bell-shaped 
cells were septate and frequently branched, (Fig. 31 and 32). The intitial hyaline 
hyphae o f  5-day old colonies gradually changed olivaceous brown with time and it 
was from such light brown hyphae that conidiophores arose. Conidiophores arose 
from the end o f  a hyphal cell very near to the septum o f the adjoining cell (Fig. 33).
Conidiophore were septate, 1-12, but frequently three (3) and measured 24.2- 
163.9 um  (avg. 54.9um) by 3.2-5.4um (avg. 3.8um) diameter. Conidiophores were 
light brown but became paler towards the tip and showing conidial scars, they were 
straight to slightly bent especially at the tip (Figs. 33,34). Colonies o f  the isolate from 
banana measured 4.5mm on CDA after nine days and 1.9cm in diameter after six 
weeks on media. Colonies were erumpent and appeared dome-shaped when viewed 
from the side.
Conidial production was observed on the sixth day o f  incubation and became 
profuse on the eighth day, with the colonies appearing light grey (Fig. 22b). Whole 
colonies sporulated as in the case o f the plantain isolate. Cercospora-type conidia 
were formed in culture similar to those found in the isolate from plantain.
Conidia were pale greenish or olivaceous, but not quite colourless, obclavate 
to cylindro-obclavate, 1-13-septate (commonly 3-8-septate), straight or mildy curved, 
with a short obconically truncate base having a visible and slightly thickened hilum
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1.7-3.5um diam. (mean 2.6um), ocassionally bulbous tip or cells o f  the conidia (Fig. 
35). Commonly obtuse tip 1.6-3.5um diam. (mean 2.6um), 31.0-158.9um long (mean 
78.4um), 3.5-4.5um diam. (mean 3.6um) at the broadest point near the base, (Fig. 25.). 
Sporulating colonies developed very whitish superficial hyphae with time as a 
consequence o f  conidia germination in culture (Figs. 29,30). Below these white mass 
o f  hyphae was a very hard rind within which were embedded the dumb-bell-shaped 
hyphal cells and spherical perithecia-like bodies, the walls o f  which were dark-brown 
with pseudoparechymatous cells.
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FIG. 31. Hyphae o f  M. fijiensis  from LTB showing dumb-bell- 
shaped cells (DBSC) arrowed, and hyaline hyphae (HH) 
(x 500)
FIG. 32. Hyphae o f  M. fijiensis from LTB, on CDA after 48 
hours. Culture from ascospores. (x 500).
Note the beginning o f dumb-bell-shaped cells development 
(arrowed).
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FIG. 33. Conidiophore (cd) o f  banana M. fijiensis  in culture. 
Note conidial scar (CS) (x 500, x 1000).
{
*
FIG. 34. Conidiophore (cd) o f banana M. fijiensis  bearing a 
young conidium (c) in cultue.(x 500).
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FIG. 35. Conidia of banana M. fijiensis  in culture. Note 
thickened hilum (H) arrowed, (x 500, x 1000).
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Crushing these colonies revealed hyaline, rectangular shaped cells believed to 
be spermatia. The dark-brown dumb-bell hyphae in association with the round 
perithecia-like bodies are responsible for the very dark appearance o f the undersurface 
o f  the fungal colony.
The cultural characteristics o f the fungal colony, in addition to the appearance 
o f the conidia produced in culture, were similar to those o f  Mycosphaerella fijiensis  
described for the isolate from plantain. Based on these observations, M. fijiensis  was 
suspected and consequently identified.
4.4. PATHOGENECITY STUDIES
4.4.1. PLANTAIN ISOLATE INOCULATED ON PLANTAIN
After 12-22 days (mean 18 days) o f  inoculating leaves, faint, minute reddish- 
brown specks, rarely visible to the naked eye were observed on the abaxial (under) 
leaf surface (Fig. 36A). W ithin days, the specks elongated and became clearly visible 
on the lower leaf surface than on the adaxial (upper surface) with their long axes 
parallel to the leaf veins. These specks referred to as first streaks were more densely 
aggregated, with some adjacent streaks coalesced (Fig. 36A). The streaks then turned 
dark-brown from reddish-brown (Fig. 36B) ;and consequently became clearly visible 
on the adaxial leaf surface, with majority o f  the streaks coalesced. These symptoms 
marked the second-streak stage. The dark-brown symptoms broaden w ith the infected 
portions heavily water-soaked especially during the early hours o f  the day. The water- 
soaking effect disappeared with time on the same day, which is characteristic o f a 
first-spot stage (Fig. 36C.). Subsequently, 6-16 days (mean 11 days) after the 
appearance o f  the initial specks, the spot became depressed, marking the beginning o f 
a second-spot stage. This was characterised by a dark-brown or black colour o f  the 
central portions o f the spot with the immediate margins appearing very black (Fig. 
37B). Immediately beyond this black margin was a yellow transition zone (Fig. 38).
The centre o f  the spot became dry within a further 7 days and appeared grey or 
buff-coloured. The spot was now surrounded by a narrow well-defined black margin
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(Fig. 37A, 38) and between this black border and the healthy green tissue o f  the leaf 
was a bright yellow transitional zone. This marked the third or mature-spot stage.
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FIG. 36: (A,B,C) Different stages o f  black sigatoka symptoms on 
greenhouse-grown plantain leaves inoculated with 
conidia o f  M. fijiensis  isolated from plantain.
A: - Initial specks (Is) and first - streak (Fs) symptoms.
Note: Other halves o f leaves that were not inoculated 
serve as control treatments throughout this experiments.
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B: - Second-streak (Ss) symptoms on abaxial leaf surface. 
Note: Other halves o f  leaves that were not inoculated serve as 
control treatments throughout this experiments.
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C: - First-spot stage symptoms (arrowed) on abaxial leaf surface. 
Note: Other halves o f  leaves that were not inoculated serve as
control treatments throughout this experiments.
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FIG. 37: (A,B) Some stages o f  black sigatoka on greenhouse- 
grown plantain inoculated w ith M. fijiensis  conidia 
isolated from plantain.
A :L j, showing specks and streaks.
L2  showing mature spot with grey centres.
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Fig. 37. B - leaf showing second-spot symptoms. Note halo- 
margin.
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FIG. 38. Array o f inoculated plantains showing different stages 
o f black sigatoka symptoms. L j-second spot symptoms. 
Note black spot with halo-margin. L2 , L3 , L4 showing 
mature spot symtomps with grey centres.
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4.4.2. BANANA ISOLATE INOCULATED ON LOCAL TALL BANANA
Symptoms were similar to those found on plantain inoculated with conidia 
derived from plantain but with variations in the time intervals. Initial specks were 
evident between 20-31 days (mean 23 days) after inoculation o f  leaves (Fig. 39A). 
These specks became clearly visible and elongated on the abaxial leaf surface o f  the 
inoculated leaves, and densely aggregated, with adjacent streaks coalesced (Fig. 39B). 
These symptoms marked the first-streak stage. Streaks turned dark-brown from 
reddish-brown (Fig. 39C) within four days and became clearly visible on the adaxial 
leaf surface, with majority o f the streaks coalesced. This stage is referred to as the 
second-streak stage.
The dark-brown portions o f  the leaf broaden and became heavily water-soaked 
especially during the early hours o f  the day. The water-soaking disappeared with time 
on the same day. This observation was similar to a first-spot stage. The spot became 
depressed 13 days after the appearance o f  the initial specks w ith the infected portion 
becoming dark-brown or black at the centre, w ith the immediate margins appearing 
black (Fig. 40A). Beyond this black margin was a yellow transition zone. The spot 
within 5 days on the average, became dry and appeared grey or buff-coloured. The 
spot was now surrounded by a narrow well-defined black margin (Fig. 40B), and 
between this black border and the healthy green tissue o f  the leaf was a bright yellow 
transitional zone. This was the final or mature spot stage o f the disease (Fig. 40C).
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Ljl
FIG.39 (a,b,c) Symptoms on greenhouse-grown LTB, inoculated 
with isolate from Local Tall Banana.
A - initial speck symptoms (arrowed)
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Fig.39 B - First-streak symptoms on abaxial leaf surface 
(arrowed).
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C - Second-streak symptoms. Note the coalescing o f  streaks, 
with water soaking (W), which is not clearly visible in 
pictures.
Note the left half o f  leaves that serve as controls are 
without symptoms.
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FIG. 40: (A,B,C,) Symptoms on greehhouse-grown LTB,
inoculated with M. fijiensis  isolate from Local Tall 
Banana.
A - Second streak symptoms with water-soaking (W) 
and first-spot symptom (Fs). Note the black spot with 
the forming halo-margin.
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Fig. 40B - Second spot stage symptom. Note black spot with the 
immediate halo-margin.
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Fig. 40C - Mature or third-stage spot with grey centres (arrowed). 
Note the left half o f all leaves that serve as control are
without symptoms.
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4.5. CROSS-INFECTION STUDIES
4.5.1. PLANTAIN ISOLATE ON LTB
Symptom expression was similar to those observed in pathogenecity studies. 
There were however variations in the incubation period as well as other stages o f  the 
disease. Initial specks were observed 29 days on the average after inoculation and 
were evident only on the abaxial leaf surface (Fig. 41 A). Symptoms passed through 
the first and second streak stages. Within 44 days (on the average) after inoculation, 
the symptoms entered the first-spot stage (Fig. 41B). Symptoms progressed through 
the second spot stage, (Fig. 42A) and 51 days after inoculation entered the third or 
mature spot stage (Fig. 42B and C). Mature spot stage was attained 22 days after the 
appearance o f initial specks (Table 1).
4.5.2. ASCOSPORES FROM PLANTAIN INOCULATED ON LTB
Initial specks were evident 21 days after inoculation and symptoms passed 
through all the stages described above until mature spots were formed (Figs. 43 
A,B,C).
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Table 1: Time Course of Disease Progression on Inoculated Plants in the Greenhouse
A. PATHOGENICITY STUDIES
TYPE OF INOCULATION
Plantain isolate on plantain 
Banana isolate on LTB
INCUBATION PERIOD 
(DAYS)
RANGE AVERAGE
12-22
20-31
18
23
AVERAGE 
NUMBER OF 
DAYS TAKEN 
FOR MATURE 
SPOTS TO 
APPEARFROM 
DAY OF 
INOCULATION
36
31
B. CROSS-INFECTION STUDIES
Plantain isolate on LTB 
Banana isolate on plantain 
Plantain ascospores on LTB
25-36
12-38
29
26
21
51
51
DAYS TAKEN 
FOR MATURE 
SPOTS TO 
APPEAR AFTER 
INCUBATION 
PERIOD
18
8
22
25
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FIGS. 41 (A,B>): Black sigatoka symptoms on greenhouse grown Local Tall 
Banana inoculated with plantain isolate o f  M. fijiensis  
A - initial specks (Is) and first-streak (Fs) symptoms
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7ig. 41B - second-streak stage developing into first-spot stage symptoms. 
Note the coalescing o f  streaks and water soaking (W) effect.
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FIGS. 42.(A,B,C): Black sigatoka symptoms on greenhouse grown Local 
Tall Banana inoculated with plantain isolate o f M. fijiensis.
A - Right (R), first-spot stage (Fs) and second spot (Ss) stage 
symptoms. Left (L) mature (third-spot) stage symptom. Note grey 
nature of symptom.
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FIG. 42B: - Mature spots - Note grey nature o f spot and the adjoining halo- 
margin.
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FIG. 42C: - Mature spots o f black sigatoka disease - Note grey nature o f  spot 
and the adjoining halo-margin.
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FIGS. 43 (A,B,C) Black sigatoka symptoms on greenhouse grown Local Tall 
Banana inoculated with ascospores o f  M. fijiensis  ejected from 
plantain.
FIG. 43A. - Disease specks (rusty spots) on abaxial leaf surface.
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FIG. 43B. - Left (L), uninoculated leaf on LTB. Right (R) inoculated leaf 
showing second spot stage symptom. Note black spots with 
surrounding yellow leaf tissue.
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FIG. 43C - Mature-spot stage symptoms.
Note grey centres (arrowed), with adjoining halo margin.
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4.5.3. BANANA ISOLATE INOCULATED ON PLANTAIN
On the average, it took 26 days for initial specks to appear on inoculated leaves (Table 1). 
Symptoms passed through the various stages described for the pathogenecity studies until mature 
spots were formed 25 days after the appearance o f initial specks (Fig. 45, 46).
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FIG.44 (A, B, C ) : Black sigatoka symptoms on greenhouse grown plantain 
inoculated with banana isolate o f M fijiensis
FIG. 44A. Second streak symptoms (arrowed) on abaxial leaf surface.
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.44B First-spot symptoms with water soaking effect (W) and second- 
spot symptoms on leaf. Note dark spot with adjoining halo-margin.
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FIG. 44C - Leaves o f plantain showing second-spot (Ss) and 
mature-spot (Ms) symptoms.
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FIG. 45. Mature-spot (Ms) o f  black sigatoka symptoms on 
greenhouse grown plantain inoculated with banana 
isolate o f  M. fijiensis. Note the dried centre with 
adjoining yellow margin.
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4.6. EFFECT OF ENVIRONMENTAL FACTORS ON DISEASE 
EXPRESSION
Inoculated plants left on the open greenhouse bench without incubation never 
developed any form o f symptoms throughout the course o f the experiment. This was 
a clear indication that high relative humidity was necessary for disease development.
4.7. CONTROL EXPERIMENTS
Uninoculated plants left in the open greenhouse in addition to the other 
uninoculated halves o f  all inoculated leaves that were incubated never developed 
black sigatoka symptoms. This has proven the absence o f  natural inoculum in the 
greenhouse.
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DISCUSSION 
FIELD SYMPTOMS
Leaf symptoms on plantain and banana in the field were similar with those 
described for black sigatoka by Meredith and Lawrence (1969) and later by Oduro et 
al. (1991). Symptoms on all greenhouse inoculated plants in this study were also 
similar to those described for black sigatoka and essentially passed through all the 
various stages described in literature (Meredith and Lawrence, 1969). The 
predominance o f spotting at the leaf tips and margins in the field is due to pronounced 
water congestion within the leaf tissues along the margins especially during the night. 
This water congestion might have increased spore germination, infection and 
pathogen development within leaf tissues. Thus, based on leaf symptoms in the field 
and the greenhouse, black sigatoka was identified to be present on both plantain and 
the local tall banana in Ghana.
SINGLE CONDIA ISOLATION
The single conidium isolation o f the pathogen from infected plantain and 
banana leaves was made impossible as a result o f  the abundant spores o f  other 
associated fungi such as Cordana musae and Chloridium  spp. This difficulty was 
complicated by the fact that, the conidia o f Mycosphaerella fijiensis  were seldom 
found in abundance but rather scattered on the leaf surface. This might be due to 
spore dispersal either by wind and/or by water. The concurrent presence o f  reddish- 
brown Chloridium  speckles that resemble the first streaks symptoms o f black sigatoka 
also impeded isolation by confusing the symptoms. The repeated washing o f  infected 
leaves under tap water could also have removed conidiophores and hence retarded 
subsequent conidia production on incubated leaves under bell jars. These difficulties 
accounted for the inability to isolate M. fijiensis  by single spore isolation. The 
presence o f the three fungal species. M. fijiensis, Cordana musae and Chloridium spp 
on both plantain and banana, and above all their presence on green leaf tissues might 
have contributed to the disease (Wardlaw, 1972). This complex might increase
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disease severity or otherwise, hence the need for further investigations to identify their 
roles separately and when in combination with M. fijiensis  in black sigatoka disease 
development.
INDUCED MYCELIAL GROWTH
Mycelial growth was not recorded from the cut edges o f  both infected plantain 
and banana leaves that were plated on both PDA and CDA. The inability in this case 
might be due to the very slow growth o f  the fungus coupled with scorching o f  the cut 
edges o f the leaf discs, which might have led to the death o f  the pathogen in  these 
sections. These notwithstanding, inexperience in this isolation technique might also 
have contributed to this failure.
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ISOLATION BY ASCOSPORE DISCHARGE FROM INFECTED BANANA 
AND PLANTAIN LEAVES
Ascospore discharge from diseased banana and plantain leaves into sterile 
distilled water (SDW), PDA and CDA was found to be a successful method o f 
isolating Mycosphaerella fijiensis  Morelet in this study and as was reported by Stahel, 
(1937); and Meredith and Lawrence (1970); Vasquez et al, (1988); Natural, (1988); 
and Foure', (1988).
The quick discharge o f ascospores (15 minutes) from wet infected leaves into 
SDW and their subsequent early germination (1 ^2  hours) in SDW as opposed to 12- 
18 hours on PDA and CDA might have a lot o f  epidemiological implications in the 
field. The early germination o f ascospores in water might result in early disease 
establishment as the spore germ tubes can penetrate stomata and induce disease within 
a short period o f time. It also emphasises the necessity o f water for spore germination 
(Goos and Tschirch, 1963; Stover, 1980; Jacome and Schuh, 1992).
The role o f  Leptosphaeria spores ejected together with ascospores o f  M. 
fijiensis from both plantain and banana, though not investigated in this study needed 
further investigation. This is o f importance since these spores also germinated very 
fast (30 minutes) and with multiple germ-tubes. As reported by Stahel (1937) for 
Yellow Sigatoka and by (Meredith and Lawrence, 1970) for black sigatoka, it seems 
this fungus association is a constant feature o f  both Yellow and Black sigatoka 
diseases.
CULTURAL CHARACTERISTICS OF BANANA AND PLANTAIN 
ISOLATES IN SDW AND ON PDA
The observed ascospore dimensions (13.8um avg. by 3.2um), appearance, 
septation for the plantain isolate and those for the banana isolate (13.9um by 3 .2um 
avg.) agreed perfectly with each other in this study and lie within the ranges reported 
for Mycosphaerella fijiensis Morelet (Meredith and Lawrence, 1970; Natural, 1988). 
The bipolar nature o f ascospore germination in SDW for both plantain and banana 
isolates was similar and compared with those reported for both Mycosphaerella
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musicola and M. fijiensis on water agar and PDA (Leach, 1941, Meredith and 
Lawrence, 1970; Natural, 1988). The absence o f branching and sporulation o f  both 
plantain and banana isolates in SDW as opposed to those on media during this study 
might be due to the absence o f  nutrients in SDW.
The time taken for ascospores to germinate (12-15 hours) on PDA for both 
isolates, and by multiple germtube development was in agreement w ith that found for 
M. fijiensis  germinating on water agar within 24 hours (Vasquez et al, 1988). A 
further indication o f  the necessity o f  nutrient for the growth o f  the isolates. The longer 
time taken for ascospores to germinate on media compared to that in SDW gave an 
indication to the slow growth o f the fungus on media in general as reported by 
Meredith and Lawrence (1970). This observation was substantiated by the finding that 
fungal colonies o f both isolates in this study became visible to the naked eye on media 
only after 5 days as opposed to 2-3 days reported by (Meredith and Lawrence, 1970) 
and subsequently grew very slowly with a very compact nature as was reported by 
Stover, (1937), and Meredith and Lawrence, (1970).
The presence o f  dumb-bell-shaped cells in culture as being responsible for the 
dark nature o f  under surface o f  colonies characteristic for cultures o f  Mycosphaerella 
fijiensis and M. musicola (Meredith and Lawrence, 1970) were observed. The 
presence o f dumb-bell-shaped cells as an indication o f pathogenic isolates o f  
Cercospora spp. (Stover, 1976) was also observed in this study for both isolates and 
later proven in pathogenecity studies in the green house. The absence o f sporulation o f 
both plantain and banana isolates on PDA in this study in contrast w ith other findings 
(Meredith and Lawrence, 1970; Natural, 1988) might be due to the fact that there 
might be different strains o f the fungus in Ghana, and which cannot sporulate on PDA 
but rather on CDA. These speculations may be confirmed by the observation in this 
study that non-sporulating colonies transferred from PDA onto CDA produced typical 
Cercospora-type conidia whereas the vice versa did not. This anomaly in sporulation 
was also found to be consistent with other Cercospora spp that were found to show
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difficulty in sporulation (Garman, 1920; Jenkins, 1930; Nagel, 1934; Calpouzos, 
1954; Kilpatrick and Johnson, 1956).
The absence o f  any visual differences between the colonies o f both isolates in 
this study coupled with the fact that they both did not sporulate on PDA gave another 
indication that they both might belong to the same Mycosphaerella sp., however no 
tentative identification could be done due to the absence o f  conidia on PDA.
Cultural Characteristics of Plantain and Banana Isolates on CDA
Fungal colonies o f  both the plantain and banana isolates on CDA were the 
same in every respect as on PDA; slow growth, hyaline hyphae changing to light 
brown, presence o f dumb-bell-shaped hyphal cells, perithecia-like bodies with 
associated spermatia-like cells were similarly observed. Based on these, the two 
isolates appeared to be coming from the same Mycosphaerella sp.
However unlike on PDA, profuse sporulation o f  both plantain and banana 
isolates was observed on CDA within one week. This observation indicated the 
suitability o f  CDA for growth and sporulation o f  the fungal isolates, which is in 
contrast to the reported 3 weeks (Natural, 1988) and 5 days ( Meredith and Lawrence, 
1970) needed for sporulation on PDA. This is the first time in literature that CDA was 
used to culture either M. musicola or M. fijiensis.
Conidiophores with evident spore scars and septation in this study conformed 
to those described for M. fijiensis  (Meredith and Lawrence, 1970) and those observed 
on infected leaf tissues from the field in this study.
In this study, the conidia in culture were found to be o f  the Cercospora-type. 
The thickened hilum which is reported to be characteristic for M. fijiensis  and the 
most definitive distinguishing character between M. musicola and M. fijiensis  pointed 
to this fact (Meredith and Lawrence, 1970; Jones and Mourichon, 1993).
Thus, based on the observation o f  infected leaf tissues under the microscope, 
the observation o f  fungal structures; conidiophores and conidia on infected leaves, 
Mycosphaerella fijiensis (Morelet) was suspected. In addition, based on the colony
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appearance, conidiphores and conidia with thickened hilum produced on CDA, and 
which was found to be similar for both plantain and banana isolates in this study and 
which compared with those reported by Meredith and Lawrence (1970), 
Paracercospora fijiensis  (Morelet) Deighton (anamorph o f  Mycosphaerella fijiensis) 
was identified as the causative organism for the plantain and Local tall banana leaf 
spot disease in Ghana.
The findings in this study have scientifically strengthened the report o f  Oduro 
et al. (1991) who, based on symptoms alone, reported that the new plantain disease in 
Ghana is black sigatoka with M. fijiensis Morelet as causal agent.
This study further proves that the symptoms found on the Local tall banana in 
Ghana is also black sigatoka caused by M. fijiensis  Morelet.
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Pathogenicity Studies
In those inoculations where the plantain isolate o f  M. fijiensis  was inoculated 
on plantain and the banana isolate o f  M. fijiensis on Local tall banana in the 
greenhouse, typical black sigatoka symptoms were recorded on all leaves inoculated 
and incubated under polythene bags. These symptoms passed through the six stages 
reported for black sigatoka disease (Meredith and Lawrence, 1970).
The use o f high inoculum concentrations as well as inoculating plants at an 
early stage did not affect symptom expression.
The symptom development in the greenhouse as depicted by photographs 
followed essentially the patterns described by Meredith and Lawrence (1970) for 
black Sigatoka.
Though differences existed between the incubation periods (Table 1), this was 
believed to be either due to the different genetic constitutions between plantain and 
banana, and/or the different times at which the experiments ran which could affect 
disease development.
The longer incubation period (23 days) observed when banana isolate was 
inoculated on the Local tall banana might be due to the period o f  time (December) in 
which the inoculations were carried out. This period also recorded the period o f  least 
Relative humidity o f 35%. However the shorter period (8 days) taken for specks to 
develop into mature spots might be attributed to the high day temperatures and light 
intensity during this period. Light is reported to increase disease severity o f  black 
sigatoka.
The shorter incubation period (18 days avg.) observed when the plantain 
isolate o f M. fijiensis  was inoculated on plantain might be attributed to the seeming 
susceptibility o f  plantain, and secondly the favourable environmental conditions o f 
high humidity prevailing in the greenhouse during this period o f  time. The longer 
period of 18 days taken for spots to develop from streaks on plantain might be due to 
the favourable environmental conditions at this time, thus making the plants grow 
more vigorously and enabled the fungus to grow favourably within the leaf. This
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vigorous growth might have delayed the rate o f  cell necroses, thus delaying spot 
development.
Cross-Infection Studies
On the whole, the cross-inoculation studies in the greenhouse progressed 
through the different stages o f symptom development for black sigatoka disease and 
was similar to those found during the pathogenecity studies above. Symptoms were 
first visible as reddish-brown specks on the under surface o f  all inoculated and 
incubated leaves and became visible on the adaxial leaf surface only at the second 
streak stage.
Incubation periods, though varied for the different inoculations (Table 1), 
might be due to the genetic differences existing among the different varieties.
The wider range for the incubation period, 25-36 days, (29 days on average) 
when the plantain isolate was inoculated on local tall banana could be due to 
environmental influence since majority o f these inoculations were done in November 
which also recorded one o f  the lowest R.H. and high temperatures.
Control Treatments
The absolute absence o f symptoms on the uninoculated controls was believed 
to be due to the unavailability o f infective propagules o f  the pathogen in the open 
greenhouse environment coupled with the unfavourable environmental conditions 
such as absence o f  leaf wetness in the open greenhouse. This observation o f  the 
absence o f  spores in the air was further proven when it was observed that 
uninoculated leaves that were incubated under polythene bags never developed any 
form of symptoms.
Absence o f symptoms on inoculated, but unincubated, leaves might be due to 
the death o f the fungus on the leaves. Since the leaves were not incubated, the high 
humidity required for conidia to germinate was not produced, hence stomatal 
penetration was impossible. The overall observations on the control treatments in this
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study agrees with those found by Goos and Tschirch (1963) on greenhouse-grown 
banana inoculated with Cercospora musae Zimm.
GENERAL: 
Effect of environment (R.H) on symptom expression/ development
Environmental conditions such as temperature and relative humidity influence 
incubation period and subsequent disease development. This was evident in 
inoculations that were carried out when the greenhouse relative humidity was on the 
high side (87%). This condition which enabled the formation o f  water films on leaves, 
had the shortest incubation period o f  12  days whereas those inoculations that were 
carried out when the relative humidity was low (35%) had longer incubation periods 
o f 25-38 days. Further prove was given when there was absence o f  symptoms on 
plants left on the open greenhouse bench, without incubation in polythene bags. Other 
reported incubation periods for M. fijiensis  on Musa  spp in the greenhouse include 14 
days on banana (Jacome and Schuh, 1992) and 22 days for some Musa  spp (Vasquez 
et. al., 1988). Foure (1988) reported 14-24 days for ground mycelium o f M  fijiensis  
on plantain whilst for ascospores, 10-32 days were reported. Though the incubation 
periods reported in this study varied from those reported elsewhere, they were both 
found to be comparable, and any differences might be due to the genetic differences 
among the varieties used as well as the different conditions under which the 
experiments were carried out. Other differences might also be attributed to different 
strains o f M. fijiensis used in the inoculation.
The wide variations observed in the incubation periods and rate o f  disease 
development in this study might also be due to the activity o f  the fungus in the leaf 
after penetration. It is believed that the fungus remains in a latent state o f  varying 
duration within the leaf. Goose and Tschirch (1963), and Leach (1946) concluded that 
such latency could be correlated with anatomical features o f the leaf and the number 
of infections per unit o f leaf area. These in this particular case might be different for 
both plantain and banana.
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The difference between disease symptoms on small plants from those on older 
plants (Simmonds, 1939) were similarly observed in this study as early disease 
symptoms initially appeared oval on three-month old plants.
It was generally observed that once the first symptoms appeared on susceptible 
inoculated plants, time for disease development to the mature spot stage varied. This 
might be due to the internal water relations o f  the plants which are more crucial than 
the external environmental conditions. Once the fungus is inside the leaf tissue, the 
external environmental conditions do not affect the activity o f  the fungus within the 
leaf and hence it is capable o f inducing disease. (Wardlaw, 1961, Goose and Tschirch, 
1963).
Difficulty was encountered in reisolating the fungus from infected plants in the 
greenhouse and this could be attributed to the method o f incubation adopted.
The absence o f misting equipment did not allow maintenance o f  water films 
on infected leaves for an appreciable length o f  time which is a necessary requirement 
for conidia production and perithecia maturation on infected leaf (Jacome and Schuh, 
1992). Consequently, no conidia or ascospores were produced on the greenhouse 
inoculated plants in this study. Inoculated plants were left in the open greenhouse 
since it became difficult to enclose whole plants under polythene bags due to their 
large sizes.
It appeared from the greenhouse studies that in Ghana, plantain seemed to be 
more susceptible than banana to black Sigatoka disease since the shortest period o f  
inoculation, 12 days (Table 1). were observed on plantain when inoculated w ith both 
the plantain and banana isolates o f  M. fijiensis  isolated in this study. This observation 
further confirms the fact that the isolates infecting both plantain and banana came 
from the same species.
In general, the greenhouse inoculations revealed and established the fact that 
the isolates obtained from both plantain and banana are pathogenic to the respective 
plants from which they were isolated and also cross-inoculated and produced typical 
black sigatoka disease symptoms.
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The observation in this study that ascospore discharge from wet infected 
leaves occurred in less than 15 minutes, and those from plantain infect the local tall 
banana is o f much epidemiological importance.
Since ascospores could serve as the primary inocula it is recommended that 
control strategies should be that o f  initially removing and burning all leaves from 
both banana and plantain showing mature (stage six) spots and subsequently 
controlling the development o f streaks to mature spots by fungicidal spray.
In Ghana, since control is mainly carried out on plantain to the neglect o f  the 
grooves o f  local tall bananas, this study has revealed that such control attempts will 
yield no long-term dividends since the pathogen on the local tall banana will build up 
and eventually attack plantain.
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RECOMMENDATIONS
It is recommended that:
1. Further work be carried out to determine the major spore-type initiating infections
in Ghana
2. Investigation o f  the role o f  Cordana musae, Chloridium  spp and Leptosphaeria  spp
in disease severity in Ghana be initiated
3. Further research into any possible pathogenic variability o f  M. fijiensis  in Ghana
and its implication be carried out
4. Control strategies should be expanded to cover all local tall banana grooves in the
country.
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Appendix 1
Ascospore dimensions of Plantain M. fijiensis on CDA
Length(um)
13.8 13.8
12.77 13.11
13.8 13.8
13.8 13.8
13.46 13.8
13.8 12.08
13.11 13.8
12.1 13.8
12 .1 12.77
14.15 13.11
12 .1 13.8
10.35 12.08
13.8
15.53
15.53
18.98
13.8
10.7
13.8
13.11
14.11
16.25
11.04
15.53
16.56
14.15
13.8
10.35
13.8
12.42
13.8
13.46
13.8
13.8
13.46
13.8
13.8
12.08
14.15
12.77
13.8
13.8
13.8
14.15
13.46
Average =13.8
Breadth ('Diameter') (urn)
3.45 2.8
3.8 3.11
3.45 3.45
3.11 3.45
3.11 3.11
2.7 3.45
2.7 2.8
3.8 2.42
3.45 3.8
3.8 3.11
3.11 2.8
3.45 2.42
3.11 3.45
3.45 3.11
3.11 3.45
3.45 2.42
2.42
3.45
3.45
3.45
3.11
3.11
3.45
3.45
3.45
3.45
2.8
3.11
2.8
2.42
2.42
3.45
3.45
3.45
2.8
2.8
2.42
3.45
3.11
3.11
3.11
2.8
3.11
2.8
2.8
Average = 3.2
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APPENDIX 2
Ascospore dimensions of banana isolate of M. fijiensis on CDA.
Lenethfum'l Breadth ('Diameter') ('um')
14.08 14.8 2.5 3.86
14.77 16.53 3.9 3.11
15.11 16.53 2.57 2.85
14.77 13.8 3.9 3.9
15.8 14.35 3.42 2.9
15.8 14.2 3.0 3.9
13.8 14.85 2.5 3.75
12.08 10.8 3.8 2.9
15.8 10.8 3.9 3.9
15.8 16.21 3.75 2.9
13.8 16.11 2.85 3.75
15.11 16.7 3.85 2.5
14.8 13.46 3.85 3.45
16.46 14.8 2.8 2.5
15.15 14.8 3.8 2.5
13.8 14.78 2 .11 2.45
13.8 13.8 3.8 2.45
14.8 3.7 3.2
14.78 2.5 3.2
14.15 3.9
13.08 3.45
13.8 2.5
13.8 2.9
15.8 3.9
13.46 3.78
13.8 3.5
14.8 2.58
16.42 2.58
13.9 3.8
13.08 3.16
13.9 2.8
12.35 3.45
15.8 2.85
14.15 3.85
16.7 3.45
15.53 3.48
14.04 3.11
16.7 2.86
16.7 3.86
15.11
Average = 3.2
Average =13.9
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APPENDIX 3
Conidia lengths for plantain isolate of M. fijiensis on CDA (jam)
31.1
108.7
72.5
84.5
44.9
100.1
79.4
80.0
361.1
69.0
48.3
88.0 
77.8
96.6
86.3
96.6
41.4
103.5
86.3
96.6
41.4
103.5
86.3
86.3
43.1
34.5
87.7 
110.4
96.6
56.4
141.5
60.5
48.3 
66.8
41.4 
123.2 
158.7 
100.1
98.3
31.6
100.1
100.4 
69.0
103.5 
46.4
44.9
55.2 
61.1
86.3
86.8
Average = 78.1
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APPENDIX 4
Conidia lengths of banana isolate of M. fijiensis on CDA (pm).
59.0 86.3
44.01 69.0
86.3 65.0
158.9 72.5
82.8 75.9
72.5 77.6
82.8 75.9
50.1 63.8
138.0 100.1
100.1
110.4
31.0
69.0
62.1
86.3
38.0
100.1
113.9
107.0
93.2
89.7
103.5
79.4
44.9
46.6
60.4
75.9
104.2
36.2
69.0
93.2
62.1
69.0
72.5
48.3
148.4
103.5
86.3
36.2
65.6
82.8
Average78.4
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APPENDIX 5
Conidia breadths (diameters) (pm) for the plantain isolate o f  M. fijiensis  
CDA.
Tip Broadest Point Hilum
1.76 3.45 2.76
2.1 3.45 2.42
2.1 3.8 1.73
2.1 3.45 2.2
2.42 3.45 3.45
2.42 3.8 3.11
3.11 3.8 3.45
3.11 4.14 2.76
2.42 3.45 3.11
2.42 3.8 1.73
2.1 3.45 2.42
2.76 4.49 3.45
3.45 3.8 3.11
1.73 3.45 3.11
2.1 3.8 3.11
2.42 3.45 3.45
2.76 3.8 3.11
2.76 3.45 1.73
2.1 3.45 2.1
2.42 ' 3.45 2.1
2.1 3.45 3.11
1.73 3.45 3.11
2.73 3.45 2.42
2.76 3.8 2.42
3.11 3.45 1.73
2.42 3.45 2.42
2.76 3.45 2.1
3.45 3.45 2.76
2.10 3.45 2.42
1.73 3.45 3.11
2.1 3.45 2.42
2.1 3.45 2.76
2.1 3.8 1.73
2.1 3.45 2.42
2.42 3.45 2.1
2.1 3.8 1.73
2.1 3.45 2.1
2.1 3.45 2.42
2.42 3.45 1.73
2.42 3.45 2.1
Average 2.4 Average = 3.6 Average = 2.5
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APPENDIX 6
Conidia breadths (diameters) (pm), for the banana isolate o f M. fijiensis 
CDA
l i e Broadest Point Hilum
3.1 4.49 3.1
3.1 3.8 2.73
3.42 3.45 3.42
2.42 3.8 3.1
3.11 3.45 1.73
3.11 3.8 2.1
3.42 3.45 2.42
2.42 3.45 1.73
3.1 3.45 2.76
2.76 3.45 2.42
3.45 3.45 3.11
1.63 3.8 2.42
2.2 3.8 2.76
2.42 3.45 2.1
2.76 3.45 2.42
2.76 3.45 1.73
3.1 3.45 3.45
3.42 3.45 3.11
2.1 3.45 1.73
2.73 3.45 2.1
1.73 3.45 2.1
2.76 3.45 3.11
2.76 3.8 2.42
3.45 3.45 2.42
2.76 3.45 3.11
2.42 3.45 3.11
3.11 3.45 3.11
2.1 3.45 3.45
1.73 3.45 2.42
2.1 3.8 1.73
2.1 3.45 3.11
2.42 3.45 2.76
2.1 3.8 3.45
2.1 3.8 3.11
2.1 4.14 3.45
2.42 3.45 2.2
2.1 3.8 1.73
2.42 3.45 2.42
2.1 3.45
3.45
2.76
Average 2.4 Average = 3.6 Average
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APPENDIX 7
Conidiophore Dimensions of plantain isolate of M. fijiensis on CDA.
Length (urn) Breadth fum'i Setrtation
3.45 163.9 13
3.45 31.1 3
3.8 51.8 4
3.8 48.3 3
4.49 65.6 4
4.49 48.3 1
3.8 76.6 4
3.8 57.8 5
3.8 51.8 4
3.14 24.2 3
3.8 51.8 3
4.14 74.9 4
4.14 38.0 6
3.45 44.9 3
5.18 43.8 1
3.11 51.8 1
3.8 25.5 2
3.11 62.1 0
3.45 56.9 2
5.18 48.3 4
5.18 31.1 3
3.11 25.9 1
3.11 86.3 1
3.8 75.9 5
4.14 65.6 5
3.8 51.8 0
3.45 59.0 1
3.11 57.3 4
3.8 53.5 3
3.45 41.4 3
3.45 48.3 3
3.83 3
Average 3.8 Average = 54.7 Average
University of Ghana          http://ugspace.ug.edu.gh
APPENDIX 8
Conidiophore dimensions of banana isolate of M. fijiensis on CDA
Length(um) Breadth(um) Septation
52.8 3.8 3
40.6 3.21 3
75.9 3.45 4
44.9 5.18 3
43.8 3.21 12
25.5 5.18 4
51.8 5.4 1
31.1 3.21 5
62.1 3.21 4
56.9 3.8 4
45.3 4.36 3
75.9 3.8 3
86.3 3.45 4
25.9 3.21 6
31.1 3.8 3
65.6 3.8 1
51.8 3.45 1
53.0 3.45 0
59.0 3.8 0
41.4 4.0 2
48.3 4.0 2
53.5 3.9 1
54.3 3.24 3
163.9 3.9 1
31.1 4.14 4
51.4 3.14 5
40.3 3.45 1
41.0 5.18 5
76.6 3.21 4
48.3 3.8 3
51.8 3.45 3
24.2 3.45 3
Average 54.9 Average = 3.8 Average
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