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STUDIES ON SOME PHYSIOLOGICAL CHARACTERISTICS AND PATHOGENICITY
OF SCLEROTIUM ROLFSll SACCo CAUSING COLLAR ROT AND CORMEL ROT OF
COCOYAMS (COLOCA.SIA AND XANTHOSOMA SPECIES) IN GHANA.
IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE M. PHIL.
DEGREE OF THE UNIVERSITY OF GHANA, THE DEPARTMENT OF
BOTANY, UNIVERSITY OF GHANA, LEGON.
SEPTEMBER, 1997.
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DECLARATION
I hereby declare that, except for reference to other peoples' work which have been duly cited,
this work is the result of my own original research work and that this thesis has neither in whole
nor in part been presented for another degree elsewhere.
PROF. G.C. CLERK
(SUPERVISOR)
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DEDICATION
Dedicated to the Almighty God
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ACKNOWLEDGEMENT
I wish to express my si.icere gratitude to my supervisor, Professor G.C. Clerk for his
endless interest, patience, constructive criticisms and guidance, without which this study
would not have reached its conclusion.
Thanks to Prof. A.A. Oteng-Yeboah, Head of the Botany Department and the other
professors and lecturers in the department for their interest in the project.
I also thank Mr. K.B. Affrim of the Oil Palm Research Institute, C.S.I.R, Kade, who
collected the cormels of cocoyams used in this work on my behalf from the Agriculture
Research Station of the University of Ghana at. Kade.
Special thanks go to Miss Cynthia Amoabeng of the Noguchi Memorial Institute of
Medical Research, who typed this thesis and all my friends and colleagues for their
support and encouragement during this study.
Finally, my special acknowledgement are due to my mother, brother and sister for their
wonderful support.
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ABSTRACT
The physiological and morphological characteristics of five strains of Sclerotium rolfsii,
namely, XLL, XAl, XA2, ELI and EL2, which were identified as strains by the
aversion test, were studied. The susceptibility of five varieties of cocoyam, 'Amankani
fitaa', 'Arnankani fufuo', 'Arnankani kyirepe' and 'Amankani pa' of Xanthosoma
sagittifolium and a single variety of Colocasia antiquo rum to these strains, was also
investigated. The appearance of the strains on Potato Dextrose Agar plates was similar
and mycelial dry weights in Potato Dextrose Broth after 6 days' incubation were
statistically similar. However, extensional growth on PDA was fastest in Strains ELI
and EL2, moderate in Strains XLL and XAI and slowest in Strain XA2. Cultures of
Strain XLL produced a mean number of 185 larger and heavier sclerotia per Petri plate
whereas the remaining four strains formed 324 to 364 smaller and lighter sclerotia per
Petri plate.
In broth media with different carbon compounds - fructose, glucose, maltose, starch and
sucrose - and with different nitrogen compounds - Ammonium chloride, Ammonium
nitrate, Asparagine, Peptone and Sodium nitrate - a clearly discernible order of ability
to utilize the two groups of compounds was noticed. The order of the strains in
descending order of efficiency was EL2 > ELI> XLL > XAI > XA2. Among the carbon
compounds the least utilized was starch. The best carbon compound varied with the
strains. Peptone was universally a good nitrogen source.
IV
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Over a concentration range of O.S to 2.0%, the greatest amount of pectolytic enzymes
was produced in both the glucose and pectin media. Filtrates of Strains XLL, XA 1 and
EL2 showed greater pectolytic Enzymes Activity than those of Strains XA2 and ELI.
But Strains XAI and XA2 showed greater ability to break down cellulose than the other
three strains.
The pathogenicity tests did not show a direct relationship between ability to produce
pectolytic enzymes and cellulose-degrading enzymes and infection of the host plants.
Thus, Strains XA 1 and XA2 caused the greatest rot in wound-inoculated cormels, while
infection of 3-month old plants proceeded fastest in soils inoculated with Strain ELI and
EL2.
None of the coco yam varieties showed resistance to the S. rolfsii strains, and they only
showed varying degrees of vulnerability. However, Colocasia antiquo rum which
supported more than 2-4 times the number of sclerotia formed by the strains than
Xanthosoma sagittifolium would playa greater role in the survival of S. rolfsii in the
field.
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LIST OF TABLES
Page
1. Growth of the five strains of S. rolfsii on Potato Dextrose Agar (PDA)
at 30°C. 48
2. Formation of sclerotia by the five strains of S. rolfsii growing on
PDA at 30°C. 49
3. Growth of the five strains of S. rolfsii in Potato Dextrose Broth (PDB)
at 30°C. 52
4. Growth of different S. rolfsii strains in liquid medium with different
Carbon sources at 30°C for 6 days 55
5. Growth of different S. rolfsii strains in liquid medium with different
Nitrogen sources at 30°C for 6 days. 59
6. Growth of the five strains of S. rolfsii in 30ml of PDB with different
concentrations of Glucose at 30°C recorded after 6 days of incubation. 65
7. Growth of the five strains of S. rolfsii in 30ml of PDB with different
concentrations of pectin at 30°C recorded after 6 days of incubation 67
8. Growth of the five strains of S. rolfsii in 30ml of PDB containing a
combination of Glucose and Pectin in different ratios at 30°C recorded
after 6 days of incubation. 69
9. Extent of maceration of discs of stem tuber of Irish Potato (Solanum
tuberosum) by filtrate of 6 day-old cultures of strains each provided
with different concentrations of Glucose. 71
10. Extent of maceration of discs of pericarp of cucumber (Cucumis sativus)
by filtrate of 6 day-old cultures of strains each provided with different
concentrations of Glucose. 72
11. Extent of maceration of discs of stem tuber of Irish Potato (Solanum
tuberosum) by filtrate of 6 day-old cultures of strains each provided with
different concentrations of Pectin. 73
12. Extent of maceration of discs of pericarp of cucumber tCucumis sativus)
by filtrate of 6 day-old cultures of strains each provided with different
concentrations of Pectin. 74
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13. Extent of maceration of discs of stem tuber of Irish Potato (Solanum
tuberosum) by filtrate of 6 day-old cultures of strains each provided
with Glucose and Pectin in different proportions. 76
14. Growth of the five strains of S. rolfsii in medium with cellulose
(filter paper) as carbon source incubated at 30"e for 7 weeks. 78
15. Growth of the five strains of S. rolfsii in medium with cellulose
(filter paper) and 0.1 % (w/v) Glucose as carbon source incubated
at 300e for 7 weeks. 79
16. Formation of infection cushion on surface of Local Variety Tomato
fruits by the five strains of S. rotfsii incubated at 30De for 4 days. 88
17. Infection of varieties of Xanthosorna sag ittifolium and Coiocasia antiquo rum
growing in soil inoculated with different S. rolfsii strains. 91
18. Rotting of wound-inoculated cormels of 'Amankani fitaa' by the five strains
of S. rolfsii at 30De in 8 days. 102
19. Rotting of wound-inoculated corrnels of 'Arnankani fufuo' by the five
strains of S. rolfsii at 300e in 8 days. 103
20. Rotting of wound-inoculated corrnels of 'Arnankani Kyirepe' by the five
strains of S. rolfsii at 300e in 8 days. 104
21. Rotting of wound-inoculated cormels of 'Amankani pa' by the five
strains of S. rolfsii at 300e in 8 days. 105
22. Rotting of wound-inoculated cormels of Colocasia aniiquorum by the
five strains of Sirolfsii at 30"e in 8 days. 106
23. Degradation of tissues of the apical and basal regions of the corrnels of the
different cocoyam varieties by the different S. tolfsii strains at 300e over
8 days. 109"
24. Degradation of tissues of the apical and basal regions of the corrnels of the
different cocoyam varieties by the different S. rot/hi strains at 300e
over 8 days. 1 10
25. Formation of sclerotia by the different S. rolfsii strains on blocks of tissue
of connels of the (1jfferent cocoyam varieties at 30°C. 115.....
26. Percentage germination of sclerotia formed by S. rolfsii strains on different
coco yam varieties at 30oe. (Percentage germination on PDA plates). 116
VII
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LIST OF FIGURES
. I
1. Mean dry weigh t of mycelia (. .) of the five isolates of S. rolfsii grown
at 30DC for 12 days and pH of the medium €>.~ during growth of the fungi.
Data for pH curves presented in Appendix A. 53
2. Mean Dry wt. of mycelium of different S. rolfsii strains (arranged in
descending order of response) grown in 30mlliquid medium with different
sources of carbon (1, Fructose; 2, Glucose; 3, Maltose; 4, Starch and 5,
Sucrose) and Nitrogen (1, Ammonium chloride; 2, Ammonium nitrate; 3,
. Asparagine; 4, Peptone; 5, Sodium nitrate) at 3CY'Cfor 6 days. 60
3. Histograms of Mean % loss in dry weight of filter paper in media inoculated
with S. rolfsii strains. Superiority of Strains XAI and XA2 to the other
strains in the use of cellulose as carbon source. 80
4. Checker-board showing results of pairing of the five strains of S. rolfsii
on Potato-Dextrose Agar (PDA) at 300C to test for aversion phenomenon.
(+ ;incidence of aversion :-; no aversion). 82
5. Infection of Petioles. of 3 month-old cocoyam varieties after inoculation of
the soil with mycelia of the different S. rolfsii strains. Percentage of
petioles infected computed from data in Table 17. 94
6. Rotting at apical region (mi) and basal region (-) of wound-inoculated
cormels of cocoyam varieties* by the five strains of S. rolfsii at 30"C
for 8 days; 107
*1. X. sagittifolium var. 'Amankani fitaa'
2. X. sagittifolium var. 'Arnankani fufuo'
3. X. sagittifolium var. 'Amankani kyirepe'
4. X. sagittifolium var. 'Arnankani pa'
Colocasia antiquo rum
7. Percentage weight loss of mini-blocks (lOxlOx5mm) of apical region (tlll4) and
basal region ( ) of cocoyam varieties* placed on cultures of the different
S. rolfsii strains for 8 days. 111
*1. X. sagittifolium var. 'Arnankani fitaa'
2. X. sagutifolium var. 'Amankani fufuo'
3. X. sagittifolium var. 'Amankani kyirepe'
4. X. sagittifolium var. 'Arnankani pa'
5. Colocasia antiquo rum
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LIST OF PLATES
Page
1. Photograph showing sclerotia formed by cultures of the S. rolfsii strains
growing on PDA and incubated at 30"C for 12 days (x 1/3). (from
left: Strains XLL, XA1, XA2, ELI, EL2). 50
2. Photograph of cultures of S. rolfsii Strain EL2 supplied with different
Carbon sources and grown at 30°C for 6 days (x 1/3). (Carbon source
from left: Sucrose, Glucose, Fructose, Starch, Maltose). 56
3. Photograph of cultures of S. rolfsii Strain XA 1 supplied with different
Carbon sources and grown at 30°C for 6 days (x 1/3). (Carbon source
from left: Sucrose, Glucose, Fructose, Starch, Maltose). 57
4. Photograph of cultures of S. rolfsii Strain EL2 supplied with different
Nitrogen sources and grown at 30°C for 6 days (x 1/3). (Nitrogen source
form left: Peptone, Asparagine, Ammonium nitrate, Ammonium chloride,
Sodium nitrate). 61
5. Photograph of cultures of S. rolfsii Strain XA 1 supplied with different
Nitrogen sources and grown at :woC for 6 days (x 1/3). (Nitrogen source
from left: Peptone, Asparagine, Ammonium nitrate, Ammonium chloride,
Sodium nitrate). 62
6. Photograph showing aversion between S. rolfsii Strains ELI and XA2
growing on PDA at 30°C (LEFT) and the absence of aversion between
two inocula of Strain ELI (RIGHT) (x 5/9). 83
7. Photograph showing aversion between 5, rolfsii Strains ELI and XLL
growing on PDA at 30"C (LEFT) and the absence of aversion between
two inocula of Strain EL I (RIGHT) (x 5/9), 84
8. Photograph showing aversion between S. rolfsii Strains XLL and XAI
(LEFT) and XLL and XA2 (RIGHT) growing on PDA at 30°C (x 5/9). 85
9. Photograph of two Petri plates showing similar patterns of aversion
between S. rolfsii Strains XA I and XA2 growing on PDA at 30°C (x 5/9). 86
10. Photograph showing 3 month-old plants of Xanthosoma sagiuifolium
variety 'A mankani fufuo' at the ti me of the pathogen icity test with
the different S. rolfsii strains (x 114). 95
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11. Photograph showing 3 month-old plant of Xanthosoma sagittifolium
variety 'Arnankani pa' at the time of the pathogenicity test with
the different S. rolfsii strains (x 5/9). 96
12. Photograph showing infection of leaves of 3 month-old plant of Xanthosoma
sagiitifolium variety 'Amankani pa' 4 days after introduction of the soil
with S. rolfsii strain EL2 (x 5/9). 97
13. Photograph showing total destruction of the leaves of 3 month-old plant of
Xamhosoma sagiuifolium variety 'Arnankani pa' 20 days after inoculation of
the soil with S. rolfsii Strain EL2 (x 5/9). 98
14. Photograph showing total destruction of the leaves of 3 month-old plants
Xanthosoma sagiuifolium variety 'Amankani fufuo ' 20 days after inoculation
of the soil with S. rolfsii Strain XA2 (x 1/6). 99
15. Photograph showing mini-blocks of corrnel of Xanrhosomasagittifolium variety
'Arnankani fufuo' covered by mycelia of the different S. rolfsii strains
after 8 days at 300e (x 1/3). (From left: Strains XLL, XA I, XA2,
ELI, EL2). 112
16. Photograph showing growth of S. rolfsii Strain EL2 and Strain XA2 on
30 x 30 x 5mm blocks of Colocasia antiquorum (left) and Xanthosoma
sagittifolium variety 'Amankani firaa' (right), respectively (x 2/3). 117
17. Photograph showing extent of sclerotium formation at 30De in 8 days by
s. rolfsii Strain EL2 and Strain XA2, growing on 30 x 30 x 5mm blocks of
Colocasia antiquorum (left) and Xanthosoma sagittifolium variety
~Amankani pal' (right), respectively (x 8/9). 118
18. Photograph showing extent of sclerotium formation at 300e in 8 days by
S. rolfsii Strain ELI and Strain XA I growing on :W x 30 x 5mm blocks of
Colocasia antiquorum (left) and Xanthosoma sagittifolium variety
'Arnankani pa' (right) respectively (x 8/9). 119
x
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TABLE OF CONTENTS
I INTRODUCTION AND LITERATURE REVIEW
II MATERIALS AND GENERAL METHODS
I MATERIALS .. '.' . 12
a Strains of Sclerotium rolfsii Sacc . 12
b. Cocoyam varieties . . . . . . . . . . . 12
1. Peptiole of the Cocoyam . . . . . . . . . . . . . . . . . . . . . . . . . 13
11. Carmel of the Cocoyam . . . . . . . 13
c. Potato Tubers of Irish Potato (Solanum tuberosum L.) . 13
d. Fruits of Cucumber (Cucumis sativus L.) . 13
e. Fruits of Tomato (Lycopersicum esculentum Mill) . 14
f. Chemicals .... 14
2. GENERAL METHODS 14
a. Isolation of the S. rolfsii Strains . . . . . . . . . . . . . . . . . . . . . . . .. 14
b. Maintenance of stock Cultures and preparation of inocula . . . . . . . . .. 15
c. Culture Media 15
1. Potato Dextrose Agar , 15
11 Potato Dextrose Broth 15
11l Synthetic Media for production of Pectolytic Enzymes , 15
IV. Synthetic Medium for assessment of production of
Cellulolytic Enzymes 17
d. Methods of Sterilization 18
e. Replication. . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
f. Determination of Utilization of different Carbon Sources by the
different S. rolfsii strains. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 19
g. Determination of Utilization of different Nitrogen sources 20
h. Assessment of Radial Growth on Solid Agar Medium . 20
1. Determination. of the dry weight of mycelium . . . . . . . . 21
J. Preparation of O.1M Citrate Buffer at pH 5.0 21
k. Measurement. .of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 22
l. Method for.accurate estimation of number of sclerotia formed on
agar medium plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 23
m. Method for determining fresh weight of mature sclerotia 23
n. Method for determining volume of mature sclerotia 23
o. Determination of sclerotium diameter . . . . . . . . . . . . . . . . . . . . .. 24
p. Determination of relative concentration of pectolytic enzymes in culture
filtrates of the S. rolfsii strains 24
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q. Determination of ability of the ~. rolfsii strains to utilize
Cellulose '.' '. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 25
r. Test of phenomenon of aversion among S. rolfsii strains 25
s. Study of infection cushion formation by the S. rolfsii strains . . . . . . .. 26
t. Study of infection of cocoyam petioles by S. rolfsii . . . . . . . . . . . . .. 27
u. Study of rotting of cocoyam cormel tissue by S. rolfsii strains by
inoculating whole cormels . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 27
v. Estimation of degradation of coco yam cormel tissue on the basis of loss
in dry weight " 28
w. Study of production of sclerotia on cocoyam corme1 tissue 29
x. Sclerotium germination test 29
y. Experimental Precautions 30
z. Statistical Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 31
III EXPERIMENT AL DETAILS 32
EXPERIMENT 1
COMPARATIVE RATE OF GROWTH OF THE DIFFERENT STRAINS OF
S. ROLFSII SACC .AND PRODUCTION OF SCLEROTIA ON PDA . . . . . .. 32
EXPERIMENT 2
COMPARATIVE RATE OF GROWTH OF THE DIFFERENT STRAINS OF
S. ROLFSII IN POTATO DEXTROSE BROTH (PDB) . . . . . . . . . . . . . . .. 33
EXPERIMENT 3
GROWTH OF THE DIFFERENT STRAINS OF S. ROLFS!! IN LIQUID
MEDIUM WITH DIFFERENT CARBON SOURCES 34
EXPERIMENT 4
GROWTH OF THE DIFFERENT STRAINS OF S. ROLFSII IN LIQUID
MEDIUM WITH DIFFERENT NITROGEN SOURCES . . . . . . . . . . . . . .. 35
EXPERIMENT 5
MACERATION OF DIFFERENT PLANT TISSUES BY PECTOL YTIC
ENZYMES IN CULTURE FILTRATES OF THE FIVE STRAINS. . . . . . .. 36
EXPERIMENT 6
USE OF CELLULOSE AS CARBON SOURCE BY THE FIVE STRAINS 37
EXPERIMENT 7
A TEST OF PHENOMENON OF AVERSION 39
EXPERIMENT 8 ' ,~
FORMATION OF INFECTION CUSHIONS ON TOMATO FRUITS BY
THE FIVE STRAINS .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 40
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EXPERIMENT 9
INFECTION OF COCOY AM PLANTS GROWING IN S. ROLFSII-
INOCULATED SOILS 41
EXPERIMENT 10
ROTTING OF WOUND - INOCULATED CORMELS OF COCOY AM VARIETIES
BY THE FIVE S. ROLFSII STRAINS . . . . . . . . . . . . . . . . . . . . . . . . .. 42
EXPERIMENT 11
DEGRADA TION OF BLOCKS OF TISSUE OF CORMELS OF THE
DIFFERENT COCOY AM VARIETIES BY THE FIVE STRAINS OF S. ROLFSII 43
EXPERIMENT 12
FORMA TION OF SCLEROTIA BY THE FIVE S. ROLFSII STRAINS GROWING
ON CORMEL TISSUES OF THE DIFFERENT COCOY AM VARIETIES 44
IV RESULTS
EXPERIMENT I
COMPARATIVE RATE OF GROWTH OF THE DIFFERENT STRAINS
OF S. ROLFSII SACCo AND PRODUCTION OF SCLEROTIA ON PDA . . .. 46
EXPERIMENT 2
COMPARATIVE RATE OF GROWTH OF THE DIFFERENT STRAINS
OF S. ROLFS!! IN POT ATO DEXTROSE BROTH (PDB) 51
EXPERIMENT 3
GROWTH OF THE DIFFERENT STRAINS OF S. ROLFSII IN LIQUID
MEDIUM WITH DIFFERENT CARBON SOURCES 54
EXPERIMENT 4
GROWTH OF DIFFERENT STRAINS OF S. ROLFSII IN LIQUID
MEDIUM WITH DIFFERENT NITROGEN SOURCES. . . . . . . . . . . . . .. 58
EXPERIMENT 5
MACERATION OF DIFFERENT PLANT TISSUES BY PECTOLYTIC
ENZYMES IN CULTURE FILTRATES OF THE FIVE STRAINS. . . . . . .. 63
EXPERIMENT 6
USE OF CELLULOSE AS CARBON SOURCE BY THE FIVE STRAINS 77
EXPERIMENT 7
A TEST OF PHENOMENON OF AVERSION 81
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EXPERIMENT 8
FORMATION OF INFECTION CUSHIONS ON TOMATO FRUITS BY
THE FIVE STRAINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 87
EXPERIMENT 9
INFECTION OF XANTHOSOMA SAGITTIFOLIUM AND COLOCASIA
ANTIQUORUM PLANTS GROWING IN S.ROLFSII-INOCULATED SOILS 89
EXPERIMENT 10
ROTTING OF WOUND - INOCULATED CORMELS OF COCOY AM
VARIETIES BY THE FIVE S. ROLFSII STRAINS . . . . . . . . . . . . . . . .. 100
EXPERIMENT 11
DEGRADATION OF BLOCKS OF TISSUES OF CORMELS OF THE
DIFFERENT COCOY AM VARIETIES BY THE FIVE STRAINS OF
S. ROLFSII 108
EXPERIMENT 12.
FORMATION OF SCLEROTIA BY THE FIVE S. ROLFSII STRAINS GROWING
ON CORMEL TISSUES OF THE DIFFERENT COCOY AM VARIETIES .. 113
V. DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 120
VI. SUMMARY......................................... 131
VII. LITERATURE CITED 137
APPENDIX A 143
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I
INTRODUCTION AND LITERATURE REVIEW
Sclerotium rolfsii Sacco is a widely occurring facultative soil parasite of economic
importance, principally in tropical and subtropical regions that are subjected to both
abundant rainfall and high temperatures (Weber, 1931). It has a wide host range. It
causes damping - off of seedlings and collar, stem or root rot of older plants. The
fungus has since been isolated from innumerable host plants. As far back as 1931 Weber
(1931) listed 189 host species from different plant families covering a fern family and
8 monocotyledonous and 42 dicotyledonous families. Several new host plants have since
been recorded. Majority of the hosts are crop plants of great economic importance, and
most of the host plants are annuals or herbaceous perennials.
In the United States nearly every southern state from Florida to California has recorded
its presence, and it has even been reported from Canada (Erdman, 1961).
According to Erdman (1961) the fungus has been recorded in South America from
Argentina, Brazil, Colombia, British Guyana, Surinam and Trinidad. There are many
reports from the West Indies, including Barbados, Bermuda, Cuba, Dominican Republic,
Jamaica, Puerto Rico, and St. Vincent (Erdman, 1961). The few records from Europe
include those from Germany, Italy and Russia (Erdman, 1961), and it occurs, as
expected, in numerous warm countries in Africa including Benin, Congo, Egypt, The
Gambia, Ghana, Malagasy Republic, Nigeria, Sierra Leone, South Africa, Togo, Uganda
and Zimbabwe (Erdman, 1961).
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In the Pacific area it has been found in Australia, Indonesia, Java, Malaysia, the
Philippines and Sumatra (Erdman, 1961).
In Ghana it has been recorded on several plants of economic importance including
Albemuschus esculentus L., Allium cepa L., Arachis hypogea L., Elaeis guineensis Jacq,
Nicotiana tabacum L., Phaseolus Spp, and Xanthosoma sagittifolium Schott (Leather,
1959). It has been estimated that the damage caused by the fungus in Ghana ranges from
5 to 30 per cent (Addison and Chona, 1971).
s. rolfsii invades the host both through wounds, natural opemngs and by direct
penetration of intact surface tissue of the host as, for example, recorded by Darkwa
(1965). The rate of infection by mycelium entering through wounds is higher than that
of mycelium attacking by direct penetration. The delay in infection during direct
penetration is due to two causes. First, penetration process which is known to be
principally mechanical rather than chemical (Hawker, 1950) would need some time to
be accomplished. Also a process which takes up even more time precedes penetration
in S. rolfsii and several other soil pathogenic fungi, such as Rhizotonia solani (Darkwa,
1965; Flentje, 1957; Khadga, Sinclair and Exner 1963; Ullstrup, 1936). The growing
hyphae collect into aggregations called infection cushions, which adhere tightly to the
host surface. From beneath the infection cushions emerge penetrating hyphae which
breach the covering layer of the host organ and invade the host.
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S. rolfsii is able to thrive in the soil for a long time in the absence of a host as a
saprophyte provided organic matter is present in the soil. It also freely forms tiny
sclerotia which are capable of withstanding adverse conditions. The fungus, thus, has
two vegetative phases that are ecologically distinct. First, there is the mycelial
development that forms the heavy white growth from which the fungus gains the common
name" white mould". This migh t also be referred to as the growth phase or pathogenic
phase of the fungus. Secondl y, there is the abundant production of sclerotia which
enables the fungus to survive adverse environmental conditions (Boyle, 1961).
Formation of sclerotia by S. rolfsii is influenced by various environmental factors.
Abeygunawardena and Wood (1957) found that the mean number of sclerotia per Petri
plate on agar medium of initial pH of 1.9, 2.4, 2.8, 4.2, 5.1, 7.8 and 8.0 was 0, 480,
1810, 1320, 1230, 1100 and 0, respectively. Out of a number of carbon sources tested,
abundant sclerotia were formed on galactose, mannitol and sodium carboxymethyl -
cellulose media of the same concentration.
Wheeler and Sharan (1965) obtained the greatest sclerotium formation with 0.1 %
Glucose and the sclerotium number decreased with increasing concentration of glucose
to zero at 3.0 per cent glucose. Increasing the concentration of KH2P04, during the same
investigation, reduced the amount of sclerotia formed. Initiation of sclerotia was
significantly greater at 0.2 % KH2P04 than at 0.0001 % KH2P04. In contrast sclerotium
formation was significantly greater at 0.5 % NaN03 than at either 0.25 or 1.0% NaN03•
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Furthermore, Wheeler and Waller (1965) found that sclerotium initiation appeared to be
regulated by the growing hyphae at the periphery of the mycelial mat. In Petri dishes
of 5, 7, 9 and 13cm diameter, initials did not appear until the lateral extension of the
mycelium was restricted. Also faster growing mycelia produced greater number of
sclerotia than slower growing ones. Thus, cultures kept continuously at the favourable
temperature of 2ye produced a maximum sclerotium initials of 900 per Petri plate at
Day 12 while those kept initially at 15°e for 5 days and then transferred to 25°e
produced maximum initials of 600 at Day 18.
II, ;
Studies by Boswell (1958) on the sclerotia of S. rolfsii is related to the continunity of the
rind of these bodies. An intact rind was associated with slow, depauperate germination.
According to Boswell (1958) the sclerotia became wrinkled during drying and the surface
layers showed considerable cracking. He found that sclerotial dormancy could be broken
by any of the several types of mechanical or chemical methods of scarification.
The growth of S. rolfsii in nature is almost always prominent at or near the soil surface,
and host organs in that zone are commonly attacked by the fungus. Its distribution in the
superficial zones of soil has led to the suggestion that it has a high demand for oxygen.
On the other hand, because S. rolfsii requires an external source of thiamine or at least
one of the moieties which form thiamine, namely pyrimidine and thiazole, it is
encouraged to grow at or near the soil surface which is rich in organic matter.
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One of the valuable crops attacked by S. rolfsii in Ghana is cocoyam, Xanthosoma
sagittifolium Schott and Colocasia antiquo rum Schott. The fungus causes both cormel
and root rot. The disease takes the form of a wet root rot, yellowing and wilting of
leaves and failure to form cormels, followed in severe cases by death. It affects plants
of all ages. It is also the cause of post-harvest rot of the cormels. Decaying cormlets
are usually coated with a typical mycelial felt of the fungus.
Cocoyam, Cassava (Manihot esculenta Crantz), yam (Dioscorea spp Linn) and sweet
potato (Ipomea batatas Poir) provide the main source of carbohydrate for a large
proportion of people of the tropics, especially in the wetter tropics areas, including
Ghana (Doku, 1966). When cereals are in short supply, as they occasionally are, these
root crops supply the. sole source of carbohydrates.
Generall y, cocoyams are not grown on a large scale. However, they are of immense
importance and are- grown in every farm, garden, or small holding. The total annual
production must, therefore, be considerable (Doku, 1966). Among root crops, cocoyams
can be regarded as important because they keep well, both in the field and in store and
are, therefore, available throughout the year.
Due to its excellent storage quality, cocoyam is most often used as travelling food by
local people. When famine threatens, cocoyams, Xanthosoma sagittifolium and Colocasia
antiquorum, are the crop which are most frequently sought for to alleviate the situation.
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Infact when people talk of famine in this country they refer primarily to a shortage of cocoyams
(Doku, 1966).
The uses to which cocoyam can be put are numerous. The tender leaves are the main if not the only
source of spinach for the inhabitants of the humid forest of Ghana and in fact, most Ghanaians. The
protein content per 100g dry weight of the leafofcocoyam is 22.17g (Liefstingh, 1963). The
cormels supply easily digestible starch and are known to contain substantial amount of protein,
vitamin C, thiamine, riboflavin and niacin (Cobley and Steele, 1976; Maduewesi and Onyike, 1980).
The leaves and tender parts of the stem are relished by livestock. The peelings of the cormels are
also fed dried or fresh to goats, sheep, cattle and pigs.
The cormel is used in different forms as described by Karikari (1971). It is peeled and boiled and
pounded into 'fufu' that is taken with soup. The boiled carmel is often also eaten directly with stew.
The unpeeled carmel is roasted and the skin removed when ready for eating. The boiled or roasted
form may also be mashed and palm oil added and taken with either fish or roasted groundnut.
The Akan name 'Okumkom' meaning, killer of all hunger, which is applied to cocoyam no doubt
portrays its importance to the whole country and to the Akans in particular who form the major tribe
of this country (Karikari, 1971).
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Karikari (1971) listed seven cocoyam varieties found in Ghana and described them as
follows:
'Arnankani pa' (proper cocoyam) or 'Amankani kokoo' (red cocoyam). Large plant
182.9 - 243.8cm high; leaf sagitate, dark green lighter on the under surface, prominent
venation, petiole reddish-purple, tubers pink. The plant flowers in the wetter districts.
This is the commonest variety and is said to possess all the desirable properties of
cooking, texture and taste.
'Amankani fufuo ' (Light-coloured cocoyam). This is similar to 'Arnankani pa'. Leaf
not quite so dark, petiole purple, tubers white.
'Arnankani fitaa' (White cocoyam). Above ground parts resemble" Amankani fufuo'
but the plant is much more delicate. Tubers are white and the cormels have one or
several constrictions. They are smaller than in the preceding two varieties. 'Amankani
serwah' (Serwah was the name of a very beautiful Ashanti Queen). This resembles the
'Arnankani fitaa' except that the cormel are very white and the skin splits and become
peelable.
'Amankani Kyirepe' (hard cocoyam). This resembles the 'Arnankani antwibo' variety.
It is, however, rich in sugar and is therefore used in sweeting foods. It contains some
poisonous properties and therefore needs to be boiled for 12 hours before been eaten.
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'Amankani antwibo ' (cocoyam which can choke one). Similar to 'Arnankani fitaa'.
Petioles are very pale green and corrnels pink and unconstricted. The central stem of the
plant is edible.
Colocasia antiquorum is known as 'Kooko ' in Twi. Petioles are pale green. The tubers
are white and smaller than the 'Amankani ' varieties. The central stem is edible. Before
the advent of the 'Amankani ' varieties it was the favoured variety but is seldom eaten
now except in cases of scarcity. However, because of its softness, it is still preferred
by old people who find it easier to eat.
For such a valuable: crop any disease that could decimate the produce should, naturally
be a matter for concern.
Nakata (1925) stated that S. rolfsii is a group species comprising numerous biological
forms which can be distinguished by the phenomenon of aversion. Aversion was
reported to occur between different strains of tile fungus but not between two cultures
of the same strain. When two colonies of the same strain are started at opposite sides
of an agar plate, the colonies grow towards each other and meet. Sclerotia are then
produced along the meeting zone due to obstruction to extensional growth. However,
when two different strains are raised in similar manner, their colonies never meet. Their
advancing edges eventually corne to a stop as they get near to each other and become
separated by a distinct inhibition zone. Each colony thereby forms its own row of
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sclerotia along the boundary resulting in two rows of sclerotia separated by the zone of
inhibition.
The presence of distinct strains of s. rolfsii with different physiological characteristics
must be the underlying cause for the differences in reports on the physiology of the
fungus by different workers. Taking the response to nutrients as an example.
Higgins (1927) found maltose and lactose among several carbohydrates and a few organic
acid tested, to support the poorest growth. In contrast, Johnson and Joham (1954) found
that S. rolfsii used fructose, galactose, glucose, lactose, mal tose, raffinose, and starch
among other carbohydrates to more or less the same degree. However, cellulose agar,
gummastic, gum guaiac, gum arabic and rhamnose were not used by the fungus.
Abeygunawardena and Wood (1957) found fructose to be the best carbon source for
growth while Townsend (1957) found sucrose to be the best at low concentrations but
glucose to be the best at high concentrations with lactose supporting poor negative
growth. Darkwa (1965) found quite a different order of preference. Of the
carbohydrates he tested, the order of utilization was starch, maltose, fructose, glucose,
cellulose, lactose, glycerol and galactose. Galactose was barely used.
Some differences are also found in results of investigations on the use of Nitrogen
compounds for growth by S. rolfsii. H iggi ns (1927) observed that S. rolfsii did not
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appear to utilize readily inorganic nitrogen either as nitrates or ammonium salts.
Abeygunawardena and Wood (1957) also found the organic nitrogen asparagine and
peptone to be more readily utilized than ammonium salts and sodium nitrate. But
Townsend (1957) found asparagine, peptone and potassium nitrate to support good
growth and Darkwa (1965) reported that asparagine, peptone, urea, ammonium chloride
and ammonium nitrate were by far better Nitrogen sources than alanine and glycine.
This important knowledge of presence of distinct strains should be considered in any
S. rolfsii - host relationship. It is well-known that many bacterial and fungal parasites
are themselves an assemblage of strains differing genetically and liable to variation. The
existence of distinguishable S. rolfsii strains in Ghana has hitherto not been examined.
The effect of different strains on crops has, therefore, also not been studied. The aim
of the project was, first, to investigate the possible existence of S. rolfsii strains in
Ghana. If they do exit, it will be important to find out whether the seven cocoyam
varieties in Ghana are equally susceptible to the different S. rolfsii strains. A particular
variety that would exhibit tolerance or resistance to as many S. rolfsii strains as possible
would be a good choice for cultivation in Ghana, as the existing cultural and chemical
methods of control either have not made any impact on the disease in Ghana so far or
have never been tried because of the cost involved. Posnette (1945) could not reduce the
incidence of the disease by application of woodashe.
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There are many other suggested ways of combating the fungus. The most recommended
cultural practices are burning of crop thrash with the inoculum, and digging in the top
soil to depths which would deprive the fungus of oxygen.
Chemical control is also possible. The growth of S. rolfsii was inhibited by atrazine
(2-chloro-4-ethylamino-6-isopropyl-amino-S-triazine), (Curl and Funderburk, 1966) and
by paraquat (1, l-dimethyl-4, 4-bipyridium salt) (Rodriquez - Kabana et al., 1967).
Curl and Funderbunk (1966) found that sclerotia production by S. rolfsii was reduced in
atrazine - treated soil. Clerk and Bimpong (1969) showed that prometryne (4, 6-bis
(isoproylamino-2-methylthio - 1,3,5 - triazine) and ramrod (2 - chloro - N -
isopropylacetanilide) were more toxic than atrazine.
This thesis contains mainly results of experiments set out to study the
a. physiology of different isolates of S. rolfsii from the Legon-Achimota area,
b. identify strains among the isolates, and
c. study pathogenicity of the identified strains with special reference to collar rot and
cormel rot of cocoyam in cocoyam varieties, 'Arnankani fitaa' (white cocoyam);
'Amankani fufuo' (light coloured cocoyam); 'Amankani Kyirepe' (hard cocoyam);
'Arnankani pa' (proper cocoyam) of Xanthosoma sagittifolium and Colocasia antiquo rum
(kooko).
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II MATERIALS AND GENERAL METHODS
I MATERIALS
a Strains of Sclerotium rolfsii Sacc
The five different strains of Sclerotium rolfsii used were obtained at different areas in
Legon and Achimota and given code numbers according to their sources.
Strain XLL from Xanthosoma sagittifolium Schott at University of Ghana, Legon.
Strain XAI from Xaruhosoma sagittifolium at Achimota, 20km west of Legon.
Strain XA2 from Xanthosoma sagittifolium at Achimota.
Strain ELI from Elaeis guineensis Jacq at Sinna's Garden, Faculty of Agriculture,
University of Ghana, Legon.
Strain EL2 from Elaeis guineensis at Forecourt, Faculty of Agriculture, University of
Ghana, Legon.
Stock cultures were maintained on Potato Dextrose Agar Slants in MaCartney tubes and
stored in the refrigerator. They were sub-cultured at fortnightly intervals.
b. Cocoyam varieties
Five varieties of local cocoyams were used in this investigation. Four of these, namely
'Amankani fitaa', 'Amankani fufuo', 'Arnankani Kyirepe', 'Amankani pa' are varieties
of Xanthosoma sagittifolium Schott and the remaining one Colocasia antiquorum.
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They were supplied by the Agriculture Research Station of the University of Ghana at
Kade, Ghana. Both the cormels and petioles were used in some of the experiments.
i. Peptiole of the Cocoyam
The cocoyam varieties were raised in garden loam soil in black polythene bags (36.5 x
22.5cm) with drainage holes at the bottom for 12 weeks and then used. The ages of the
leaves and, therefore, the petioles were related to the positions of the leaves on the plant.
ii. Cormel of the Cocoyam
The corrnels of the different cocoyam varieties were purchased when needed from a
particular cocoyam seller at the Central Market, Tema with whom a standing order had
been placed.
c. Potato Tubers of Irish Potato (Solanum tuberosum L.)
Tubers ofIrish Potato (Solanum tuberosum L.) used in the preparation of Potato Dextrose
Agar and Potato Dextrose Broth and in 'Pectolytic Enzyme Tests' were purchased from
the Makola Market in Accra when needed and used the same day.
d. Fruits of Cucumber tCucumis sativus L.)
Fresh Fruits of Cucumber (Cucumis sativus L) used in 'Pectolylic Enzyme Test' were
purchased from Makola Market in Accra on the day they were to be used.
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e. Fruits of Tomato (Lycopersicum esculetuum Mill)
Fruits of tomato (Lycopersicum esculentum Mill) were used in studying the formation of
infection cushions of the different S. rolfsii strains, Firm unblemished ripe fruits were
purchased from Makola Market in Accra on the clay they were to be used.
f. Chemicals
All chemicals used in the investigation were from British Drug House, Poole, England
and OXOlD Limited, Basingstoke, Hampshire, England.
2. GENERAL METHODS
a. Isolation of the S. rolfsii Strains
Mature brown-coloured sclerotia were carefully removed with a pair of sterile fine
forceps from the host plant and placed in distilled water to wash off any adhering soil
particles. They were then surface-sterilized by immersing them in Sodium hypochlorite
(1.0%) for three minutes, rinsecl after that in three changes of sterile distilled water and
inoculated onto sterile Potato Dextrose Agar in sterile Petri dishes. The growing
mycelium was subcultured after 4 days. A second sub-culturing was again done to
ensure a completely pure culture.
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b. Maintenance of stock Cultures and preparation of inocula
Stock culture of each S. rolfsii strain was maintained on Potato Dextrose Agar slants in
MaCartney's tubes in the refrigerator, and subcultured fortnightly. Whenever an
experiment was to be carried out, the fungus was raised on Potato Dextrose Agar in
sterile Petri dishes for 4 days and the mycelium then used as the inoculum. The
inoculum was always taken from the advancing edge of the culture. The culture was
allowed to grow for 12 days in cases where mature sclerotia were required as inoculum.
C. Culture Media
i. Potato Dextrose Agar
An amount of 200g of peeled Irish potato was thoroughly washed, cut into small pieces
and boiled in 500ml of distilled water for 15 minutes. The extract was then strained with
muslin cloth and made up to one litre with distilled water. Fifteen grams of Agar and
109 of Dextrose were added. The mixture was stirred and warmed in a water bath at
80°C to melt the agar before the medium was autoclaved.
ii Potato Dextrose Broth
Prepared in the same way as Potato Dextrose Agar without the addition of Agar.
iii Synthetic Media for production of Pectolytic Enzymes
The different S. rolfsii strains were raised in several synthetic liquid media and the
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The standard medium consisted of:
Peptone 1.0%
MgS04.7H20 0.5%
KH2P04 0.1 %
Thiamine 0.005%
Distilled water 1L
The various media were made by adding different concentrations of either Glucose or
Pectin, referred to as Glucose and Pectin media, respectively.
The Glucose media contained the following Glucose concentrations:
Medium 1 0.5%
Medium 2 1.0%
Medium 3 1.5%
Medium 4 2.0%
The Pectin media contained the following Pectin concentrations:
Medium 1 0.5%
Medium 2 1.0%
Medium 3 1.5%
Medium 4 2.0%
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Other media, Glucose-Pectin Media, contained both Glucose and Pectin in different ratios
as follows:
Medium 1 Glucose 0.75 %, Pectin 0.25 %
Medium 2 Glucose 0.50%, Pectin 0.50%
Medium 3 Glucose 0.25%, Pectin 0.75%
Several 250ml Erlenmeyer flasks each containing 30ml of the test medium were plugged
with non-absorbent cotton wool covered with aluminium foil and then autoclaved. The
aluminium foil prevented entry of condensed water into the medium and it was removed
after the medium had been inoculated. This procedure was followed throughout this
investigation. Each flask was inoculated after cooling with a 3mm - disc of the
mycelium of the fungus.
iv, Synthetic Medium for assessment of product ion of Cellulolytic Enzymes
The relative amounts of cellulolytic enzymes produced by the different S. rolfsii strains
were assessed using the following medium useel in cellulolytic enzyme studies by Ryan,
Beadle and Tatum (1943)
Potassium nitrate (KNOl) S.Og
Potassium dihydrogen phosphate (KH2P04) l.Og
Magnesium sulphate (MgS04' 7H20) 0.5g
Thiamine O.Smg
Yeast extract l.Og
*Micronutrient l Oml
Distilled water lL
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"Micronutrient Solution
O.lOmg
O.Olmg
Ferrous phosphate (FeP04.2H20) O.20mg
Manganesium sulphate (MnS04.5H20) O.02mg
O.02mg
Zinc sulphate (ZnS04.7H20) O.15mg
Distilled water lL
Aliquots of 20ml were dispensed into several 250ml Erlenmyer flasks and one 7cm -
..
diameter WhatmanNo.3 filter paper put into each. The flasks were plugged with non-
absorbent cotton wool and the medium autoclaved.
d. Methods of Sterilization
All media and distilled water used were sterilized by autoclaving at lTkg/crrf steam
pressure at 121°C for 15minutes. All glassware were sterilized by heating at 160°C
for 18 hours in an electrically heated oven (Gallenkamp Model, Town and Mercer Ltd.,
Croydon, England).
Inoculation was done in the inoculating room under the Laminar Microflow Chamber.
The air conditioner and the Laminar Flow Cabinet were switched on for about 30minutes
to reduce air microflora in the room before inoculation was done. Inoculating pins,
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inoculating loops and cork borers were flame - sterilized pnor to use. Surface
sterilization of tomato fruits, cocoyam cormels and blocks of tissue of coco yam cormels
was done by immersing the materials in 5.0% Sodium hypochlorite for five minutes.
They were then rinsed in three changes of sterile distilled water.
e. Replication
Four replicates per treatment were used in all the various experiments performed.
f. Determination of Utilization of different Carbon Sources by the different S. rolfsii
strains
The ability of the 'different S. rolfsii strains to utilize different carbon sources was
investigated. The common quantity of carbon source in laboratory media, 1.0%, was
used in this test. Five separate preparations of a basal medium containing Peptone, 1%;
MgS04.7H20, 0.1 %; KH2 P04, 0.1 %; Thiamine, 0.005 % and 1L distilled water were
made and to which was added either 1.0% fructose, glucose, maltose, starch or sucrose.
Each preparation was tben divided into five lots and allocated to the five S. rolfsii strains.
The medium for each was then dispensed in quantities of 30ml into four 250ml
Erlenmeyer flasks. All the flasks were then autoclaved , cooled and each inoculated with
3mm - mycelium disc of the test S. rolfsii strain. The inoculated flasks were incubated
at 30°C for 6 days and the mycelium harvested with filter paper funnels. The dry weight
of the mycelium was then determined as described in section 2(i).
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The pH of the culture filtrate was then measured. A reserve medium in each case was
autoclaved, cooled and the pH measured to provide the initial pH of the medium.
g. Determination of Utilization of different Nitrogen sources
The preceding procedure was followed to determine the ability of the different S. rolfsii
strains to utilize Ammonium chloride, Ammonium nitrate, Asparagine, Peptone and
Sodium nitrate. Each of these Nitrogen sources was incorporated at a concentration of
0.1 % into a basal medium of Glucose, 1.0%; MgS04.7H20, 0.1 %; KH2P04, 0.1 %;
Thiamine, 0.005 % and lL distilled water.
The inoculated 250ml - Erlenmeyer flasks each containing 30ml of the test medium were
also incubated at 30°C for 6 days. The dry weight of the mycelium produced, the initial
pH of the medium and the final pH of the culture filtrate were determined as in the
previous experiment.
11. Assessment of Radial Growth on Solid Agar Medium
About 20m1 of the medium was poured into each of 9.0cm - diameter sterile Petri dishes
and allowed to solidify. Two diameters at right angles to each other were drawn at the
bottom of the Petri dishes. Each plate was inoculated at the intersection of the two
diameters with a 3mm - disc of the mycelium from the growing edge of 4 - day old of
the culture of the fungus.
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The diameter of each growing culture was measured along the two diameters and the
mean for the replicates calculated.
i. Determination of the dry weight of mycelium
The mycelium of cultures in liquid medium was collected on a funnel-shaped previously
dried and weighed Whatman's No. 1 filter paper. The filter paper with the harvested
mycelium was dried in an electrically heated oven (GallenKamp oven 300, plus series)
at 60°C for 24 hours. The filter paper carrying the dried mycelium was weighed after
being allowed to cool in a desiccator. The dry weight of the mycelium was then
calculated.
J. Preparation of O.JM Citrate Buffer at pH 5.0
From the Henderson - Hasselbach equation of buffers:
pH = pKa + log [Salt]
[Acid]
pKa of citric acid at pH 5 is 4.7
(Segel, 1975).
Substituting pH = 5 and pKa = 4.7 into equation
5 = 4.7 +log [Salt]
[Acid]
. ;; ,~i '
[Salt] = log:' 0.3
[Acid]
= 1.99
= 2.0
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Therefore O.lM Acid should react with O.2M salt. For the same molar concentration,
therefore, the volume ratio will be 1:2 for the Salt and Acid, respectively.
[Salt] - b/lO = b Molarity of Salt x Vol. Of salt (V-Salt) I
[Acid] aiiOO a Molarity of Acid x Vol. Of Acid (V-Acid) 2
For same malority and a final buffer volume of lOOml
[Salt] Vsalt = 1
[Acid] Vacid 2
2Vsalt Vacid
(b = no. of moles of salt; a = no. of moles of acid). Therefore, l Ornl of 1M Citric acid
was mixed with 20ml of 1M disodium hydrogen phosphate (NazHP04) and the volume
topped to 100mi with sterile distilled water to give O.lM Citrate buffer of pH 5.0.
The pH of the prepared 100ml stock of O.lM citrate buffer was confirmed with a pH
meter.
k. Measurement of pH
The pH of all media, culture filtrates and the O.IM citrate buffer was measured using a
pH meter (TOA pH meter, HM-60s. aSK - 11478, OGAWA SEIKO co. LTD., Japan).
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I. Method for accurate estimation of number of sclerotia formed on agar medium
plates
Some of the S. rolfsii strains produced a large number of sclerotia on agar medium
plates. In order to ensure an accurate counting, the culture was divided into sectors with
lines drawn at the bottom of the Petri dish with a felt pen into eight equal sectors. It was
, !'
then easier to count 'accurately the sclerotia in each sector using a Tally counter and the
total number calculated.
Ill. Method for determining fresh weight of mature sclerotia
A reliable method of determing fresh weight of the mature tiny sclerotia adopted in this
investigation was to weigh together 100 randomly selected sclerotia. Four determinations
were made for each S. rolfsii strain and the mean per 100 sclerotia calculated. The
weighing was done with an Electronic Balance ER - 108A, A & D Co. Ltd, Tokyo
Japan .
.t:t. Method for determining volume of mature sclerotia
The procedure usedwas similar to that of weight determination. One hundred mature
sclerotia were randomly selected from the culture plate and put in a lOml measuring
cylinder containing 5ml of distilled water. The subsequent rise in the level of the water
represented the volume of the sclerotia. Four determinations were made for each
S. rolfsii strain and the mean per 100 sclerotia calculated.
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o. Determination of Sclerotium diameter
The diameter of the sclerotium was measured under the microscope using the eye piece
graticule (GRATICULE LTD., TONBRIDGE, KENT ENGLAND).
p. Determination of relative concentration of pectolytic enzymes in culture filtrates of
the S. rolfsii strains
The experiment studied the time it took for maceration of thin discs of Irish potato tuber
and pericarp of cucumber immersed in the culture filtrates to occur following the method
used by Cole and Wood (1961). For each test, the replicate culture filtrates were pooled
and used. ., i
Plugs, 1.0cm in diameter were removed with a Number 8 cork borer from either the
potato tuber or cucumber pericarp and placed separately in distilled water in Petrin
dishes. In each case, 0.5mm - thin discs were carefully cut with a surgical blade from
the plug. The discs were washed in sterile distilled water and drained on filter paper.
An amount of 2ml of the test filtrate in a mini - Petri dish (6cm diameter) was adjusted
to pH 5.0 by adding 0.5ml of O.lM citrate buffer of pH 5.0, and six discs were then
introduced. Each disc was picked and gently pulled from two opposite ends at 5 minutes
intervals using two pairs of blunt forceps. The occasion came when the disc broke apart
when it had been macerated by the constituent pectolytic enzymes of the culture filtrate.
The time was noted. The Enzyme Activity was calculated by the formula:
1
x 100
mean time of maceration of the six discs
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q. Determination of ability of the S. rolfsii strains to utilize Cellulose
The experiment measured indirectly the relative level of cellulolytic enzymes In the
culture filtrate of the vanous S. rolfsii strains. Several 7 ern-diameter discs of
Whatman's number 3 filter paper were oven dried at 60°C for 24 hours and weighed
singly. Each was placed in a 250ml Erlenmeyer flask containing 20ml of the medium
described in Sectio~ 2(c)iv. to whieh 1.0% Glucose was added as a booster. The flasks
were autoclaved and the medium in each was inoculated with a 3mm mycelial disc. The
non-absorbent cotton wool plugs were covered with cellophane to prevent excessive water
loss. The flasks were. incubated at 30°C for 7 weeks. The remaining fragments of the
decomposed filter paper were carefully collected into a pre-weighed aluminium foil cap,
from each t1ask, dried at 60°C for 24 hours, and weighed. The weight of the constituent
minerals of the medium was substracted from the computed dry weight of the filter paper
used by the fungus, (Garrett, 1962; Forbes and Dickson, 1977).
r. Test of phenomenon of aversion among S. rolfsii strains
About 20ml of Potato Dextrose Agar was poured into each of 9. Oem diameter sterile
Petri dishes and allowed to solidify. Aversion was determined by inoculating one plate
with two 3mm diameter mycelial inocula, one at the edge of the plate and the other
diametrically opposite at the other edge.
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The inocula belonged to two different strains or were of the same strain. The inoculated
plates were incubated at 30°C until the plates were covered. They were then examined
to see whether the two colonies grew into each other-which would occur where there was
no aversion - or just stopped short when the growing edges come close to each other
leaving a zone of bare agar between them indicating incidence of aversion.
s. Study of infection cushion formation by the S. rolfsii strains
Infection cushion formation by different S. rolfsii strains was compared using
the mean number formed on unit tomato fruit surface area of 5x5mm and
11 the mean diameter, measured with the aid of an eye-piece graticule of 50
randomly selected infection cushions.
Surface-sterilized mature semi-ripe tomato fruits were placed in plastic boxes (24.0 x
12.0 x 6.0cm) with humid internal atmoshpere maintained with miostened filter paper.
The fruits were surface-inoculated by placing mature sclerotia on the intact surface of
each. Four sclerotia were placed equidistantly on each fruit. After four days, tiny
squares of epidermal strips (5x5mm) were removed, stained with cotton blue in
lactophenol and mounted on glass slides in drops of plain lactophenol, keeping the
external surface uppermost. The infection cushions on the epidermal strips were
observed under the microscope and the details mentioned above were recorded.
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t. Study of infection of cocoyam petioles by S. rolfsii
Three-month old cocoyam plants were inoculated by the different strains of S. rotjsii.
Cultures of the test fungus were grown in Potato Dextrose broth in 250ml Erlenmeyer
flasks each containing 30ml of the medium. The culture filtrate was poured off after 6
days' incubation. Sterile distilled water was then used to rinse the mycelium to remove
traces of the acidic filtrate and poured off. The mycelium was used to inoculate the
growing potted cocoyam plants in garden loam soil of pH 5.0 and 25.0% WHC.
The content of one flask was stirred into the soil of one pot (36.5 x 22.5cm) and the
mouth of the pot covered with black polythene bag for two days to enable the fungus to
become established in the soil. The cumulative number of petioles which had been
infected were counted after 4, 8, 12, 16, 20 and 24 days.
tJ. Study of rotting of cocoyam cormel tissue by S. rolfsii strains by inoculating whole
cormels
Mature cocoyam cormels were inoculated with the different strains of S. roljsii and the
rotted areas measured after 8 days.
Mycelium was raised in Potato Dextrose Broth at 30°C for 4 days and used as inoculum.
The culture filtrate was poured off and the mycelium rinsed with sterile distilled water.
The mycelium was then transferred into a sterile Petri dish containing 10mi sterile
distilled water and macerated with a pair of blunt forceps.
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The cocoyam cormels were surface-sterilized and placed in 24.0 x 12.0 x 6.0cm plastic
boxes with tight-fitting lids. A plug, 3mm deep, was then removed with a Smm - cork
borer from opposite sides, at both the apical and basal halves of the cormel. Into each
of the four cavities was placed the same amount of macerated mycelium using a
microspatula. The plug of cocoyam tissue removed was replaced and the wound sealed
with vaseline. The inoculated cormels were incubated at 30°C for 8 days. Each cormel
was then cut transversely with a sharp scapel along the plane of the two opposing plugs.
The radii of the rot along the surface and depth of the rot from the inoculum were
measured.
v. Estimation of degradation of cocoyam cormel tissue on the basis of loss in dry weight
Cormels of the cocoyam were peeled and each was cut transversely into two halves.
Blocks, measuring 1.0 x 1.0 x O.Scm, were cut from both halves and surface sterilized.
Five blocks were selected from each batch and dried at 80°C for 24 hours. They were
then weighed individually and the mean dry weight per block for the two batches
calculated. Blocks for the test were surface-sterilized with 5% Sodium hypochlorite for
S minutes and rinsed in three changes of sterile distilled water and drained on sterile
filter paper. The blocks were then placed on 4-day old PDA culture plates of S. rolfsii,
five per Petri plate, and incubated at 30°e. One Petri plate of every treatment was
withdrawn after 2, 4, 6 and 8 days. The adhering mycelium on the blocks was carefully
removed with a pair of fine forceps and the blocks were oven dried at 80°C for 48 hours
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and weighed. The mean dry weight for each batch and percentage loss in dry weight
were calculated.
w. Study of production of sclerotia on cocoyam cormel tissue
Flat blocks of peeled corrnels, measuring 30 x 30 X 5mm, were cut and surface sterilized
with 5% Sodium hypochlorite for S minutes and rinsed in three changes of sterile
distilled water. Each block was placed in a sterile empty Petri dish and inoculated with
a 3mm mycelial plug of a 4-day old S. rolfsii culture growing on PDA. The preparation
was incubated at 30°C and inspected everyday.
Recordings were made of the following:
time of first appearance of sclerotia.
11 time melanin deposition was observed in the earliest formed sclerotia.
111 number of sclerotia formed on each block in 12 days; and
IV volume of 100 mature brown sclerotia.
x, Sclerotium germination test
S. rolfsii cultures were raised on blocks of cocoyarn cormel tissue in Petri dishes at 300C
for 12 days. Forty mature and brown sclerotia were then selected randomly and seeded
on fresh PDA Petri plates and incubated at 30°C. The cumulative number of germinated
sclerotia after 12, 24, and 36 hours was recorded.
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y. Experimental Precautions
1. Glassware cleaned with detergents were rinsed several times with tap water to
remove all traces of the detergent and then air-dried.
11. Petri dishes were only half-opened when pounng media In order to avoid
con tamination.
111. There were usually four replicates for the various treatments. Whenever two
replicate cultures became contaminated the entire set of four was discarded and
the experiment repeated.
IV. The Laminar Microt1ow Chamber in the inoculating room was cleaned with
disinfectant and switched on for 30 minutes immediately before being used.
v. Mycelium serving as inoculum for all experiments was always obtained from the
advancing edge of 4-day old cultures.
VI. Oven-dried filter paper usually lost some weight because of the heating. Filter
paper used throughout the investigation for harvesting mycelium from broth
media was, therefore, heated at 60°C for 24 hours prior to use.
Vll. Filter paper with oven-dried mycelium was conveyed to the balance room in a
closed desiccator to avoid absorption of moisture.
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z. Statistical Analysis
Standard error of means was calculated using the formulae:
Variance
n - 1
Standard Error of mean (S. E. M) ~v/n
Significance of the difference between the means in the Tables of results was determined
by means of Con fidence Limits at 9S %.
Confidence Limit = mean +(1.96 x Standard Error of Mean) (Kershaw, 1973).
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TIl
EXPERIMENTAL DETAILS
The results of the experiments described here have been presented in the same order and
under the same heading in Chapter IV - RES ULTS.
EXPERIMENT 1
COMPARATIVE RATE OF GROWTH OF THE DIFFERENT STRAINS OF
S. ROLFSII SACC AND PRODUCTION OF SCLEROTIA ON PDA
Potato Dextrose Agar (PDA) was useel in studying the radial growth of the five S. rolfsii
strains: XLL, XA1, XA2, ELI and EL2. Agar plates in 9cm diameter Petri dish were
inoculated at the center with a 3mtn - diameter mycelium of the test strain and incubated
at 30°C. There were four replicates for each strain. The diameter of each culture along
two predetermined diameters drawn at the bottom of the Petri dish was measured at 12hr
intervals until either the plate was completely covered or growth stopped. The mean of
the 8 diameter measurements for each treatment at each recording time was calculated
(Table 1). The plates were then left at the end of the growth studies for a total of 12
days to enable sclerotia to develop. All the sclerotia, both brown mature and pale
immature, on each plate were then coun ted and the mean per plate calculated (Table 2).
A photograph was taken of the samples of the five treatments to show the formation of
sclerotia by the five strains (Plate I).
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One hundred of the mature sclerotia were then picked at random and weighed together
to obtain the fresh weight, This was done for all the four replicate plates and the mean
per hundred sclerotia was calculated (Table 2),
The volume of the hundred sclerotia selected from each plate was also determined by
water displacement method. The mean per hundred sclerotia was then calculated (Table
2). Finally the diameter of SO mature sclerotia of each isolate was measured and the
mean calculated in each case (Table 2).
EXPERIMENT 2
COMPARATIVE RATE OF GROWTH OF THE DIFFERENT STRAINS OF
S. ROLFSII IN POTATO DEXTROSE BROTH (PDB)
Since the surface growth method does not take into account mycelium growing in the
agar medium, Potato Dextrose Broth was used in a subsequent experiment to study again
growth of the five S. rolfsii strains. At desired intervals the mycelia were harvested,
dried at 60°C for 24 hours and weighed. They were harvested on pre-weighed filter
paper and the difference in weight represented the dry weight of the mycelium.
Twenty-one 250ml: Erlenmeyer flasks each containing 30ml of the medium, were
prepared for each strain. Twenty of the flasks were each inoculated with one 3mm -
diameter culture disc. The remaining flask was set aside after autoclaving the media and
used to determine the initial pH.
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The inoculated flasks were incubated at 30De and four flasks of each strain were
withdrawn after 4, 6, 8, 10 and 12 days in that order. The cultures were each harvested
separately, dried and weighed and the mean dry weight calculated (Table 3)
The culture filtrates of the four replicates of each treatment were pooled and the pH
measured with a pH meter. The values of the pH obtained are presented in Appendix
A and the data used to plot a graph alongside the graph of the dry weight of the
mycelium (Fig 1)
EXPERIMENT 3
GROWTH OF THE DIFFERENT STRAINS OF S. ROLFSII IN LIQUID MEDIUM
WITH DIFFERENT CARBON SOURCES
The ability of the five. strains to use different carbon sources was next investigated as
they may most likely use the different compounds to varying degrees.
The dry weight of mycelium of each strain produced in six days was recorded. The
carbon sources tested were fructose, glucose, maltose, starch and sucrose at a
concentration of 1.0%. As in Experiment 2, the cultures were raised in 250ml
Erlenmyer flasks each containing 30ml of the liquid medium and the inoculated flasks
were incubated at ~O~,C. The mycelia were harvested separately after 6 days on pre-
weighed filter paper .and dried at 60De for 24hours. The pH of the filtrate at the end of
the incubation period and the pH of an autoclaved uninoculated medium representing the
initial pH were measured with a pH meter.
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In addition to the dry weights obtained which are presented in a tabular form in Table
4, photographs were taken of the cultures. Photographs of growth of Strain EL2 and
Strain XA 1 in media containing the different Carbon sources have been presented as
examples (Plates 2 and 3).
EXPERIMENT 4
GROWTH OF THE DIFFERENT STRAINS OF S. ROLFS!! IN LIQUID MEDIUM
WITH DIFFERENT NITROGEN SOURCES
The ability of the five strains to use different Nitrogen sources was also studied. The
same procedure was followed using the same basal medium containing 1.0% Glucose as
Carbon source and 0, 'I% of either ammonium chloride, ammonium nitrate, asparagine,
peptone, or sodium nitrate as Nitrogen source. The dry weight of mycelium produced
after 6 days of incubation at 30°C was determined as in Experiment 3. The initial pH
of the autoclaved uninoculated medium in each case and the final pH of each filtrate were
measured. Data of the dry weight and the pH obtained were put together in a composite
table (Table 5). To; illustrate the appearance of some of the cultures, photographs of
cultures of Strains EL2 and XAI in media containing the different Nitrogen sources have
,~.';
been presented (Platesd and 5).
It was observed that the strains showed the same order of ability to use both Carbon and
Nitrogen sources. The results of both Chapter 3 and 4 were therefore put together in Fig
2 to show the corresponding order of the ability of the isolates to use the Carbon and
Nitrogen sources. ;,.: ,
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EXPERIMENT 5
MACERA TION OF DIFFERENT PLANT TISSUES BY PECTOLYTIC ENZYMES
IN CULTURE FILTRATES OF THE FIVE STRAINS
The production of pectolytic enzymes into culture filtrates is one of the parameters by
which a fungus species or strain can be characterized. The amount of pectolytic enzymes
secreted by the different S. rolfsii strains into the culture filtrate was examined. The
procedure used has been described in General Methods Section 2 (p). Thin (lmm thin)
discs, 10mm in diarrieter , of either Irish potato tuber or cucumber pericarp were put in
separate filtrates of 6 day old cultures of the test fungi grown in different types of media
at 30De. The times, taken by the constituent pectolytic enzymes of the filtrate to macerate
~.
a total of six discs were recorded and the mean calculated. The mean enzyme activity
(lit x 100) calculated, where t = mean time of maceration. The fungi were grown in
media with either Glucose or Pectin as Carbon sources of different concentrations as
follows:
~ media with Glucose concentration of 0.5, 1.0, 1.5, and 2.0 per cent.
~ media with Pectin concentration of 0.5, 1.0, 1.5 and 2.0 per cent.
~ media with a combination of Glucose and Pectin:
0.75 % Glucose and 0 r25 % Pectin;
0.5 % Glucose and 0.5 % Pectin;
0.25 % Glucose and 0; 75 % Pectin.
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The pH of the test media was standardized by adjusting with 0.1 M citrate of pH 5.0.
The mycelium in each case was harvested as in the preceding experiments and the dry
weight recorded. The final filtrate pH was measured at the end of the growth period.
The results of these various tests are recorded in Tables 6 to 13.
EXPERIMENT 6
USE OF CELLULOSE AS CARBON SOURCE BY THE FIVE STRAINS
Since cellulose is a major component of plant cell walls the ability of the five strains to
use cellulose as carbon. source was investigated. Filter paper was used as the cellulose.
The basal medium described at Section 2 (q) of the General Methods was used. Several
250ml Erlenmyer's flasks each containing 30ml of the basal medium were autoclaved.
Discs of 7cm - diameter filter paper were autoclaved separately and one was put in the
autoclaved basal medium in each flask and then inoculated with 3mm - diameter culture
discs.
At the end of the incubation period, the mycelium and sclerotia were carefully removed
with a pair of forceps and the remains of the filter paper harvested by picking with a
pair of fine forceps into previously weighed aluminium foil cups. The cups were put in
an oven of 60°C for 24 hours to dry the fragmented filter paper and weighed to obtain
the dry weight.
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Because of the way the fragmented filter paper was harvested it was possible that some
pieces might have been left unharvested. It was therefore considered necessary to repeat
the experiment to confirm the results.
The results of the first and the second experiments are shown in Table 14.
A third experiment on cellulose utilization was carried out. In that experiment an attempt
was made to hasten growth of the fungi by providing a supplementary readily absorbable
Carbon source to the cellulose.
The same experiment was designed but in this case 0.1% glucose was added to the
medium as a soluble carbon source. The experiment was repeated as in the original
cellulose test. The two sets of results are shown in Table 15. Histograms of all the
results of the cellulose experiments appear in Fig 3.
As in the earlier experiments the pH of the filtrate was measured after harvesting the
remains of the filter paper.
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EXPERIMENT 7
A TEST OF PHENOMENON OF AVERSION
It has already been mentioned in the Introduction and Literature Review that a reliable
method of identifying strains of S. rolfsii is by the demonstration of the phenomenon of
aversion. The results of the various preceding experiments have shown differences in
the characteristics of isolates and this experiment was conducted to varify whether the
isolates were indeed strains.
Several PDA plates were prepared and the five isolates were paired in all combinations
as shown in Fig. 4.
For each test, two 2mm culture discs, of two different strains or of the same strain were
placed near the edge of the agar plate diametrically opposite each other. The plates were
incubated at 30°C for 12 days. They were then withdrawn and examined for the
incidence of aversion. The observations are recorded in Fig. 4
Plates 6, 7, 8 and 9 are photographs of some of the preparations.
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EXPERIMENT 8
FORMATION OF INFECTION CUSHIONS ON TOMATO FRUITS BY THE FIVE
STRAINS
Having established that the five isolates were indeed strians they were then used in
various pathogenicity tests. In this first experiment, the formation of infection cushion
by the different strians was studied.
Firm ripe fruits of tomato (local variety) were surface sterilized and dried with sterile
filter paper. They were then placed in empty Petri dish lower halves which were
standing in a shallow-pool of water in plastic humidity chambers.
Four mature sclerotia were picked from 12 day old cultures and placed equidistantly on
the upper hemisph~re of each fruit. The lids of the humidity chambers were replaced
and the preparations incubated at 30°C for four days.
The mycelium grew on the surface of the fruits and formed infection cushions. Infection
cushion formation by the different strains was assessed by:
~) determining the number of infection cushions per unit area of 5x5mm, and
~) determining the mean diameter of the infection cushions.
At the end of the incubation period tiny strips (SxSmm) of the tomato epidermis were
gently removed and placed in lactophenol cotton blue in watch glasses to stain the
hyphae.
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They were then mounted on a slide in drops of plain lactophenol with the outer surface
of the strips uppermost.
All the infection cushions on each strip were counted and the diameters of fifty randomly
selected infection cushions were measured with the aid of an eye - piece graticule. The
mean number of infection cushions per unit area of (5xSmm) and the mean diameter of
, ,I
the infection cushions in each case are shown in Table 16.
EXPERIMENT 9 "
INFECTION OF COCOYAM PLANTS GROWING IN S. ROLFSII -
INOCULA TED SOILS
Four varieties of Xanthosoma sagittifoliurn: 'Amankani fitaa', 'Amankani fufuo",
'Amankani kyirepe' and 'Amankani pa' and one variety of Colocasia antiquo rum were
grown in separate pots in garden loam soil. The plants were allowed to grow for three
months and then inoculated. The inoculum for each pot consisted of mycelium raised
in 30ml broth medium (PDB) (in a 250ml Erlenmeyer flasks) for six days at 30°C.
Because of the drift of the culture filtrate to acidic pH during growth of S. rolfsii,
the mycelium was first washed with sterile distilled water before using as inoculum.
The cumulative number of petioles infected was recorded after 4,8,12,16 and 20 days.
The results are tabulated in Table 17 and the progress of petiole infection presented
graphically in Fig 5.
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In the chapter of Results, Plates 10 and 11 show photographs of the three month old
'Amankani pa' and 'Arnankani fufuo ' plants respectively prior to inoculation. Plates
12 and 13 are photographs of 'Arnankani pa' plants on the 4th and 20th day,
respectively, after inoculation with S. rolfsii Strain EL2 and Plate 14 shows dead plants
of 'Amankani fufuo' on the 20th day after inoculation with Strain XA2.
EXPERIMENT 10
ROTTING OF WOUND - INOCULATED CORMELS OF COCOYAM VARIETIES
BY THE FIVE S. ROLFSII STRA INS
Using the procedure described at Section 2 (u) of Materials and General Methods,
surface - sterilized corrnels of the five cocoyam varieties were inoculated in a series of
tests by the five S. rolfsii strains. With each S. rolfsii strian, five cormels of each of the
varieties Xanthosoma sagittifolium - 'Arnankani fitaa '. 'Arnankani fufuo', 'Amankani
kyirepe' and 'Arnankani pa' and corrnels of Colocasia antiquo rum were inoculated.
Each cormel was inoculated at both the apical and basal regions. The inoculated cormels
were put in plastic humidity chambers and incubated at 300e for four days. Transverse
sections of the cormels through the site of inoculation were made and the dept of rot and
the diameter of rot immediately beneath the carmel covering were measured and the
mean calculated for each treatment. The results obtained are tabulated in Tables 18 to
22 and also presented together as histograms in Fig. 6 for purposes of comparison.
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EXPERIMENT 11
DEGRADATION OF BLOCKS OF TISSUE OF CORMELS OF THE DIFFERENT
COCOYAM VARIETIES BY THE FIVE STRAINS OF S. ROLFSIl
Another way of assessing the ability of facultative parasites to attack host organs is to
inoculate different organs in vitro. This experiment as in the preceding experiment used
both the apical and basal regions of the corrnels separately. Mini-blocks, measuring
10xlOx5mm, were cut from the two regions, surface sterilized and placed on growing
mycelium of the test S. rolfsii strain in petri dishes. Five mini-blocks were placed on
the culture in each petri dish. The plates were incubated at 300e for 8 days. Identical
samples of the mini-blocks were earl ier used to determine the original mean dry weight
of the blocks.
After the incubation period the mycelium covering the blocks were carefully removed
with a pair of fine forceps and the decomposing blocks were carefully lifted with a
spatula and transferred to aluminium foil cups and dried in the oven at 800e for 48
hours.
The mean dry weight and percentage Joss in dry weight are shown in Table 23 and 24
and Fig. 7.
Plate 15 shows the mini-blocks of Xanthosoma sagittifolium, 'Amankani fufuo' variety
over-grown by mycelia of S. rolfsii Strains XLL, XA 1, XA2, ELl and EL2.
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EXPERIMENT 12
FORMA TION OF SCLEROTIA BY THE FIVE S. ROLFSII STRAINS GROWING
ON CORMEL TISSUES OF THE DIFFERENT COCOY AM VARIETIES
In Section 2 (w) of Material and General Methods, the methods used in this experiment
have been described. Large flat cocoyam blocks, measuring 3x3xO.5cm, taken from the
two carmel regions (apical and basal) were surface sterilized and each placed in a
sterilized Petri dish. Each block was inoculated with one 3mm- diameter culture and the
inoculated blocks were incubated at 30°C over a total period of 12 days. The plates
were examined daily so that the day sclerotia started to appear and the day melanin
deposition in the sclerotia could be detected were recorded.
The results presented in Table 25 and 26 cover:
a) mean time (hours) of first appearance of sclerotia.
b) mean time (hours) after which browning of the first-developed sclerotia occurred.
~) the number of sclerotia formed on each block after 12 days,
~) the volume of 100 mature sclerotia in each treatment.
~) the germination capacity of the mature sclerotia of each treatment placed on PDA plates
and incubated at 30°(, for 36 hours as described in Section 2 (x) of Materials and General
Methods.
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The photographs of plates 16 and 17 show blocks of Colocasia antiquorum and
Xanthosoma sagittifo li11m , 'Arnankani fitaa' inoculated with S. rolfsii Strain EL2 and
Strain XA2, respectively, after incubation for 4 days and 8 days. Plate 18 also shows
blocks of Colocasia antiquorum ane! Xanthosoma sagittifolium, 'Amankani pa' variety,
8 days after inoculation with S. rolfsii Strain Ell and Strain XA 1, respectively.
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IV RESULTS
EXPERIMENT I
COMPARATIVE RATE OF GROWTH OF THE DIFFERENT STRAINS OF S.
ROLFSII SACCo AND PRODUCTION OF SCLEROTIA ON PDA
There were differences in the rates of growth of the five Strains as shown in Table 1.
The five strains could be separated into three categories of fast-growing, slow-growing
and intermediate. The fast-growing ones were Strains ELI and EL2 which covered the
agar medium of 9cm in diameter in 72 hours, while the slow-growing Strain XA2 took
96 hours to cover the plate. Strains XLL and XA I formed the intermediate group which
covered the PDA plate in 84 hours.
The mycelia of all .the strains appeared similar in density (Plate 1). Also extensional
growth was linear in all the strains. S. rolfsii is a fast-growing fungus (Darkwa, 1965)
and it was noteworthy that within the first 12 hours of incubation the diameter of the
culture increase from the initial 3mm of the disc of inoculum to 11-12mm.
The data in Table 2 and the photograph (Plate 1) show that the five S. rolfsii strains
differed in the number of sclerotia they formed. The diameter, volume and weight of
the sclerotia also differed with the strains. Strain XLL formed significantly fewer
sclerotia of 185 per Petri plate than the rest where the number ranged from.324 to 364
per Petri plate: on the other hand, Strain XLL with the smaller number of sclerotia had
the heavier sclerotial weight and the greatest sclerotia diameter and volume. The value
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of all three parameters were significantly different from those of the rernammg four
strains. However, unlike the number of sclerotia formed, these parameters differed
among those four strains. By the calculated confidence limits at 9S %, the four strains
could be placed in descending order of magnitude or EL2, XA2, XAI and ELI.
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TABLE 1 Growth of the five strains of S. rolfsii on Potato Dextrose Agar (PDA)
at 30°C
Period of Mean Diameter + Standard Error (S.E.) em of strain
Incubation
XLL XAI XA2 ELI EL2
in Hours
12 1.1 +0.02 l.1 +0.02 1.1 +0.02 1.1±0.03 l.2+0.02
24 2.0+0.04 1.9+0.02 1.8+0.02 2.4+0.02 2.3+0.02
36 3.3+0.04 2.7+0.03 2.6+0.03 3.9+0.03 3.9+0.02
48 4.5+0.03 4.0+0.03 3.8+0.02 5.4+0.02 5.3+0.01
60 5.7+0.04 5.3+0.03 5.1 +0.04 7.2+0.03 6.7+0.02
72 6.8+0 ..04 7.0+0.04 6.5 +0.04 9.0+0.00 9.0+0.00
84 9.0+0.03 9.0+0.03 8.8+0.02 *
96 9.0+0.0
Measurements stopped after plate hac! been covered.
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TABLE 2 Formation of Sclerotia by the five strains of S. rolfsii growing on
PDA at 30°C
Mean values of parameters + S. E. recorded for
sclerotia of 12 day-old cultures
S. rolfsii Total No. of. Weight of 100 Volume of 100 Sclerotium
strain Sclerotia/ Sclerotia (X 10-2mg) Sclerotia (X 10-2 crrr') Diameter (,um)
plate*
XLL 185+20.5a 27.8+0.002e1 20.0+0.00d 84.6+0.3d
XAI 356+42.5b 23.6+0.002b J3.0+0.03a 65.3+0.3b
XA2 324+ 12.5b 25.5 +O.OOlc 15.0+0.03b 75.9+0.3c
ELI 332+44.0b 21.7 +0.002a J3.0_±_0.03a 58.3+0.3a
EL2 364+28.0b 26.0+0.002c J8.0_±_O.03c 76.9+0.3c
* Corrected to the nearest whole number.
By the calculated confidence limits at 95 %, means in the vertical rows bearing the same
letters are not significantly different.
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PLATE 1. Photograph showing sclerotia formed by cultures of the S. rolfsii strains growing
on PDA and incubated at 300e for 12 clays (x 1/3)
(From left: Strains, XLL, XAl, XA2, ELI, EL2)
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EXPERIMENT 2
COMPARATIVE RATE OF GROWTH OF THE DIFFERENT STRAINS OF S.
ROLFSII IN POTATO DEXTROSE BROTH (PDB)
In this experiment in which the entire mycelium produced was considered, the results of
the growth test changed slightly. The data in Table 3 show that whereas S. rolfsii Strains
ELI and EL2 again had the greatest mean mycelium dry weight of O.31g in 6 days, the
mean dry weight of 0.26g of Strain XAI also after 6 days was lower than the mean dry
weight of the mycelium of 0.28g of Strain XA2 which in Experiment 1 grew slowest on
the PDA plate. Anyway, all the mean mycelial dry weights after 6 days were not, by
the calculated Confidence Limits at 95 % R.H significantly different from each other.
All the strains grew .rapidl y and attained the highest mycelial dry weight in 6 days. The
dry weight declined, due to autolysis, after that, as is clearly shown in Fig. 1. The rate
of autolysis, however, differed according to the species. By the graph (Fig. 1) the
decline was gentle in Strian XLL and sharper in Strains ELI and EL2. Using the data
in Table 3, the calculated percentage loss in mean dry weight from the 6th to 12th day
in Strain EL2, ELI XAl, XA2 and XLL was 16.1, 12.9, 11.5, 10.7 and 3.5 per cent,
respectively. The pH of the culture media from the 4th day to the end of the incubation
period was very acidic, the range of the initial pH was pH 5.3 to 6. 1 and range during
growth of the cultures, was 2.4 to 3.3. It was observed that, although the pH was still
acidic, it rose slightly from the lowest by the 6th day (a range of pH 2.4 to 2.7, to a
range of pH 2.6 to,3;2 by the 12th Day).
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TABLE 3 Growth of the five strains of S. rolfsii in Potato Dextrose Broth (PDB)
at 30°C
Period of Mean Dry weight + S.E (g) of mycelium in 30ml PDB of Strain
Incubation
XLL XAI XA2 Ell EL2
in Days
4 0.05+0.0b 0.04+0.0a 0.16+0.0c 0.18+0.01c 0.18+0.01c
0.29+0.01a 0.26+0.01a 0.28+0.02a 0.31±0.02a 0.31 +O.Ola
8 0.28+0.01ab 0.25+0.0Ia 0.28 +0.02ab 0.30±0.01b 0.2 7 + 0.01 ab
10 0.28+0.02a 0.25+0.01a 0.28+0.01a 0.28+0.01a 0.27+0.01a
12 0.28+0.01b 0.23 +0.0 la 0.25 +0.0 lab 0.27 +0.02ab 0.26+0.01ab
By the calculated confidence limits at 9S %, means in the horizontal rows bearing the same
letters are not significantly different.
52
------ ---- I t
STRAIN X LL
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1---------------------0--- -0- - - -0-_ ---00'00 .... --,2
O· 35 I--------------------- :-"T=-R""':"A-:N-:-~IX-:-A:-:l~ 7
S
2:
- ):J
w0
2:
LL 0·00
0
_J 0·35
2:
0
(T')
Z
...... <,
0'1 <,
2: <,
:J <,
_J <.
W
U
>- '" "<, '0----0---- -0- - - - -0-_ - - -02: 0·00
LL
0 0'35
l-
I
l') <,
W <,
~ <,
>- <,
0:: <,
0 <,
<,
Z
w« '-''0- _ _.-0- - - - --0
2: - - - -0- - - - -0-
0·00 L~---------------------------------------------------------~20'35 ----- ~~~~~7
STRAIN EL 2
<,
"'o., - - --0- _- - -0- - --~---~
L-------------------- ~----~~2
0·00 2 it 6 8 10 12
PERIOD OF INCUBATION (DAYS)
FIG.!
Mean dry weight of mycelia (. .) of the five
isolates of S. rolfsii grown at 30°C for 12 days and
pH of the medium
the fUngio (0----0) during growth of
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EXPERIMENT 3
GROWTH OF THE DIFFERENT STRAINS OF S. ROLFSIl IN LIQUID MEDIUM
WITH DIFFERENT CARBON SOURCES
The results of studies on the utilization of the di fferent carbon sources by the five S.
rolfsii strains presented in Table 4 could be summarised as follows:
a. taking all the carbon sources together, the strains could be arranged in the
following descending order EL2 > EL 1> XLL > XA 1> XA2 according to their
mean dry weight as can be seen in Fig. 2 in the results of Experiment 4.
b. starch was least used by all the strains, followed by fructose.
c. glucose, maltose and sucrose were superior to fructose and starch and the strains
used them to almost the same degree. The best two carbon sources for growth
of Strains XAl, ELI and EL2 were maltose and sucrose; and those for Strain
XLL were glucose and sucrose while the best carbon source for Strain XA2 was
maltose followed by glucose and sucrose which supported the production of the
same mean dry weight of mycelium.
Evidently, the five carbon sources were utilized differently by the five S. rolfsii strains.
Plates 2 and 3 show the growth of Strains XA2 and EL2 in media of the different
carbon sources at 300e over 6 days.
54
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Table 4 Growth of different S. ,olfsii strains in Liquid MeJ
Mean Dry Weight of myce
Carbon Sources Mean Initial pH pH of filtrate (sh.
of medium
XLL XA1
Fructose 4.6 0.16 + O.OOb 0.13 + 0.001
2.1 2.3
I
Glucose 4.8 0.22 + O.Olb 0.14 + 0.01
2.0 1.8 I
Maltose 4.7 0.20 + O.Olb 0.16 + 0.011
2.2 2.1
Starch 4.7 0.15 + O.Olab 0.12 + 0.00
2.3 2.1
Sucrose 4.8 0.22 + O.Olb 0.15 + 0.01
2.0 l.8
By the calculated confidence limits at 95 %, means in horizontal rows
55
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lID with different carbon sources at 30°c for 6 days
ium (g) + S.E produced in 30ml of medium and final
vn below the dry weight) of culture of strain
XA2 ELI EL2
0.11+0.01a 0.18 ± O.OOc 0.21 + 0.02c
2.0 2.4 2.4
0.13 + O.Ola 0.24 ± O.OOb 0.26 + O.Olb
1.9 2.0 2.0
b 0.14 + O.OOa 0.25 ± O.Olc 0.27 + O.Olc
2.2 2.2 2.0
0.10 + 0.02a 0.17 + 0.1 Ob 0.20 + O.Olb
2.3 2.4 2.2
0.13 + O.OOa 0.25 + O.Olbc 0.28 ± O.Ole
1.8 2.1 2.2
rearing the same letters are not significantly different.
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Plate 2 Photograph ·of cultures of S. rolfsii Strain EL2 supplied with different Carbon
sources and grown at ]O°C for 6 days (x 1/]).
(Carbon source from left: Sucrose, Glucose, Fructose, Starch, Maltose).
56
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Plate 3 Photograph of cultures of S. rolfsii Strain XA I supplied with different Carbon
sources and grown at 30°C for 6 clays (x 1/3).
(Carbon source from left: Sucrose, Glucose, Fructose, Starch, Maltose).
57
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EXPERIMENT 4
GROWTH OF DIFFERENT STRAINS OF S. ROLFSII IN LIQUID MEDIUM
WITH DIFFERENT NITROGEN SOURCES
The results showed that the response of the five S. rolfsii strains to the five Nitrogen
sources supplied showed practically the same pattern as was observed in Experiment 3.
Strains EL2 grew best in all the different media, followed by Strains ELI, XLL, XAI,
XA2 in that order. The results of tests of the use of both the carbon and nitrogen
sources have therefore, been illustrated together by histograms in Fig. 2, for purposes
of comparison.
'e'
The other salient points of results tabulated in Table 5 are:
a. Strains XLL, XA 1, XA2 and EL2 grew best in the Peptone-medium, while Ammonium
chloride, Ammonium nitrate, Sodium nitrate and Peptone supported practically the same
extent of growth of Strain ELI.
b. different nitrogen sources were found to support the poorest growth in different strains.
Ammonium chloride and Sodium nitrate were least used by Strains XLL, XAl, and
XA2.
The smallest mean I"l/ycelium dry weight of Strain ELI occurred in the Asparagine
medium. However, the value of O.IOg was close to those of the other media of 0.12 and
0.13g. With Strain EL2, the smallest mean mycelium dry weight occurred in the Sodium
nitrate medium. Plates 4 and 5 show growth of Strains XA 1 and EL2 in media of the
different nitrogen sources at 30°C over 6 days.
S8
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Table 5 Growth of different S. rolfsii strains i~
Mean Dry Weight of
l
Nitrogen Mean Initial pH of filtrat
Sources pH of medium
XLL XAl I
I
Ammonium 4.6 0.06 + O.OOa 0.06 1
Chloride I
2.2 2.0
I
Ammonium 4.7 0.08 + O.Ola 0.07 J-I
nitrate
2.4 2.1
Asparagine 4.6 0.09 + O.OOa 0.08 1·
2.4 2.2
Peptone 4.9 0.11 + O.Olab 0.10 1
2.2 2.0
Sodium nitrate 4.6 0.07 + O.Ola 0.06 :!
2.9 2.6
By the calculated confidence limits at 95 % ~ means in horizont
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1
Illiquid medium with different Nitrogen sources at 30°C for 6 days
1
1================================
1 ~ mycelium (g) + S.B produced in 30ml of medium and final
: te (shown below the dry weight) of culture of strain
XA2 ELI EL2
1
1 + O.Ola 0.05 + O.OOa 0.13 ± 0.03ab 0.15 + O.Olb
2.1 2.3 2.3
1
1 + O.Ola 0.06 + O.Ola 0.12 ± O.OOb 0.14 + O.Olb
2.2 2.2 2.3
, + O.Ola 0.07 + O.Ola 0.10 ± O.Ola 0.16 + O.Olb
2.2 2.5 2.7
+- O.OOb 0.09 + O.GOa 0.12 ± O.Olb 0.17 + O.OOe
2.1 2.2 2.3
+- O.Ola 0.05 + O.Ola 0.12 ± O.Olb 0.13 + O.OOb
2.5 2.7 2.9
tal rows bearing the same letters are not significantly different.
59
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Mean
Dry wL of
mycelium
( 9 )
·m30ml.of
medium
O·3~----------------------------------------
Mean
Dry wt.of
mycelium
( g)
In 30ml. of
medium
o·o~~~~~~~L---~~~~~~~--~~~
2 4 5 2
Strain Strain ELI Strain
FIG.2 Mean Dry wt. of mycelium of different
of response) grown in 30ml liquid medn
2, Glucose; 3, Maltose; 4, Starch and
2, Ammonium nitrate; 3, Asparagine; 4
60
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C - SOURCES
N - SOURCES
1
1 XLL Strain
_§_. rolfsii strains (arranged in descending order
urn with different sources of carbon (1, Fructose;
5, Sucrose) and Nitrogen (1 Ammonium chloride;
., Peptone; 5, Sodium nitrate') at 30°C for 6 days.
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Plate 4 Photograph of cultures of S. rolfsii Strain EL2 supplied with different Nitrogen
sources and grown at 30°C for 6 days (x l/J).
(Nitrogen source from left: Peptone, Asparagine, Ammonium nitrate, Ammonium
chloride, Sodium nitrate).
61
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Plate 5. Photograph of cultures of S. rolfsii Strain XA I supplied with different Nitrogen
sources and grown at 30°C for 6 clays (x 1/3)
(Nitrogen source from left: Peptone, Asparagine, Ammonium nitrate, Ammonium
chloride, Sodium nitrate),
62
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EXPERIMENT 5
MACERATION OF DIFFERENT PLANT TISSUES BY PECTOLYTIC ENZYMES
IN CULTURE FILTRATES OF THE FIVE STRAINS
The maceration of plant tissues by pectolytic enzymes of facultative parasites and the
degradation of pure pectin by pectolytic enzymes in visco meters are equally reliable and
the former was used to compare the amount of pectolytic enzymes produced by different
S. rolfsii strains. The experiment examined at the same time the influence of the growth
medium on enzyme production and the rate of maceration of tissues of different plant
species. Tables 6 to 13 contain results of the various tests.
63
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Growth of the S. rolfsii strains in different Glucose media (Table 6)
a. In media with different glucose concentrations of 0.5, 1.0, 1.5 and 2.0 per cent, the
mean dry weight of mycelium formed in 6 days by each S. rolfsii strain increased with
increasing glucose concentration.
b. At glucose concentrations of 1.0, 1.5 and 2.0 per cent, Strain EL2 produced the highest
mean mycelial dry weight, although not statistically different from the amount produced
by some of the other strains.
c. At all glucose concentrations, Strain XA2 produced the least mean mycelial dry weight,
although the difference between that value and those of some of the strains was not
statistically significan t.
d. The pH of all the media drifted from an initial pH 6.0 - 6.2 to a final pH 2.0 - 3.4.
64
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I
I
I
Table 6 Growth of the five strains of S. rolfsii II
I
I
I
I
I
recorded after 6 days of incubation II
I
I
I
I
I
I
I
I
I
Mean Dry Wei! II
I
Glucose (sh II
I
Concentration IInitial pH I
I
(%: IV-I/v) XLL XAI II
I
I
I
I
I
,
I
0.5 6.2 0.26 + O.Olb 0.21 II
I
I
I
3.2 3.0 II
I
I
1.0 6.2 I0.34 + O.Ola 0.31 I- I
I
I
2.6 2.5 II
I
I 1.5 6.1 0.46 + O.Olb 0.43 -=
I
I
I
I 2.4 2.3
I
I
I
I
I 2.0 6.0 0.53 + O.Olb 0.47 .:
I
I
I
I
I 2.0 2.1
I
I
I
I
I
I
I
I
,
I By the calculated confidence limits at 95%, means in horizontal
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
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sii in 30ml of PDB with different concentrations of Glucose at 30°C '
eight of mycelium + S. E (g) of strain and final pH of filtrate
'shown below the dry weight) of culture of strain
~l XA2 ELI EL2
I + O.Olab 0.18 + O.OOa 0.23 ± O.Olb 0.23 + O.Olab
3.2 3.2 3.4
I + O.Ola 0.31 ± O.Ola 0.31 + O.Ola 0.35 + O.Ola
2.6 2.6 3.0
,
' 3 -+ O.Olb 0.38 ± O.Ola 0.46 ± O.Olb, 0.48 + O.Olb
2.4 2.4 2.7
" 7 + O.Ola 0.45 ± O.Ola 0.55 ± O.Olb 0.57 + O.Olb
,
2.1 2.1 2.3
,
" tal rows bearing the same letters are not significantly different.
,
65
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Growth of S. rolfsii strains in different Pectin media (Table 7)
a. As with the glucose media, the mean dry weight of mycelium formed in 6 days by each
strain increased with increasing pectin concentration from 0.5 to 2.0 per cent.
b. Strains XAI and XA2 produced the lowest mean mycelial dry weights at all pectin
concentrations. The rematrung three strains produced practically comparable mean
mycelial dry weight in each concentration of pectin.
c. The pH of all the media drifted from an initial pH 4.0 - S.O to a final pH 2.6 - 3.5.
66
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~ I
Table 7 Growth of the five strains of S. rolfsii in : I
recorded after 6 days of incubation
I
I
Mean Dry Weight oi I
Pectin (shown
Concentration Initial pH ----------- I
(%: w/v) XLL XAI
I
0.5 5.0 0.24 + O.Olb 0.17+0.
I
3.4 3.4
1.0 4.6 0.25 + O.Olc 0.22 + O. I
3.3 3.3
I
1.5 4.2 0.27 + O.OOb 0.25 + O. I
3.0 3.0
2.0 4.0 0.39 + O.OOe 0.30 + O.
2.7 2.6
By the calculated confidence limits at 95 % ~ means in horizontal ro-
67
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30ml of PDB with different cOllcentrations of Pectin at 30°C
of mycelium + S.B (g) of strain and final pH of filtrate
n below the dry weight) of culture of strain
ELI ------------------XA2 EL2
O.Ola 0.16 + O.OOa 0.25.±. O.OOb 0.25 + O.OOb
3.4 3.4 3.5
O.OOb 0.19 + O.Ola 0.23 .±. O. OOc 0.28 + O.OOd
3.4 3.4 3.5
O.Olab 0.23 + O.Ola 0.28 -± a.OOe 0.28 + O.OOe
3.1 3.3 3.2
J.OOa 0.28 + O.Ola 0.35 -± o.on. 0.38 + O.Olbe
2.8 3.0 2.9
ows bearing the same letters are not significantly different.
7
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Growth of S. rolfsii strains in media with different Glucose-Pectin combinations
(Table 8)
The combination of glucose and pectin used were: 0.75 % Glucose and 0.25 % Pectin;
0.50% Glucose and 0.50% Pectin; and 0.25 % Glucose and 0.75 % Pectin. Growth of
the S. rolfsii strains in the different media differed.
a. A combination of 0.5 % Glucose and 0.5 % Pectin supported the greatest growth 111
Strains XLL, XAl, XA2 and EL2.
b. Combinations of 0.25 % Glucose and 0.75 % Pectin supported the greatest growth in
Strains ELI. However, the highest value in all cases was not statistically different from
the value next to it,
c. The pH of the media became strongly acidic (luring growth of the fungi.
68
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Table 8 Growth of the five strains of S. rolfsii in 30m
in different ratios at 30°C recorded after 6 da
Medium with Mean Dry Weigh'
Concentration (shov
-(-%--w-/-v-) -o-f-------- Initial pH
Glucose Pectin XLL XA1
0.75 0.25 4.6 0.39 + O.Olb 0.32 +
2.7 2.7
0.5 0.5 4.1 0.42 + O.Olb 0.37 +
2.7 2.6
0.25 0.75 4.0 0.34 + 0.02a 0.35 +
3.0 2.8
By the calculated confidence limits at 95 %, means in horizont;
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nl of PDB containing a combination of Glucose and Pectin
lays of incubation
ht of mycelium + S.E (g) of strain and final pH of filtrate
own below the dry weight) of culture of strain
XA2 ELI EL2
+ O.Ola 0.30 + O.Ola 0.39 + O.Olb 0.41 + O.Olb
2.6 2.8 2.8
+ O.Ola 0.36 + O.Ola 0.43 + O.Olb 0.43 + O.Olb
2.5 2.9 2.9
+ O.Ola 0.33 + O.Ola 0.44 + O.02b 0.42 + O.Olb
2.9 3.2 3.2
ital rows bearing the same letters are not significantly different.
69
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Enzyme activity of filtrates of S. rolfsii strains growing in Glucose and Pectin media
(Table 9-12)
The Enzyme Activity determined by maceration of discs of tuber of Irish potato in
culture filtrates of all the S. rolfsii strains was far greater, ranging from 6.8 to 8.4, in
culture filtrates of the 2.0% Glucose medium than in the remaining three media of lower
glucose concentrations 'as shown in Table 9. These showed comparatively low Enzyme
Activities of 1.6 to 2.2. Using the results of the 2.0% Glucose medium tests to compare
the S. rolfsii Strains, Strain EL2 produced the greatest Enzyme Activity of 8.4 and
Strains XAI and ElJllthe lowest of 6.S.
The data in Table 1.0 showed that the same trends were also observed when discs of
cucumber peri carp were used as the test tissue. The greatest Enzyme Activity in each
case occurred in the culture filtrates of the 2.0% Glucose medium, showing a range of
11.7 to 13.3. The Enzyme Activities of filtrates of the other glucose concentrations were
from 2.9 to 6.4.
The strains fell into two groups at any of the glucose concentrations. Higher levels of
Enzyme Activity were recorded for Strains XLL, XAI and EL2 and lower levels for
Strains XA2 and EL 1.
Results of experiments carried out with culture filtrates of fungi growing in pectin media
using Irish Potato tuber and cucumber pericarp tissues are shown in Tables 11 and 12.
The greatest Enzyme Activity occurred in filtrates of the 2.0 % pectin media. Also, there
was generally greater Enzyme Activity in filtrates of Strains ELI and EL2 than those of
Strains XLL, XAI and XA2 in the 0.5, l.0 and 2.0% pectin media.
:l 70
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Table 9 Extent of maceration of discs of stem tuber of Irish :F
I
of strains each provided with different concentratio:
Glucose Mean time of maceration of 6 discs (min) by
concentration filtrate of strain
(%) in growth
medium XLL XAI XA2 ELI EL2
0.5 45.0 55.8 64.2 55.4 51.0
1.0 55.6 54.4 62.1 63.3 61.5
1.5 52.1 59.6 55.8 57.1 51.9
2.0 14.0 14.8 13.1 14.8 11.9
71
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Potato (Solanum tuberosum) by filtrate of 6 day-old cultures
ons of Glucose
Enzyme activity of strain
XLL XAI XA2 ELI EL2
2.2 1.8 1.6 1.8 2.0
1.8 1.8 1.6 1.6 1.6
1.9 1.7 1.8 1.8 1.9
7.2 6.8 7.6 6.8 8.4
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Table 10 Extent of maceration of discs of pericarp of
strains each provided with different concentr.
Glucose Mean time of maceration of 6 discs (min) b
concentration filtrate of strain
(%) in growth
medium XLL XAI XA2 ELI EJ
0.5 18.7 20.2 33.9 21.9 21
1.0 17.3 19.8 24.8 30.4 2C
1.5 15.6 21.8 26.7 28.1 IS
2.0 7.5 7.7 8.5 7.9 7.
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tf cucumber (Cucumis sativust by filtrate of 6 day-old cultures of
:rations of Glucose
by Enzyme activity of strain
EL2 XLL XAI XA2 ELI EL2
21.0 5.3 5.0 2.9 4.6 4.8
20.2 5.8 5.1 4.0 3.3 5.0
19.8 6.4 4.6 3.8 3.6 5.1
7.7 13.3 13.0 11.7 12.7 13.0
72
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Table 11 Extent of maceration of discs of stem tuber of Iri~
of strains each provided with different concentr~
Pectin Mean time of maceration of 6 discs (min) by
concentration filtrate of strain
(%) in growth
medium XLL XA1 XA2 ELI EL2 I
0.5 10.2 10.0 10.0 8.1 8.1
1.0 7.5 9.6 11.5 7.5 7.3
1.5 7.5 7.3 7.3 8.3 8.5
2.0 6.7 6.5 6.9 6.3 6.2
72
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"ishPotato (Solanum tuberosumi by filtrate of 6 day-old cultures
rations of Pectin
Enzyme activity of strain
,- XLL XA1 XA2 EL2
9.8 10.0 10.0 12.3 12.3
13.3 10.4 8.7 13.3 13.7
13.3 13.7 13.6 12.0 11.7
15.0 15.4 14.4 16.0 16.2
73
I
I
I
I University of Ghana http://ugspace.ug.edu.gh
I
I
I
I
I
I
,
I Table 12 Extent of maceration of discs of pericarp o:
I
,
I strains each provided with different concent
I
I
I
I
I
I
I Pectin Mean time of maceration of 6 discs (min)
I
I
I concentration filtrate of strain
I
I
I (%) in growth
I
I medium XLL XAI XA2 ELII
I
I
I
I
I
I
,
I
I 0.5 8.1 7.7 7.9 6.9
I
I
I
I 1.0 6.0 7.3 7.3 5.4
I
I
I
I
I 1.5 5.4 5.4 5.6 5.6
I
I
I
I 2.0 5.0 5.0 5.0 5.0
I
I
I
I
I
I
I
I
I
,
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
,
I
I
,
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
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.r cucumber (Cucumis sativusi by filtrate of 6 day-old cultures of
lrations of Pectin
I
I
I
I
:l by Enzyme activity of strain
I
I
I
:EL2 XLL XA1 XA2 ELI EL2
I
: 6.3 12.3 13.0 12.6 14.6 16.0
I
I
: 5.6 16.7 13.7 13.7 18.5 17.8
I
I
: 5.4 18.5 18.5 17.9 17.8 18.5
I
I
: 5.0 20.0 20.0 20.0 20.0 20.0
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
74 III
I
I
I
I
I
I
I
I
I
I
I
I
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Enzyme activity of filtrates of S. rolfsii strains growing in Glucose - Pectin media
The Enzyme Activities of filtrates of the five S. rolfsii strains growing in media
containing different combinations of glucose and pectin are shown in Table 13. The
results indicated that the levels of pectolytic enzymes in anyone particular medium was
practically the same for all five S. rolfsii strains.
Secondly, with all the five strains, higher values of Enzyme Activity, from 14.1 to 16.6,
were found in the 0.5% of Glucose + 0.5% Pectin, and the 0.25% Glucose + 0.75%
Pectin media than in the 0.75% Glucose + 0.25% Pectin media of 7.5 to 7.9 Enzyme
Activity.
75
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Table 13 Extent of maceration of discs of stem tuber
of strains each provided with Glucose and
Medium with Mean time of maceration (
concentration (%) by filtrate of strain
of
Glucose Pectin XLL XA1 XA2
0.75 0.25 12.7 12.9 13.1
0.5 0.5 6.7 6.9 6.3
0.25 0.75 7.1 6.6 6.0
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er of Irish Potato (Solanum tuberosum) by filtrateof 6 day-old cultures
id Pectin in different proportions
1 of 6 discs (min) Enzyme activity of strain
ELI EL2 XLL XAI XA2 ELI EL2
12.7 13.3 7.9 7.7 7.6 7.9 7.5
6.7 6.5 15.0 14.6 16.0 15.0 15.5
6.5 6.9 14.1 15.2 16.6 15.4 14.5
76
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EXPERIMENT 6
USE OF CELLULOSE AS CARBON SOURCE BY THE FIVE STRAINS
Two different experiments were carried out. In one experiment cellulose, in the form
of filter paper, was added to the medium as the carbon source. In the other experiment
cellulose was supplemented with 0.1 % Glucose. Both experiments were repeated, but
the results were kept apart. The tables of results (Tables 14 and 15) therefore contain
results of the two tests.
In the experiments using cellulose only as carbon source (Table 14) the results were
consistent. S. rolfsii Strains XA 1 and XA2 utilized cellulose to a greater extent than the
remaining three strains, with Strain XA2 superior, in both tests, to Strain XAI but the
difference between the two values was not statistically significant. However, the two
values mean percentage loss in dry weight of the filter paper of 62.5 and 68.8 per cent
in the original test and 62.5 and 71.9 per cent in the second test were significantly
different from values for the remaining three strains.
Unlike the previous growth experiments, there was only a slight shift of pH of the media
to the acidic side; that is, from pH 5.3 to pH 3.9 and 4. 1. The results followed the same
trend when 0.1 % (w/v) Glucose was added to the medium as shown in Table 15, and in
Fig. 3. Strains XA 1 and XA2 used the carbon sources to a greater extent than the
remaining three strains.
Furthermore values for Strain XA2 were superior to those of Strain XA I and the pH
drifted slightly from the original pH 5.1 to a final pH of pH 3.8 to 4.0.
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Table 14 Growth of the five strains of S. rolfsii iI1
for 7 weeks
Initial Dry S. rolfsii
Test Initial pH wt. of filter strain
paper (g)
I 5.3 0.32 XLL
XAI
XA2
ELI
EL2
CONTROL
2 5.3 0.32 XLL
XAI
XA2
ELI
EL2
CONTROL
By the calculated confidence limits at 95 %, means bearir
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1medium with cellulose (filter paper) as carbon source incubated at 300(1
Final pH of Mean Dry wt. of Mean loss in Mean % loss I
filtrate remaining filter Dry wt. of in Dry wt. of I
paper (g) fi1t~r (g) fil ter paper
4.1 0.18+0.0lb 0.14 43.8
4.1 0.12+0.01a 0.20 62.5
4.1 0.10+0.01a 0,22 68.8
4.1 0.16+0.01b 0.16 50.0
3.9 0.18+0.01b 0.14 43.8
5.2 0.32+0.01c 0.0------ 0.0
4.1 0.19+0.01c 0.13 40.6
4.1 0.12 +O.Ola 0.20 62.5
4.1 0.09+0.01a 0.23 71.9
4.1 0.16+0.0b 0.16 50.0
4.0 0.18+0.01bc 0.14 43.8
5.2 0.32+0.0d 0.0 0.0
I'ng the same letters are not S.'IgnIficantly di fferent.1
I
78
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Table 15 Growth of the five strains of S. rolfsii i
source incubated at 30°C for 7 weeks
Initial Dry S. rolfsii
Test Initial pH wt. of filter strain
paper (g)
1 5.1 0.32 XLL
XAI
XA2
ELI
EL2
CONTROL
2 5.1 0.32 XLL
XAI
XA2
ELI
EL2
CONTROL
By the calculated confidence limits at 95 %, means beari
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in medium with cellulose (filter paper) and 0.1 % (w/v) Glucose as carbc
Final pH of Mean Dry wt. of Mean loss in Mean % loss
filtrate remaining filter Dry wt. of in Dry wt. of
paper (g) ~er(g) filter paper
4.0 0.18+0.00c 0.14 43.8
3.9 0.10+0.01a 0.20 62.5
4.0 0.09+0.01a 0.23 71.9
3.9 0.14+0.01b 0.18 56.3
3.8 0.16+0.01bc 0.16 50.0
5.1 0.32+0.0Id -0-.0------ 0.04.0 0.18+0.01c 0.14 43.8
3.9 0.11+0.0b 0.21 65.6
4.0 0.08+0.01a 0.24 75.0
3.8 0.14+0.01c 0.18 56.3
3.8 0.16+0.01c 0.16 50.0
5.1 0.32+0.01d 0.0 0.0
'i.ng the same letters are not sl.g' nlfican tlYdifferen t.
7Q
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L-
a~.
c c1a.
III L-
III
-0
~
-- S~
-0.
0 ..-
C 0c1"';
~ ~
:2: >-
L-
a
FIG.3 Histograms of Mean % loss in dry weight of filter paper
in media inoculated with S. rolfsii strains.
Note superiority of Strains XAl and XA2 to the other
strains in the use of cellulose as carbon source.
80
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EXPERIMENT 7
A TEST OF PHENOl'AENON OF AVERSION
The strains were paired in all sorts of permutations to test for aversion, in addition to
inoculating the same PDA plate with two inocula of the same strain. The pairings and
results are shown in Fig. 4. There was no aversion when two inocula of the same strain
grew on the plate (Plates 6 and 7). On the other hand, aversion occurred in all the
numerous pairings of two different strains (Plates 6 to 9), proving conclusively that XLL,
XAI, XA2, ELI and EL2 deserved to be recognized as true strains.
81
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Fig. 4 Checker-board showing results of pairing of the five strains of S. rolfsii on Potato-
Dextrose Agar (PDA) at 30°C to test for aversion phenomenon.
(+ ~ incidence of aversionj-; Jl0 aversion)
S. rolfsii strains
XLL XAI XA2 ELI EL2
XLL - + + + +
XAI + - + + +
XA2 + + - + +
..
"
I ELI + + + - +
EL2 + + + + -
82
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Plate 6. Photograph showing aversion between S. ro(j\ii Strains ELI and XA2 growing
on PDA at 30"C (~I4-HT) and the absence of aversion between two inocula of
Strain ELI (LEFT) (x 5/9)
83
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Plate 7· Photograph showing aversion between S. rolfsii Strains ELl and XLL growing
on PDA at 30°C (LEFT) and the absence of aversion between two inocula of
Strain ELI (RI(;HT) (x 5/9)
84
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Plate 8 Photograph showing aversion between S. rolfsii Strains XLL and XAI (LEFT)
and XLL and XA2 (RIGHT) growing on PDA at 30°C (x 5/9).
85
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Plate 9 Photograph of two Petri plates showing similar patterns of aversion between S.
rolfsii Strains XAI and XA2 growing on PDA at 30"e (x 5/9).
86
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EXPERIMENT 8
FORMATION 01' INFECTION CUSHIONS ON TOMATO FRUITS BY THE FIVE
STRAINS
There was a remarkable variation in the pre-penetration habits of the five S. rolfsii strains
as indicated by the data in Table 16. That involved both the number of infection
cushions formed per unit area and the mean diameters of the infection cushions.
Strains ELI and EL2 formed a mean number of 99 and 102 infection cushions per unit
area of 5x5mm respectively, which were vastly greater than the mean numbers of 34-39
by Strains XLL, XAI and XA2. It was observed that there was an inverse relationship
between mean number of infection cushions and mean diameters of the infection
cushions. Thus, the infection cushions of Strains XLL, XA I and XA2 had larger mean
diameters of 12.7 - 18.0j.tm which were significantly larger than the mean diameter of
11.3j.tm of strains ELI and EL2.
87
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TABLE 16 Formation of infection cushion on surface of Local Variety Tomato Fruits by
the five strains of S. rolfsii incubated at 30"e for' 4 days
Mean Total Number of infection cushions Mean Diameter (f-tm)of
S. rolfsii Strain per unit surface area of 25mm2 + S.E infection cushions ± S.E
XLL 34 + 1.I2a 18.0 + 0.15c
XAI 37 + 1.96a 12.7 + 0.13b
XA2 39 + 2.25a 13.3 + 0.12b
ELI 99 ± 3.03b 11.3 + 0.14a
EL2 102 ± 2.64b 11.3+0.11a
By the calculated confidence limits at 95 %, means in vertical rows bearing the same letters are
not significantly different.
88
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EXPERIMENT 9
INFECTION OF XANTHOSOMA SAG/ITIFOLIUM AND COLOCASIA
ANT/QUORUM PLANTS GROWING IN S.ROLFSII-INOCULATED SOILS
Three-month old plants of cocoyam varieties, X. sagittifolium var 'Amankani fitaa',
X. sagittifolium var 'Arnankani fufuo ' (Plate 10), X. sagittifolium vaL 'Arnankani
kyirepe', X. sagittifolium var. 'Arnankani pa' (Plate 11) and C. antiquo rum were
separately inoculated by the soil-inoculation method, with the different S. rolfsii strains.
In all cases, all the replicates were infected and the leaves were dead by the 20th day
after inoculation as shown in Plates 13 and 14. The plants were dug out at the end of the
experiment and bissected longitudinally and examined. The cormels of all the treatments
were heavily rotted, the rot progressing from the top downwards.
Apparently, the fungus entered the plant through the base of the outermost petiole and
migrated into the inner petioles of the plant. Also, the direction of infection of the
petioles was centripetal. The fungus moved from the outermost petiole into the cormel
and then moved both downwards into the cormel and transversely inwards to infect the
inner petioles. Plate .12 shows the progressive invasion and death of the petioles of X.
sagittifolium vaL 'Amankani pa' from the outside inwards.
89
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The plants were not infected at the same rate by the different S. rolfsii strains according
to the data in Table 17. Infection proceeded slowest in X. sagittifolium var. 'Arnankani
fufuo', in which some of the leaves still remained uninfected by the 16th day after
inoculation. On the other hand, Strains EL I and EL2 had infected all the leaves of X.
sagiitifolium var. 'Amankani fitaa' by the 12th day. This variety could be considered
the most susceptible. All the leaves of the remaining three varieties had been infected
by the 16th day by all the S. rolfsii strains. The graphs in Fig. 5 show that infection
progress fastest in the first 8 days after inoculation. None of the control plants showed
any signs of infection during the investigation.
90
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Table 17 Infection of varieties of Xanthosoma sagiuifolium I
different S. rolfsii strains
I
I
Cocoyam Variety Strain No. of petioles at No. of
time of I
inoculation 4
X. sagittifolium XLL 13 6
'Arnankani fitaa' XAI 13 5
XA2 14 6
ELI 13 6
EL2 14 7
CONTROL 12 0
X. sagiuifolium XLL 13 6
'Amankani fufuo ' XAI 13 6
XA2 14 6
ELI 15 7
EL2 13 6
CONTROL 12 0
91
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itifolium and Colocasia antiquorum growing in soil inoculated with
No. of petioles rotted in indicated number of days
4 8 12 16 20
6 10 11 13
5 9 10 13
6 11 12 14
6 10 13
7 11 14
0 0 0 0 0
6 8 10 12 13
6 8 9 12 13
6 9 10 13 13
7 10 12 14 14
6 9 10 11 13
0 0 0 0 0
91
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Table 17 (Cont.'d)
I
Cocoyam Variety Strain No. of petioles at Nc'
I
time of inoculation
4 I
x. sagiitifolium XLL 14 6 I
'Amankani Kyirepe'
XA1 13 4 I
XA2 12 5 I
ELI 13 6 I
EL2 13 5 II
CONTROL 12 0 I
x. sagittifolium XLL 12 6
'Arnankani pa'
XAI 12 5
XA2 13 6
ELI 14 5
EL2 13 7
CONTROL 12 0
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No. of petioles rotted in indicated number of days
)n
4 8 12 16 20
6 10 11 12 12
4 9 11 13
5 Q 12_, 10
6 10 12 '13
5 10 12 13
0 0 0 0 0
6 9 10 12
5 8 9 11 11
6 8 9 12 12
5 10 11 13 13
7 9 10 12 12
0 0 0 0 0
92
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Table 17 (Cont.'d)
Cocoyam Variety Strain No. of petioles at No
time of
inoculation 4
Colocasia XLL 11 5
antiquo rum XA1 14 -,J7
XA2 14 6
ELI 14 6
EL2 13 6
CONTROL 12 0
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No. of petioles rotted in indicated number of days
4 8 12 16 20
5 7 8 11
J7 10 11 14
6 9 11 14
6 9 10 13 13
6 9 11 12 12
0 0 0 0 0
93
94
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100 I
.'
......, ., . '
Z5~------------------------~----ST~RA~IN XLL
100
KEY
COCOYAM VARIETIES
zs~----~------------------~-S-TR-A-IN-X-A-1-~• X. sag'lttifoi"lum
100 t Amank ani f itaa'
t, of OX. sagittifolium
etio les -t- . ,Amankanl fufuo
,fected
() ~ sagittifolium
STRAIN XA2 . . ,
Z5~----------------------------------' tAmankanl kyir epe
100
o -X. s agit td.o,liu m
tAmankanl p a
XC. antiquorum
STRAIN EL1
2SL----------------------------------,
100
STRAIN EL2
25L_----~4~----~B-------1~2------~176------~2~O--~
I Time after Inoculation (Days)F IG.5 Infection of petioles of 3 month-old cocoyam varieties after
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Plate 10. Photograph showing 3 month-old plants of Xanthosoma sagittifolium varieity
'Arnankani fufuo' at the time of the pathogenicity test with the different S. rolfsii
strains (x 1/4).
95
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Plate 11. Photograph showing 3 month-old plant of Xanthosoma sagiuifoliutn varieity
'Amankani pa' at the time of the pathogenicity test with the different S. rolfsii
strains (x 5/9).
96
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Plate 12. Photograph showing infection of leaves of :1 month-old plant of Xanthosoma
sagittifolium varieity 'Arnankani pa' 4 clays after introduction of the soil with S.
rolfsii Strain EL2 (x 5/9).
97
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Plate 13. Photograph showing total destruction of leaves of 3 month-old plant of
Xanthosoma sagjuifolium varieity 'Amankani pa' 20 days after inoculation of the
soil with S. rolfsii Strain EL2 (x 5/9).
98
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Plate 14 Photograph showing total destruction of the leaves of 3 month-old plants of
Xanthosoma sagittifolium varieity 'Amankani fufuo" 20 days after inoculation of
the soil with S. rolfsii Strain XA2 (x 1/6).
99
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EXPERIMENT 10
ROTTING OF WOUND -INOCULA TED CORMELS OF COCOY AM VARIETIES
BY THE FIVE S. ROLFSII STRAINS
The histograms in Fig. 6 bring together the resul ts contained in Tables 18 to 22 of the
various tests which examined rotting of cormels of the five cocoyam varieties which were
wound - inoculated with the different S. rolfsii strains. Information was obtained on the
extent of rotting at the apical and the basal ends of the cormels, on the depth of rot and
on the diameter of rot.
The data in all the tables (Table 18-22) indicated that in all the cases (a) the basal region
of the cormels was rotted to a greater extent than the apical region and (b) the diameter
of the rot measured immediately beneath the carmel covering was greater than the depth
of the rot.
The diameter and depth of the rot both at the apical and basal regions were greatest in
all the coco yam varieties inoculated with S. rolfsii Strains XA 1 and XA2. These two
strains were therefore, the most potent. Strai n EL I, on the other hand, was the least
active, in all the varieties except X. sagittifolium var. 'Amankani fitaa', followed by
Strains EL2 and XLL. The diameter of the rot caused by Strain ELI was in some cases
markedly and significantly smaller statistically than those of rots caused by the rest
(Table 19 and 20),
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The cormels of the different cocoyam varieties were rotted to varying degrees by the
different S. rolfsii strains. As clearly shown in Fig.6, the broadest rot diameter occurred
in cormels of Colocasia antiquorum inoculated with any of the S. rolfsii strains. While
the deepest rot was observed in Xanthosoma saglttifolium 'Arnankani pa' cormels.
None of the control cormels showed any signs of rotting.
101
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TABLE 18. ROTTING OF WOUND - INOCULATED CORMELS OF 'AMANKANI
FITAA' BY THE FIVE STRAINS OF S. ROLFSII AT 30°C IN 8 DAYS
Rotting at inoculated Apical Rotting at inoculated Basal
s.. rolfsii strain region of cormel region of cormel
Extent of Rot (mm)+S.E Extent of Rot (mm)+S.E
Diameter Depth Diameter Depth
XLL 8.4+0.05a 3.4+0.03b 12.8+0.09a 4.0+0.03a
XAI 9.3.±O.08c 3.0+0.03a 17.7+0.07c 4.9+0.03b
XA2 13.3+0.06d 5.0+0.04cl 18.8+0.09d 6.5 +O.04c
ELI 8.8+0.06b 3.7+0.03c 15.1 +O.08b 4.8+0.03b
EL2 8.7+0.05b 3.0+0.03a IS.3+0.11b 4.9+0.03b
Control O.O+O.Oe O.O+O.Oe. O.O+O.Oe O.O+O.OJ.
By the calculated confidence limits at 95 %, means in vertical rows bearing the same letters are
not significantly different.
102
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TABLE 19 ROTTING OF WOUND - mOCULATED CORMELS OF IAMANKANI
FUFUO' BY THE FIVE STRAINS OF S. ROLFSII AT 30°C IN 8 DAYS.
Rotting at inoculated Apical Rotting at inoculated Basal
~. ro1fsii strain region of cormel region of cormel
Extent of Rot (mm)+S.E Extent of Rot (mm) +S.E
Diameter Depth Diameter Depth
XLL 11.5 +O.05b 3.8+0.03b l7.1+0.1Ic 4.6+0.03bc
XAI 12.9+0.09d 4.I.±O.03c 17.2+0.09c 4.8+0.04c
XA2 14.2j:O.44e 4.4+0.02d l8.0+0.08d 5.8+0.04d
ELI 1l.O.±O.07a 3.9+0.03b l2.7±O.10a 4.8+0.03a
EL2 12.4+0.07c 3.0.±O.02a 14.6+0.08b 4.4.±O.03b
Control O.O+O·Of O.O+O.Oe O.O.±O.Oe. O.O.±O.Oe.
By the calculated confidence limits at 95 %, means in vertical rows bearing the same letters are
not significantly different.
103
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TABLE 20 ROTTING OF WOUND - INOCULATED CORMELS OF 'AMANKANI
KYIREPE' BY THE FIVE STRAINS OF S. ROLFSII AT 30°C IN 8 DAYS.
Rotting at inoculated Apical Rotting at inoculated
S. rolfsii strain region of cormel Basal region of cor mel
Extent of Rot (mm)+S.E Extent of Rot (mm)+S.E
Diameter Depth Diameter Depth
XLL 9.8+0.06a 3.4±0.03a 13.4+0.06a 4.8+0.04c
XAI 10.8+0.06c 4.1 +0.04b 16.4+0.09c 5.0+0.03c
XA2 1l.8+0.08d 3.9±0.02b 15.6+0.09b 4.5+0.03b
ELI 10.1 +0.08ab 3.9±0.03b 15.3+0.07b 4.5+0.03b
EL2 1O.4+0.07b 3.9±0.03b 13.6+0.08a 4.9+0.04a
Control O.O+O.Oe O.O+O.OC 0.0+0.0 d_ 0.0+0.0d.
By the calculated confidence limits at 95 %, means in vertical rows bearing the same letters are
not significantly different.
104
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TABLE 21 ROTTING OF WOUND - INOCULATED CORMELS OF 'AMANKANI PA'
BY THE FIVE STRAINS OF S. ROLFSJ/ AT 30°C IN 8 DA YS.
Rotting at inoculated Apical Rotting at inoculated
S. rolfsii strain region of corrnel Basal region of cormel
Extent of Rot (mm)+S.E Extent of Rot (mm)+S.E
Diameter Depth Diameter Depth
XLL 10.4+0.03a 4.3+0.04c 12.7+0.20b 6.0+0.10c
j,
XAI 14.7+0.20d 6.0+0.10d 18.1+0.12d 6.8+0.07c
XA2 13.0.±O.O/'c 4.3.±O.04c 18.1 +O.20d 6.8+0.10d
ELI 1O.9+0.05b 4.0+0.03b 14.4+0.07c S.4+0.04b
EL2 lO.7+0.20ab 3.4+0.03a 11.8+0.08a 4.1 +O.04a
Control O.O+O.Oe. O.O+O.Oe O.O+O.Oe O.O+O.Oe.
By the calculated confidence limits at 95 %, means in vertical rows bearing the same letters are
not significantly different. .
105
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TABLE 22 ROTTING OF WOUND - LNOCULATED CORMELS OF COLOCASIA
ANTIQUORUft,l BY THE FIVE STRAINS OF S. ROLFSII AT 30°C IN 8
DAYS.
Rotting at inoculated Apical Rotting at inoculated
S. rolfsii strain region of cormel Basal region of cormel
Extent of Rot (mm)+S.E Extent of Rot (mm)+S.E
Diameter Depth Diameter Depth
XLL 11.2±O.07b 3.5+0.03a 17.0+0.07b 4.9+0.03c
XAI 13.0+0.07d 3.4+0.04a 17.5 +0.09c 4.4+0.05b
XA2 l7.2..:.tO.07e 4.4+0.03b 21.0+0.lld 6.0+0.03d
ELI 1O.5+0.06a 3.6+0.04a 16.2+0.11a 4.6+0.05a
EL2 12.6±O.09c 3.5+0.03a 17.1 +0.06b 4.6+0.04a
Control O.O+O.Of O.O+O.Oc O.O+O.Oe.. O.O+O.oe
By the calculated confidence limits at 95 %, means in vertical rows bearing the same letters are
not significantly differen t.
106
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20 r---------------------------~---------------------------X-LL-20STRAIN
10 10
o~--~~~~~~~~~~~--_+--~~~~~~~~~~~~~o
20 STRAIN XAl 20
10 10
,.....,. 0 0
E 20 20
E E
0 E
0::
...... 10 10 0
0 0::
....
c:.I -0
c:.I s:
Eo 0 0 -a.
·0 20 STRAIN ELI 20 c:.I0
co c
c:.I o
2: c:.I2:
10 10
o r---~~~~~~~~~~--_+---L~~~~~~~~~~~O
20 STRAIN EL2 20
Cocoyam varities
FIG.6 Rotting at apical region( ~ )and basal region (_ )
of wound - inoculated cormels of cocoyam varieties*
by the five strains of S. rolfsii at 30°C for 8 days;
*1, x. sagittifolium var. 'Amankani fitaa'
2, X. sagittifolium var. IAmankani fufuo'
3, ~ .. sagittifolium var. IArnankani kyirepe'
4, ]I. sagittifolium var. IAmankani pal
5, Colocasia antiquorum.
107 •
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EXPERIMENT 11
DEGRADATION OF BLOCKS OF TISSUES OF CORMELS OF THE DIFFERENT
COCOYAM VAR][ETIES BY THE FIVE STRAINS OF S. ROLFSII
Experiment 10 measured both diameter and depth of rots caused by the different S. rolfsii
strains in wound-inoculated cormels. Experiment II which determined the extent of
degradation of blocks of tissues of the corrnels embodied the two activities - that is -
lateral extent and depth of the rot. The results of the two experiments are, therefore,
comparable to some extent. Blocks of tissues placed, on S. rolfsii cultures (Plate 15)
provided greater surface area to volume ratio to the hyphae than in the usual inoculation
method. The result of Experiment J 1 are presentecl in Table 23 and 24 and in Fig. 7.
Blocks of tissues of the corrnels of the different cocoyam varieties were degraded at
different rates. As in Experiment 10, the blocks from the basal half of the corrnels were
degraded to a greater extent than those from the apical half. Differences in percentage
loss in dry weight by the 8th day of incubation ranged from 2.0 per cent in X.
sagittifolium var. 'Amankani fitaa' inoculated with S. rolfsii Strain XLL to 14.2 per cent
in Colocasia antiquorum inoculated with Strain XLL (Table 24). Generally, considering
degradation of both apical and basal regions, blocks of X. sagiuifolium var. 'Amankani
fitaa' lost the greatest dry weight and X. sagittlfotium var 'Amankani Kyirepe' the least
. especially by the activity of Strains XA 1, ELI and EL2.
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Table 23 DEGRADATION OF TISSUES OF THE APICAL ANI:
COCOY A 1\1 VARIETIES BY THE DIFFERENT S. RG
Weight loss (g) out of original mear
the two regions of cormels of Xa
s. rolfsii Period of 'Amankani fitaa' 'Amankani fufuo'
strains Incubation
Apical Basal Apical Basal p.
(0.52) (0.46) (0.52) (0.47) ((
XLL 2 0.06 +0.01 0.07+0.01 0.06+0.01 0.06+0.01 0
4 0.15±0.01 0.16±0.01 0.08+0.02 0.08±0.01 0
6 0.20+0.01 0.21 +0.01 0.09±0.01 0.11_±0.01 0
8 0.22+0.00 0.23+0.01 0.15±0.02 0.18_±0.01 0
XA1 2 0.06+0.01 0.09+0.01 0.08+0.00 0.09±0.00 0
4 0.10+0.01 0.13 +0.01 0.08+0.01 0.09+0.01 O.
6 0.19_±0.01 0.20+0.01 0.13 +0.01 0.15_±0.00 O.
8 0.22+0.01 0.24+0.01 0.19+0.0 0.21_±0.01 O.
XA2 2 0.05 +0.01 0.08+0.00 0.09 +0.01 0.10+0.01 O.
4 0.07 +0.01 0.12+0.01 0.15+0.01 0.14+0.01 O.
6 0.17+0.01 0.19+0.01 0.16+0.01 0.18+0.00 O.
8 0.22 +0.01 0.25 +0.00 0.19 +0.00 0.21 +0.00 O.
ELI 2 0.08 +0.00 0.09+0.00 0.07 +0.00 0.09+0.00 O.
4 0.10+0.00 0.13+0.01 0.12+0.01 0.14+0.00 O.
6 0.19+0.01 0.20+0.01 0.17 +0.01 0.19±0.01 O.
8 0.23 +0.00 0.24+0.01 0.21 +0.00 0.23 +0.00 O.
EL2 2 0.07 +0.00 0.08+0.01 0.08+0.01 0.09±0.01 O.
4 0.08+0.00 0.10+0.01 0.14+0.01 0.13 +0.00 0.(
6 0.16+0.01 0.18+0.01 0.19+0.01 0.21_±0.00 O.
8 0.23 +0.02 0.24+0.01 0.23_±0.02 0.24_±0.01 O.
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BASAL REGIONS OF THE COIU1ELS OF THE DIFFERENT
,FSII STRAINS AT 30°C OVER 8 DAYS-J
dry weight (g) in parenthesis by Weight loss(g) out of original
uhosoma sagittifolium variety mean dry weight (g) in
parenthesis by the two regions
.mankani Kyirepe' 'Amankani pa' of cormels of
Colocasia antiquorum
oical Basal Apical Basal Apical Basal
54) (0.48) (0.52) (0.49) (0.52) (0.46)
)5 +0.01 0.06_±0.01 0.06+0.02 0.08_±0.01 0.06+0.01 0.07+0.00 '
)7 +0.01 0.09_±0.01 0.10_±0.00 0.12+0.01 0.09+0.01 0.12+0.00
13+0.01 0.14+0.02 0.16 +0.01 0.18_±0.01 0.17_±0.00 0.21 +0.01
"c 6_±0.0 1 0.18 +0.01 0.18 +0.02 0.19_±0.02 0.18_±0.01 0.22+0.01
)8_±0.01 0.09+0.00 0.07 +0.02 0.09_±0.01 0.08+0.02 0.09_±0.01
;0+0.01 O.I 1+0.02 0.09 +0.01 0.12_±0.01 0.10+0.00 0.13 +0.01
[2+0.02 0.14+0.01 0.15 +0.01 0.19.1.0.01 0.15 +0.02 0.16+0.02
[7+0.01 0.]8+0.01 0.17+0.01 0.20.1.0.02 0.17 +0.01 0.20+0.01
)7 +0.0] 0.08 +0.01 0.08+0.01 0.09.1.0.01 0.08+0.01 0.10+0.01
2+0.01 0.]4+0.02 0.13 +0.02 0.13.1.0.03 0.12+0.01 0.16_±0.01
4+0.02 0.17+0.01 0.15 +0.03 0.17.1.0.01 0.19+0.01 0.22+0.02
9_±0.01 0.21 +0.01 0.18_±0.01 0.20.1.0.01 0.23 +0.01 0.26 +0.01
4+0.01 0.05 +0.01 0.06 +0.01 0.08_±O.01 0.07 +0.01 0.09 +0.01
0+0.02 0.10+0.01 0.09 +0.01 0.11_±0.02 0.10_±0.01 0.12+0.02
0+0.00 0.12+0.00 0.14+0.01 0.16_±0.01 0.12+0.01 0.18 +0.02
8_±0.02 0.19_±0.02 0.19+0.01 0.23.±0.01 0.22+0.01 0.23 +0.01
8+0.03 0.07 +0.01 0.06_±0.01 0.07.±0.01 0.05 +0.01 0.08 +0.01
9+0.02 0.10+0.03 0.08_±0.01 0.10.±0.01 0.08+0.01 0.10+0.02
2+0.03 0.11+0.01 0.16 +0.02 0.19_±O.02 0.10+0.01 0.14+0.02
6_±0.01 0.16 +0.01 0.19+0.01 0.21_±O.01 0.16+0.01 0.18+0.01
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Table 24 DEGRADATION OF TISSUES OF THE APICAL ANl
COCOYAl\1 VARIETIES BY THE DIFFERENT S. R(
% Weight loss (calculated from data in Table 23) of the
Period of
IAmankani fitaa' IAmankani fufuo'
S. rolfsii Incubation
strains
Apical Basal Apical Basal
XLL 2 11.5 15.6 .. 11.1 12.2 .
4 28.3 34.8 14.8 17.4
6 38.5 45.7 17.0 24.0
8 44.0 46.0 30.0 40.0
XA1 2 11.3 19.6 14.8 18.8
4 19.2 27.7 15.1 19.6
6 35.8 44.4 26.0 30.6
8 42.3 52.2 37.3 46.7
XA2 2 9.6 17.4 17.3 22.2
4 13.2 26.7 28.8 31.1
6 34.5 41.3 30.8 40.0
8 43.1 54.3 38.0 47.7
ELI 2 15.1 20.0 13.0 19.6
4 19.2 27.7 23.1 31.0
6 35.8 43.5 32:7 41.3
8 45.1 52.2 38.9 51.1
EL2 2 13.2 16.7 15.1 20.0
4 26.7 22.2 26.4 28.3
6 30.2 40.0 35.8 45.7
8 42.6 51.1 42.6 52.2
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lSAL REGIONS OF THE CORl\ffiLS OF THE DIFFERENT
ill STRAINS AT 30°C OVER 8 DAYS
I regions of cormels of Xanthosoma sagittifolium variety % Weight loss (calculated
from data in Table 23) of the
I 'Amankani Kyirepe' 'Amankani pa' two regions of cormels of
Colocasia antiquorum
I
I Apical Basal Apical Basal Apical Basal
I 9.3 12.5 11.1 16.3 11.8 14.6
-; _., .'-.
I 12.5 19.1 19.2 25.5 17.3 25.5
I 23.6 29.2 30.2 37.5 33.3 44.6
I 32.0 40.0 34.6 38.8 34.6 48.8
15.7 19.1 13.5 18.0 15.4
I 18.8
18.2 23.9 17.0 25.0 18.9 28.3
! 21.8 30.0 28.8 39.6 28.8 35.6
30.9 37.5 33.3 40.0 32.1 42.6
12.7 16.7 15.7 18.0 15.1 22.2
21.4 29.2 25.0 27.7 23.1 33.3
25.9 33.3 29.4 34.7 35.8 48.9
34.5 44.7 34.6 40.8 43.4 56.5
7.5 10.4 11.8 16.7 13.2 18.8
18.2 20.8 17.6 23.4 18.9 24.5
18.5 25.0 28.0 34.0 23.1 38·3
34.0 41.3 37.3 47.9 42.3 '+l·q
14.5 15.2 11.3 17.5 9.8 16.7
17.3 21.3 15.4 22.2 15.4 20.8
22.6 23.4 30.2 41.3 19.2 31.1
30.2 34.0 36.5 45.7 30.8 37.5
110
1 ----------- -----------------------
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60~----------------------1
STRAIN X Ll
60~~--~J-~--'J-.c-.-..-.t-:.-..-.-..-LI-..-.L-.-..-J.-::-..-.-.t-:.-.~-.-...A....<:.......c...~__1
30 STRAIN XA 1
0
60
',. weight
loss
of mini-blocks 30 STRAIN XA2
o ~ __ ~~~~~~~~~U---1
60~-----------------------1
30 STRA IN El1
o ~~~~L-~~~~~~~__1
60~------------------------_t
STRAIN EL2
2 3 4 5
Coco y am var iet ies
F 1G. 7. Percentage weight loss of mini-blocks (10x10x5mm)
of apical reg-ion (_~ ) and basal region ( -.-
of cocoyam varieties* placed on cultures of the
different s. rolfsii strains for 8 days.
*1, X. sagTItifolium var. 'Amankani fitaa'
2, X. sagittifolium var. 'Amankani fufuo'
3 J X. sagittifolium var. 'Amankani kyireoe'
4, X. sa£!ittifnlilln'>
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Plate. 15 Photograph showing mini-blocks of carmel of Xanthosoma sagittifolium variety
'Amankani fufuo' covered by mycelia of the different S. rolfsii strains after 8
days at 30°C (x 1/3).
(from left: Strains XLL, XA I, XA2, EL I, EL2)
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EXPERIMENT 12
FORMATION OF SCLEROTIA BY THE FIVE.5,. ROLFSII STRAINS GROWING
ON CORMEL TISSUES OF THE DIFFERENT COCOY AM VARIETIES
The importance of the relationship between S. rolfsii strains and cocoyam varieties
involves not only the susceptibility of the variety but also its role as substrate for
sclerotium formation, and hence the survival 01' the strain. Plates 16, 17 and 18 show
the sort of variation which was recorded in both growth and sclerotium formation in the
S. rolfsii strain - cocoyam variety combinations. The various data collected are tabulated
in Tables 25 and 26.
To summarise the major observations:
a. S. rolfsii Strains XLL and XA2 produced sclerotia] initial earliest on the X. sagittifolium
variety 'Amankani pat blocks, while Strains XA I, ELI and EL2 produced the sclerotial
initials earliest on Colocasia antiquorum.
b. The longest time for the sclerotial initials of Strains XLL, XA 1, ELI and EL2 to appear
were recorded in cultures on X. sagittifolium var. 'Arnankani kyirepe'.
The longest time taken by Strain XA2 to produce sclerotial initials was recorded on X.
sagittifolium var. 'Arnankani fufuo' and Colocasi a antiquorum.
c. The majority of the sclerotial initials matured in 24 hours. The longest time was 36
hours by Strain XLL sclerotial initials on X. sagiitifoilium var. 'Arnankani fitaa' and
Strain EL2 on X. sagittifolium var. 'Arnankani fitaa' and X. sagittifolium var.
'Amankani kyirepe'.
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d. There was a significantly heavier sclerotium production on the Colocasia antiquo rum
blocks (plates 17 and 18).
The highest mean numbers of sclerotia on the blocks of X. sagittifolium varieties formed
by Strains XLL, XA 1, XA2, ELI and EL2 were 105, 157, 183" 311, and 251
respectively. The corresponding mean numbers on the blocks of C. atuiquorum were
280, 870, 633, 976 and 1,023 respectively.
e. The mean volume of 100 mature sclerotia formed on C. antiquorum was either identical
to that of sclerotia on the X. sagittifolium varieties (Strains XLL, XAI and ELI) or
smaller in some cases (Strains XA2 and EL2).
f. Sclerotia of Strains XLL and EL2 had greater germination capacity than those of Strains
XAl, XA2 and ELI (Table 26).
g. Sclerotia formed on the different cocoyarn varieties did not germinate to the same degree.
Sclerotia of Strains XLL, XA 1 and EL2 formed on X. sagittifolium var. 'Amankani
kyirepe' germinated better than those formed 011 the other cocoyam varieties, while the
best substrate for Strains XA2 and EL 1 was X. sagittifolium varieties 'Arnankani fitaa'
and 'Arnankani fufuo' respectively.
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Table 25 Formation of Sclerotia by the different S. rolfsii strains on
Xanthosoma
S. rolfsii 'Amankani fi taa'
strain a. Mean Time
(RRS) of first
appearance of
sclerotia Mean No. of Mean volume
b. Mean Time Sclerotia per (ern') of 100
(RRS) after block after 12 mature scleroti: I
which melanin days
first developed
XLL a. 144+9.8 80+8.2 0.20±0.0
b. 36+6.9
XA1 a. 152+ 13.8 120+ 7.8 0.15 +0.0
b. 32+6.9
XA2 a. 132+6.9 137.±22.8 0.15±O.0
b. 24+0.0
ELI a. 150+11.5 159+ 14.2 0.15±O.0
b. 30 +6.0
EL2 a. 144_±9.8 181+11.3 0.18.±O.0
b. 36_±6.9
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on blocks of tissue of conneIs of the different cocoyam varieties at 3<FC
na sagittifolium variety
'Amankani fufuo'
a. Mean Time
(RRS) of first
appearance of
le sclerotia Mean No. of Mean volume
) b. Mean Time Sclerotia per (em') of 100
·otia (RRS) after block after 12 mature sclerotia
which melanin days
first developed
a. 150+6.0 70+ 12.9 0.20+0.0
b. 24+0.0
a. 150+6.0 157+16.1 0.15 +0.0
b. 24+0.0
a. 138+] 1.4 128+7.2 0.18+0.0
b. 24+0.0
a. 138+11.4 160+10.6 0.15 +0.0
b. 24+0.0
a. 138+6.0 240+20.5 0.20+0.0
b. 24+0.0
15
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XQ(\tho soma.. $~9kt% t;4tn V,
'Amankani kyirepe' 'Amankani pa'
a. Mean Time a. Mean Time
(RRS) of first (RRS) of first
appearance of appearance of
sclerotia sclerotia
Mean No. of Mean volume
b. Mean Time Sclerotia per (cnr') of 100 b. Mean Time
(RRS) after block after 12 mature (RRS) after
which melanin days sclerotia which melanin
first developed first developed
a. 156_±6.9 100_±31. 7 0.20+0.0 a. 132_±6.9
b. 24+0.0 b. 24_±0.0
a. 168+12.0 152+23.1 0.18+0.0 a. 126_±6.0
b. 24_±0.0 b. 24_±0.0
a. 128+6.9 183 + 15.9 0.15 +0.0 a. 126_±0.0
b. 24+0.0 b. 24+0.0
a. 156+6.9 172+11.5 0.15+0.0 a. 132_±6.9
b. 24+0.0 b. 24+0.0
a. 156+6.9 185 + 15.7 0.20_±0.0 a. 132 +6.9
b. 36+6.9 b. 24+0.0
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Colocasia antiquorum
a. Mean Time
(Hrs) of first
appearance of
sclerotia
Mean No. Mean volume
of Sclerotia (crrr') of 100 b. Mean Time Mean No. of Mean volume
TIer block mature (HRS) after Sclerotia per (crrr') of 100
after 12 sclerotia which melanin block after mature
days _ first developed 12 days sclerotia
105 + 12.2 0.20+0.0 a. 144+9.8 280+20.2 0.20+0.0
b. 24+0.0
107+7.9 0.15_±0.0 a. 120+0.0 870+82.3 0.15 +0.0
b. 24+0.0
141+21.1 0.18 +0.0 a. 138+11.4 633 +50.4 0.15 +0.0
b. 24+0.0
311+9.5 0.15 +0.0 a. 120+0.0 976+67.1 0.15 +0.0
b. 24+0.0
251 +6.9 0.20+0.0 a. 126+6.0 1023 +92.8 0.18+0.0
b. 24+0.0
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Table 26 Percentage gerrnination of sclerotia f
different cocoyam varieties at 30°C (l
Cocoyam variety Period of Percentage gel
Incubation the differ
(Hrs) XLL XAl
"Amankani fitaa' 12 88.7 33.4
24 100 42.5
36 100 60.0
,Amankani fufuo' 12 80.0 52.5
24 95.0 76.3
36 100 92.5
(j\mankani Kyirepe' 12 87.5 66.3
24 100 100
36 100 100
c)\ll1ankani pa' 12 77.5 38.8
24 97.5 56.3
36 100 63.8
Colocasia antiquorum 12 28.8 41.3
24 73.8 96.3
36 95.0 100
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formed by S. rolfsii strains on the
(Percentage gennination on PDA plates)
ermination out of a total of 40 Sclerotia formed on
-rent cocoyam varieties by the S. rolfsii strain
\l XA2 ELI EL2
4 76.3 77.5 82.5
5 86.3 85.0 96.3
0 92.5 88.8 97.5
5 56.3 80.0 76.3
3 72.5 100 100
5 92.5 100 100
3 68.8 81.3 87.5
86.3 88.8 100
91.3 93.8 100
52.5 83.8 85.0
75.5 96.3 100
82.5 100 100
76.3 58.8 67.5
77.5 80.0 87.5
77.5 80.0 95.0
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PLATE 16. Photograph showing growth of S. rolfsii Strain EL2 and Strain XA2 on
30x30x5mlll blocks of Colocasia antiquorum (left) and Xanthosoma sagittifolium
variety 'Amankani fitaa' (right), respectively (x 2/3).
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PLATE 17. Photograph showing extent of sclerotium formation at 30°C in 8 days by S. rolfsii
Strain EL2 and Strain XA2, growing on 30x30x5m1l1 blocks of Colocasia
antiquo rum (left) and Xanthosoma sagittifolium variety 'Amankani pa' (right),
respectively (x 8/9).
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PLATE 18. Photograph showing extent of sclerotium formation at 300e in 8 days by S. rolfsii
Strain ELI and Strain XA I growing on 30x30x5mlll blocks of Colocasia
antiquo rum (left) and Xanthosoma sagitiifolium variety 'Arnankani pa' (right),
respectively (x 8/9)
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V. DISCUSSION
The host range of Sclerotium rolfsii is remarkable. In most pathogenic fungi, variation
within species is common. Many parasites exist in various forms, strains and varieties
associated with specific hosts thereby widening the host range and spectrum of host
species. A typical example is Fusarium oxysporum in which the various forms are
named after their specific host plants (Booth, 1971). Even though S. rolfsii attacks not
less than 189 plant species (Weber, 1931) the possible existence of strains with specific
hosts has not received sufficient attention. This is surprising as the hosts of S. rolfsii
include dicotyledonous and monocotyledonous plants and ferns. Relevant reports have
indicated that the fungus exists in a number of distinct strains. Isolates studied by Nakata
(1927), Takahashi (1927), Epps, Patterson and Freeman (1951), Weber (1931) and
Abeygunawardena and Wood (1957) differed sufficiently in morphology and physiology.
Epps, Patterson and Freeman (1951) used the phenomenon of aversion to identify strains.
This method was used in this study and the five isolates of S. rolfsii obtained from
Achimota and Legon were identified as strains (see Fig. 4 and Plates 6-9). This showed
that some morphological and physiological differences noticed among the five strains
were reliable indicators.
Because of the lack. of sufficient number of incubators to provide a range of
temperatures, the optimum temperature for growth of S. rolfsii of 30"C reported by
Higgins (1927), Abeygunawardena and Wood (1957) and Darkwa (1965) was adopted
for the present investigation. The descending order of magnitude of growth of the five
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strains XLL, XA I, XA2, EL 1, and EL2 was the same for mycelial extensional growth
on PDA plates and mycelial dry weight in PDB. Strains ELI and EL2 grew fastest on
the PDA plates and produced the heaviest mycelia in PDB (See Tables 1 and 3). The
greatest dry weights of the mycelia of the strains, all recorded after incubation for 6
days, were, however, not statistically different (see Table 3). Furthermore, autolysis
practically proceeded at the same rate in the broth medium and the amount of mycelia
broken down by autolysis in 6 days after the peak growth had been achieved was less
than 20 percent of the maximum mycelial weight in all strains (see Fig. 1). It has always
been appreciated that. the weight of mycelium gave the better measure of growth, and the
results of the PDB experiment showing statistically similar peak dry weights, could be
used to compare growth of the strains instead of growth on the PDA plates.
There were, however, significant differences in sclerotial production and the number,
weight and size of sclerotia formed could be used to distinguish the strains. Strain XLL
stood out clearly among the strains. It formed the smallest number of sclerotia, but the
sclerotia were statistically the largest and heaviest (see Table 2). This relationship
between the number of sclerotia on one hand and the size and weight on the other hand
was noteworthy. If the larger sclerotia would produce more emerging hyphae on
germination than the smaller ones, the greater number of hyphae would compensate the
smaller number of sclerotia during establishment of Strain XLL in the habitat.
Abeygunawardena and Wood (1957) noticed somewhat similar distinguishing sclerotial
features in two strains, they studied. Strain SR4 was isolated from tomato plants in Sri
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Lanka and Strain SR7 from groundnut plants in Ghana. SR4 produced numerous
spherical sclerotia whereas SR7 produced a much smaller number of irregularly shaped
sclerotia.
There were also significant differences in mycelial dry weights when the fungi were
supplied with different carbon and nitrogen compounds. Strain EL2 consistently grew
better in all the media than the rest, with Strain ELI a close second. All the values of
Strain EL2 were significantly greater than the corresponding values of Strians XLL,
XAI, and XA2 (see Tables 4-8 and Fig. 2). It seemed possible that the original hosts
of the strains had an influence on their physiological characteristics. Those from Elaeis
guineensis fruits grew better than strains isolated from Xanthosoma sagittifolium.
Cellulolysis by the different strains was not directly related to the ability to utilize the
other carbon compounds. Weight loss determinations of cellulolysis using filter paper
showed that the lowest cellulose breakdown occurred in the XLL, ELI, and EL2 culture
vessels (see Table 14). Cellulolysis involves a chain of reactions and should not be
compared directly with utilization of soluble carbohydrates. The difference observed
might be due to differences in the rates of reactions at the different stages of cellulose
breakdown by the different strains.
What was more striking was that cellulolysis was not affected when a glucose 'Starter'
was added to the medium (see Table 15 and Fig.3). Many fungi in relevant studies had
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been induced to break down cellulose better if glucose 'starter' was introduced. For
example, filter paper of identical weight, lost 2:)5.3, 191.4 and 80.1 mg when they were
of the same media, containing a glucose 'starter', the loss in weight of the filter paper
rose to 603.1, 570.0 and 606.7mg, respectively (Forbes and Dickinson, 1977). The
implication of the observation on cellulose utilization by the S. rolfsii strains is that S.
rolfsii would use cellulose in plant tissues economical] y, and the cellulose would not be
exhausted too quickly, even if soluble carbon compounds were present. This suggests
that the slowly-used cellulose would contribute to the survival of the strains. However,
an extended survival would only be possible if there were no cellulose decomposing
organisms growing in the same habitat.
Root crop is a term used by agriculturists for all sorts of plant products which are formed
in the soil. Root crops include stem tubers such as Irish potato and yam (Dioscorea
spp.), root tubers of cassava (Manihot esculenta Crantz), tiger nut (Cyperus esculentusy,
sugar beet (Beta vulgaris) and sweet potato (Ipomoea batata Poir.), rhizomes of ginger
(Zingiber officinalei and cormels of cocoyam etc. They have very high moisture content
like succulent fruits and vegetables and are together referred to as perishables. The
internal high moisture content allows active enzymatic action which leads to tissue
degradation. In addition, the high atmospheric temperatures and relative humidities
support rapid growth and metabolism of fungi and bacteria which attack the products and
cause rotting.
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Some of the organisms responsible for the rotting infect the products after harvest. Others, on the
other hand, are carried over from the field, and referred to as field infection. Field infection is the
more serious of the two as it is responsible for both pre-harvest and post-harvest losses. Cormels
of cocoyam and their associate rot fungus, S. roljsii, belong to this category. There has been
hitherto, no studies on the susceptibility of the various cocoyam varieties cultivated in Ghana to this
widespread soil facultative parasite. Relevant studies have been duly carried out on the
pathogenicity of the five S. rolfsii strains on four varieties of Xanthosoma sagittifolium (,Amankani
fitaa', 'Amankani fufuo', 'Amankani kyirepe' and 'Amankani pa') and Colocasia antiquorum.
Although S. rolfsii belongs to the sub-division Deuteromycotina, it does not form conidia in nature.
In the absence of conidia, healthy cormels are normally attacked by actively growing mycelium in
the soil. There are two ways by which corrnels could be infected. The mycelium may penetrate the
epidermis of the petioles and grow in the petiole and subsequently invade the cormel. The mycelium
may also enter the corrnels through wounds. Plates 12, 13 and 14 show the progressive invasion of
the petioles of the growing cocoyam plants. Tables 18 to 22 also clearly indicate the rotting of
wound - inoculated cormels of all the five cocoyam varieties by the five S. roljsii strains.
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Strain XA2 caused the greatest rot followed by Strain XA I, and the least damage was
caused by Strains XLL and EL2. The rate of growth of a strain in culture alone could
not determine the extent of rot it could cause. For, in the experiments on growth of the
strains, Strain EL2 gave the highest values of mean mycelial dry weight in Potato
Dextrose Broth (see Table 3 and Fig. 1) and it utilized best the carbon and nitrogen
sources (see Table 4 and 5, and Fig. 2). It also had the greatest mean mycelial dry
weight in some of the Glucose-Pectin media (see Table 8). The activity of the fungi in
the cormels was certainly influenced by a combination of factors which deserve future
studies. Another supporting evidence for this view is that the culture filtrate of Strain
XA2 which caused the greatest rot did not show high Enzyme Activity (see Tables 9-12).
The probability that factors in infected tissues could alter observations in pure cultures
had been observed by many workers. Cole and Wood (1961), for example found that
there were more compounds in apple fruits rotted by Botrytis cinerea, Penicillium
expansum, Pyrenochaeta furfuracea and Sclerotinia fructigena than in sound apples.
There were greater:" number of these compounds in extracts of apple rotted by P.
expansum and P..furfuracea than in extracts of rots produced by B. cinerea. This shows
that tissues rotted by different fungi, and also different strains, would contain different
products from the break-down of polysaccharides which may have different effects on
further activity of the rotting organisms.
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The course of events in the rotting process of plant tissues by facultative parasites is well
known. The parasite begins to grow into healthy plant tissue and starts to produce
pectinesterase and chain-splitting enzymes. The latter cause some degradation of cell
wall pectic substances and the pectinesterase de-esterifies some of the high molecular-
weight pectinases to form pectates. The cells of the host are killed, possibly as a result
of the action of pectic enzymes on the cell walls, and phenols are brought together with
host and parasite phenolases to form coloured oxidative products some of which
inactivate the chain-splitting enzymes (Wood, 1967).
In this investigation the rot did not extend uniformly from the inoculated spot. The rot
was wider near the exterior of the cormel and lesser penetration inwards as shown by the
data in Tables 18 to 22 and in Fig. 6. Because of the nature of the rot, it was not
possible to calculate the linear rate of advance of the strains through the cormels.
Gregory and Horne (1928) working with apple fruits which are spherical derived the
relation
v = (y/R)3 (8-3y/R) 116
between V the volume of the rot as a proportion of the volume of the apple, .B the radius
of the apple and y the radius of the rot.
The slower growth of the fungi into the inner regions of the cormels might be due to
decreasing oxygen concentration with increasing depth.
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Different amounts of oxidative products might have been formed in the rotting cormels
of the different varieties resulting in difference in their susceptibility. Cormels of
Colocasia antiquo rum were the 1110stsusceptible and the greatest extent of rot occurred
when they were inoculated especially with Strains XA2 and XA 1 (see Table 22). Among
the X. sagittifolium varieties, 'Arnankani fitaa' and 'Amankani kyirepe' rotted to a
lesser extent than 'Arnankani fufuo' and 'Amankani pa' (see Tables 18-21). No variety
could, however, be regarded as resistant. The cocoyarn varieties could be considered as
showing varyi ng degrees of vul nerabi li ty .
Taubenhaus (1919) and Weber (1931) isolated cultures of S. rolfsii from a number of
different crops, and cross-inoculated from one crop to another. Their results indicated
that the original cultures varied only slightly in their virulence on any given crop
regardless of the plant from which the cultures were originally isolated. No significant
differences among isolates were indicated. In another study of Epps, Patterson and
Freeman (1951), all four strains of S. rolfsii tested on 12 crops infected all the plants.
The relative degree of susceptibility of the crops to the four strains varied only slightly.
It was stated that all four were capable of killing essentially all the plants if vigorous
inoculum was used. They furthermore reported that between two strains Band R, B
strain was slightly more virulent than the R strain, but the difference was inconsistent and
occasionally in the other direction. The results of the present investigation provide
another instance of the nature of strains of S. rolfsii.
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This VIew was supported by studies on the degradation of mini-blocks, measuring
lOxlOx5mm of the different cocoyam varieties placed on mycelia of the different s.
rolfisii strains. In that experiment the hyphae had greater access to the cormel tissues
and oxygen concentrations was not restricted. The varieties did not exhibit the same
order of susceptibility as was observed in the wound-inoculation experiments. X.
sagittifolium variety'Amankani kyirepe' again consistently showed the smallest loss in
mean tissue dry weight (see Table 24). However, in contrast to the trend in the wound -
inoculation experiments, C. antiquo rum showed a lesser loss in mean dry weight than
X. sagittifolium variety 'Arnankani fitaa' - a less susceptible variety in the earlier
experiment. Indeed, this variety showed the greatest loss in mean dry weight (see Table
24).
Infection of the 3-month old cocoyam plants also showed a different trend. This maybe
due to the fact that the course of infection was different.
Mycelium from the soil would first form infection cushions on the petioles, followed by
penetration and invasion of the tissues. The length of period for infection to occur will
depend on the efficiency of breaching the epidermis and the rate of growth and activity
in the petioles. These processes occurred fastest in X. sagittifolium variety, 'Amankani
fitaa', and slowest in 'Arnankani fufuo' and 'Arnankani pa' varieties (see Table 17).
This shows that an assessment of the degree of susceptibility of the different coco yam
varieties to S. rolfsii should be based on the various aspects of the disease.
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Although the five cocoyam varieties are close in susceptibility, some are likely to play
a more significant role in the survival of S. rolfsii in the field than others. Colocasia
antiquo rum consistently supported the production of the largest number of sclerotia when
blocks of cormels were inoculated with the various strains. The mean number of
sclerotia per block of X. sagittifolium varieties ranged from 80 to 311, compared to a
range of 280 to 1023 sclerotia per block of C. antiquorum (see Table 25 and Plates 17
and 18).
Also, among the S., rolfsii strains, Strain EL2 was the most prolific, confirming the
results in Table 2, with a mean range of 181 to 1023 sclerotia per block, and Strain XLL
the least productive with a range of 70 to 280 per block (see Table 23). Sclerotium
production should naturally be influenced by compounds in the cormels, because glucose
and other compounds have been found to influence sclerotium formation in S. rolfsii
(Abeygundawardena and Wood, 1957; Wheeler and Sharan, 1965). It is likely that C.
antiquorum cormel was a more favourable substrate. The constituents of the corme1s of
the varieties should be investigated in future studies to verify this hypothesis.
In conclusion, there are strains of S. rolfsii with distinctive characteristics in Ghana.
Any studies concerning the species must involve as many strains as possible to provide
a balanced view of. events. The five strains were not widely different in their
pathogenicity and it is reasonable to suggest that the five cocoyam varieties tested will
be vulnerable to many S. rolfsii strains in Ghana. In order to safeguard the stability of
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the soil ecosystem it is proposed that cultural practices should be used and they could be
relied upon to prevent catastrophic epidemics. The disease is easily detectable either by
the death of the large leaves or by the presence of the conspicuous clusters of sclerotia
on the infected plants. Infected plants should be removed and destroyed, the top soil in
the area should be dug in, and after harvesting, the thrash should be burnt. Since most
parts of the plant is eaten very little thrash is left behind and its disposal is less
formidable than it would seem. In addition to these field practices only sound cormels
should be stored so that the crop of the next planting season would be raised with clean
materials.
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VI SUMMARY
lea). Five strains of Sclerotium rolfsii: XLL, XAI, XA2, ELI and EL2 were studied.
(b). The five Strains inoculated onto PDA plates could be separated into three categories as
fast-growing, Strains ELI and EL2, covering the plates in 72 hours; slow-growing,
Strain XA2, covering the plates in 96 hours; and the intermediate group, Strains XLL
and XAl, covering the plates in 84 hours. All the cultures had similar appearance.
2(a). In Potato Dextrose Broth peak mean mycelial dry weight was achieved by all the strains
in 6 days. Although Strain EL2 had the highest mean mycelial dry weight after 6 days
of incubation, the mean mycelial dry weights of all the strains were found not to be
statistically different.
(b). Autolysis proceeded after the 6th day of incubation at different rates according to the
strains in the descending order of EL2, ELI, XA I, XA2, and XLL.
(c). The pH of the media drifted from an initial of pH 5.3 - 6.1 to the acidic side of pH 2.4-
3.3 during growth of the strains.
3(a). The five strains could he distinguished by the number of sclerotia formed, and by the
size and weight of the sclerotia.
(b). Strain XLL produced significantly fewer sclerotia of 185 per Petri plate whereas Strains
XA 1, XA2, ELI, and EL2 formed from 324 to 364 sclerotia per Petri plate.
(c). On the other hand Strain XLL had the largest sclerotia (mean diameter of 84.6ftm and
the heaviest sclerotia (mean weight of 100 sclerotia of 27. 8x I (J2mg). The corresponding
values for the remaining four strains ranged from 58.3 to 76.9ftl11 and 21.7xlo-2 to
26. Ox 10·2mg.
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4(a). The five strains could also be distinguished by their ability to utilize different carbon
compound. Taking all the carbon sources - fructose, glucose, maltose, starch and
sucrose - together, the strains could be arranged in the following descending order of
utilization: EL2 > ELI> XLL > XA I > XA2, on the basis of the mean dry weights of the
mycelia.
(b). Starch was least used by all the strains, followed by fructose.
(c). Glucose, maltose and sucrose were best used by each strain to almost the same degree.
(d). Maltose and sucrose were the best two carbon sources for growth of Strains XAl, ELI
and EL2.
(e). Glucose and sucrose were the best two carbon sources for growth of Strain XLL.
(f). Maltose followed by' glucose and sucrose were the best carbon sources for growth of
Strain XA2.
5(a). The order of response of the five S. rolfsii strains to the five Nitrogen sources -
Ammonium chloride, Ammonium nitrate, Asparagine, Peptone and Sodium nitrate - was
as observed with the carbon sources.
(b). Strains XLL, XAl, XA2 and EL2 grew best in the Peptone - medium, while Strain ELI
grew to the same extent in Ammonium chloride, Ammonium nitrate, Sodium nitrate and
Peptone media.
(c). Ammonium chloride and Sodium nitrate were least used by Strains XLL, XA 1, and
XA2.
(d). Strain ELI recorded smallest mean mycelium dry weight in the Asparagine medium.
(e). Strain EL2 recorded smallest mean mycelium dry weight in the Sodium nitrate medium.
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6(a). The mean dry weight of mycelium formed in 6 days by each S. rolfsii strain increased
with increasing glucose concentration over the range of 0.5 to 2.0 per cent.
(b). At glucose concentrations of 1.0, 1.5 and 2.0 per cent, Strain EL2 produced the highest
mean mycelial dry weight although not statistically different from the amount produced
by some of the other strains.
(c). Strain XA2 produced the least mean mycelial dry weight, at all glucose concentrations,
although the difference between that and those of some of the strains was not statistically
significan t.
7(a). The mean dry weight of mycelium formed in 6 days by each strain increased with
increasing pectin concentration from 0.5 to 2.0 per cent.
(b). The lowest mean mycelial dry weights at all pectin concentrations were produced by
Strains XA 1 and XA2.
8(a). Glucose - Pectin combination of 0.5 % Glucose and 0.5 % Pectin supported the greatest
growth in Strains XLL, XA I, XA2 and EL2.
(b). The greatest growth in Strain ELI occurred in combination of 0.25% Glucose and 0.75%
Pectin.
9(a). The 2.0% Glucose. medium culture filtrates of all the strains produced in a tissue
maceration method using tuber of Irish potato, the greatest Enzyme Activity ranging from
6.8 to 8.4 compared to the low values of l.6 to 2.2 at the lower glucose
concentrations.
(b). At 2.0% Glucose concentration, Strain EL2 produced the greatest pectolytic Enzyme
Activity and Strains, XA I and EL I the lowest.
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10. The 2.0% Glucose medium culture filtrates of all the strains also produced the highest
pectolytic Enzyme Activity ranging from 11.7 to 13.3 when pericarp tissue of Cucumber
was used. The pectolytic Enzyme Activity of filtrates of 0.5 to 1.5 % glucose filtrates
of 0.5 to 1.5 % glucose concentration ranged from 2.9 to 6.4.
11. The strains could be separated into two group at any glucose concentration: (i) Strains
XLL, XA1 and EL2 which showed higher pectolytic Enzymes Activity and (ii) Strains
XA2 and ELI which had lower pectolytic Enzyme Activity.
12. In pectin media, the greatest pectolytic Enzyme Activity occurred in filtrates of the 2.0%
pectin concentration, using both Irish potato tuber and cucumber peri carp tissues.
13(a) In Glucose - Pectin media the level of pectolytic enzymes in anyone particular medium
was practically the same for all the five S. rolfsii strains.
(b) In all the five strains, higher values of pectolytic Enzyme Activity, from 14.1 to 16.6,
were recorded in the 0.5 % Glucose + 0.5 % Pectin, and the 0.25 % Glucose + 0.75 %
Pectin media than in the 0.75% Glucose + 0.25% Pectin media of7.5 to 7.9 pectolytic
Enzyme Activity.
14. S. rolfsii Strains XAI and XA2 utilized cellulose to a greater extent than the remaining
three strains.
15. Addition of 0.1 % .(w/v) glucose as 'starter' to the medium did not Improve the
breakdown of the cell ulose (filter paper).
16. No incidence of aversion occurred when two inocula of the same strain grew on the PDA
plate but pairing .of two different strains showed incidence of aversion proving
conclusively that XLL, XA 1, XA2, ELI and EL2 deserved to be recognized as true strains.
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17a. Strains ELI and EL2 formed a mean number of 99 and 102 infection cushions per unit
area of 5x5mm, respectively, on tomato fruits which w=ere-va_stly-g-rea-ter than the meannumbers of 34 to 39 by Strains XLL XA 1 and XA.~2;!;:. --------
b. There was an inverse relationship between mean number of infection cushions and mean
diameters of the infection cushions. The infection cushions of Strains XLL, XA I and
XA2 had larger mean diameters of 12.7 - 18.0f-t11land Strains ELI and EL2 had mean
diameter of 11.3f-tITI.
18a. Three-month old plant of five cocoyam varieties individually innoculated, by soil-
inoculation method, with the different S. rolfsii strains were all killed by the fungi in 20
days.
b. Infection proceeded slowest in X. saglttifolium var. 'Amankani fufuo' in which some of
the leaves still remained uninfected by the 16th clay after inoculation.
c. Strains ELI and EL2. infected all the leaves of X. sagittifolium var. 'Amankani fitaa' by
the 12th day and this variety was considered the most susceptible.
d. All the leaves of the remaining three varieties had been infected by the 16th day by all
the S. rolfsii strains.
19a. Wound-inoculated cormels showed greater rot at the basal region of cormel than at the
apical region.
b. The diameter of the rot measured immediately beneath the cormel covering was greater
than the depth of the rot in all the cocoyarn varieties.
c. Strains XA1 and XA2 caused the greatest diameter and depth of the rot both at the apical
and basal regions in all the cocoyam varieties aile! were considered most potent.
d. Considering growth in cormels of all varieties, Strain ELI caused the least rot followed
by Strain EL2 and XLL.
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20. The cormels of the different cocoyarn varieties were rotted to varying degrees by the
different S. rolfsii strains. The broadest rot diameter occurred in cormels of Colocasia
antiquo rum inoculated with any of the S. rolfsii strains and Xanthosoma sagiitifolium var.
'Amankani pa' cormels showed the deepest rot.
21a. Blocks of tissue of the cormels of the different cocoyam varieties were degraded at
different rates. Blocks from the basal half were degraded to a greater extent than blocks
of the apical half.
b. Blocks of X. sagiuifolium var. 'Amankani fitaa' lost the greatest dry weight and X.
sagittifolium var. 'Amankani kyirepe' the least.
22a. Strains XLL and XA2 produced sclerotial initials earliest on the X. sagtuifotuon var.
'Amankani pa' blocks while Strains XA I, Ell alld EL2 produced the sclerotial initials
earliest on Colocasia atuiquorum.
b. The majority of sclerotial initials matured in 24 hours.
c. A significantly heavier sclerotium production occurred 011 the Colocasia antiquo rum
blocks. The mean number of sclerotia produced by Strains XLL, XA 1, XA2, ELI and
EL2 on this variety was 280, 870, 633, 976 and 1,023, respectively. The corresponding
figures on blocks of X. sagiuifolium were 105, 157, 183, 311 and 251
23. No cocoyam variety was found to be resistant to the S. rolfsii strains, at best the varieties
showed different degrees of vulnerability.
24. For a crop which is planted in small holdings and III mixed farming systems, the best
control against S. rolfsii would be achieved by cultural practices, as infected plants are
easily detectable in the farms.
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VII
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142
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Initial pH of Potato Dextrose Broth and pH of the medium during growth of
the five strains of S. rolfsii at 30uC
Period of pH of culture filtrate strains
Incubation in Days XLL XAI XA2 ELI EL2
0 5.3 5.4 5.5 6.1 6.0
4 2.6 2.6 2.6 3.0 3.0
6 2.4 • i 2.4 2.4 2.7 2.7
'1"
8 2.5 2.6 2.6 2.9 3.0
10 2.8 2.9 3.0 3.3 3.3
12 2.6 2.7 2.8 3.1 3.2
143