INTRODUCTION
Lasiodiplodia theobromae (Pat.) is
fungal pathogen of great economic importance. L. theobromae
is an opportunistic plant pathogen that causes different types of plant
diseases with worldwide distribution within tropical and subtropical regions
(Faber et al., 2007). Its host range
estimated to be more than 280 plant species (Domsch et al., 2007; Khanzada
et al. 2006; Sutton, 1980) however,
pathological effects varies among plants hosts.
In the
tropics, B. theobromae
is an economically important fungus known to cause major losses to mango,
cocoa, banana and yam farmers (Rieger, 2006; Amuse et al., 2003). The fungus is known to
cause tuber rots in yam, root rot in cassava, collar rot in peanuts, crown rot
in banana, Stem end rot in mango fruits, stem rot in
pawpaw and leaf spot in citrus (Sangeetha et al. 2011; Rossel
et al., 2008; Khanzada
et al., 2004b; Jiskani,
2002; Arjunan et
al., 1999; Sangohote, 1988). B. theobromae
is associated with die-back on mango (Khanzada et al., 2004a,b)
and pod rot of cocoa (Phillips, 2007).
Onyenka et al., (2005) reported that the fungus
is present in more than 70% of farms surveyed in Nigeria and it is linked to
colossal yield losses around 80% of marco
harvest. Jiskani (2002) and Sangchote
(1988) respectively have identified B.
theobromae
to be a virulent fungus and a common isolate found on diseased mango fruits in
Pakistan. French (2006) also reported that the pathogen infects and causes
extensive damage to mango, cocoa, banana fruits and yam tubers. Rots caused by
the fungus, particularly in the root and tuber crops often occur underground
and so diagnosis of the disease is usually delayed or under repaired. Moreover,
the wider host range (Crammer, 1979) and the host non- specificity (Mohali et al., 2005) of B. theobromae
makes control and management of the disease very difficult.
Regrettable
there are limited information about the influence of culture media
of Lasiodiplodia theobromae. The lack of information on
host range of L. theobromae
on the trees found in Aboretum of Forestry and
Environment, Rivers State University, Nkpolu-Oroworukwo,
Port Harcourt has necessitated for this research. Therefore, the present study
was undertaken to observe the influence of environmental factors on the mycelial growth of Lasiodiplodia theobromae.
This
research is aimed at investigating the mycological studies on Lasiodiplodia theobromae
the causal agent of gummosis infected African mahogany.
Specific
objectives of this research were to:
(i)
evaluate the
effect of culture media on the mycelial growth of Lasiodiplodia theobromae.
(ii)
determine effect of
temperature on the mycelial growth of Lasiodipodia theobromae.
(iii)
assess the
effect of light and darkness on mycelial growth
of Lasiodiplodia theobromae.
MATERIALS AND METHODS
Study Area
The study was carried
out at the laboratory of Forestry and Environment (Pathology Unit) and Food
Science and Technology, Rivers State University, Nkpolu
Oroworukwo, Port Harcourt, Nigeria.
Effect
of Culture Media on the Growth of L. theobromae
Effect of potato
dextrose agar (PDA) and potato dextrose agar stem exudates (PDASE) media on the
colony growth and sporulation of Lasiodiploda theobromae was evaluated. These media were poured into
9mm diameter Petri dishes and allowed to solidify. 5mm disc of the fungus was
removed with a sterile cork borer from the edges of
the fungus colony and placed in the centre of each
9mm Petri dish containing the media. The Petri dishes were then wrapped with
aluminum foil and incubated at room temperature (28+20C) in
the dark for 5, 10 and 15 days respectively. There were five replicate Petri
dishes of each medium. The colony diameter in each Petri dish was measured
after 5, 10 and 15 days respectively along two axes perpendicular to one
another. (Ukoima and Chukunda, 2016; Chukunda, 2014; Saleem and Nasir, 1991).
Effect of
light and darkness on mycelial growth of fungus L. theobromae
To study
the effect of light and darkness on mycelial growth
of isolated fungus 5 mm
culture discs were cut with the sterilized cork borer
from advancing margin of the colonies of L.
theobromae and inoculated on PDA and PDASE plates
separately at 5 days interval for 15 days. Carbon paper was used to wrap the
Petri dishes for darkness, while unwrapped Petri dishes were used for light
exposure. All the Petri dishes were incubated at 28 ± 2 °C in five replicates
under continuous light and darkness, (Kausar et al., 2009).
Effect of Temperature on the Growth of Lasiodiplodia theobromae.
Five millimeter
culture disc of L. theobromae
were cut with sterilized Cork borer from advancing margin colonies of the
fungus and inoculated on PDA and PDASE plates separately. The effect of
temperature on mycelial growth of L. theobromae was evaluated on potato
dextrose agar (PDA) and potato dextrose agar stem exudates (PDASE). The
inoculated plates were placed in an inoculating chamber and incubated at 15,
20, 25, 30, 35oC in the dark. Each treatment was replicated three
times. At each temperature the plates were arranged in a completely randomized
design (CRD). Colony diameters were measured along two axes perpendicular to
one another. The measurement of the mycelial growth
was calculated after 5, 10 and 15 days of inoculation (Ukoima
and Chukunda, 2016).
Experimental Design and Statistical Analysis
The
experiment was laid out in a Completely Randomized Design (CRD). The treatment were replicated three time. Data collected were
analyzed by analysis of variance (ANOVA) using SPSS Genstat
software as described by Steel and Torrie (1980).
Duncan Multiple Range Test at probability of 5% (DMRT) to separate the means.
RESULTS
Effect
of culture media on mycelia growth of Lasiodiplodia theobromae
The results on the
effect of culture media on the mycelial growth of L. theobromae are
shown in Table 1. The results indicated that potato dextrose agar (PDA) and
Potato dextrose agar stem exudates (PDASE) significantly (P≤0.05)
affected the growth of Lasiodiplodia theobromae.
PDA and PDASE affected the growth of L. theobromae at different days. On the 5th
days of incubation L. theobromae
growth on both media was (15.6mm ± 0.01;
18.2mm ± 0.02). However, the highest
growth was observed on the 10th day for both PDAE (28.2mm ± 0.02) followed by PDA (20.6mm ± 0.01).
DISCUSSION
The
mycelia growth of Lasiodiplodia theobromae (Table
1) were significantly affected by the culture media of potato dextrose agar
(PDA) and potato dextrose agar stem exudates (PDASE). The results indicated
that there was significant interaction between type of medium and mycelia
growth of L. theobromae.
The present research findings agreed with the reports of Alam
et al., (2001) who recorded good
mycelium growth of Botryodiplodia theobromae on
potato dextrose agar (PDA) than on potato dextrose agar stem exudutes (PDASE). Similarly,
Qureshi and Meah (1991),
observed linear growth of B. theobromae on Richard agar solution, mango leaf extract
agar on PDA. Alasoadura (1969) observed maximum stromata of B. theobromae on malt agar and oat meal agar. Sabalpara et al.,
(1991) reported that nutrient rich medium supported the size and number of pycndia produced by B.
theobromae. Saha et al., (2008) and Jash
et al., (2003) reported on the
influence of culture media and environmental factors on mycelial
growth and sporulation of Lasiodiplodia theobromae: in their findings addition of root extract
increased sporulation and mycelial growth of L. theobromae
which was in agreement to our findings. Several other researchers stated that
PDA was the best media for mycelial growth (Xu et al., 1984).
Alam et al., (2001) reported that highest mycelial
growth and sporulation of B. theobromae was recorded on PDA, which was in agreement
to the present work. Several other researchers also stated that PDA was the
best media for mycelial growth (Xu
et al., 1984; Maheshwari
et al., 1999). Kumar and
Singh (2000) also stated that L. theobromae grew well in potato dextrose medium. Result
of this study agrees with that of Karlatti and Hiremath (1989), who observed high mycelial
growth of Altermaria zinniae on
potato dextrose agar medium and recorded higher sporulation on leaf extract
dextrose agar medium.
The
mycelia growth of lasiodiplodia theobromae
(Table 3) showed a variable trend in response to temperature change using
potato dextrose agar (PDA) and potato dextrose stem exudates (PDASE) media
used. mycelia growth increased as temperature
increased from 20-35OC and then decreased with further increase
temperature. However, optimum mycelia growth of test fungus occurred at 25-35OC.
This results agreed with those reported by Quroshi
and Meah (1991) and Alam et al., (2001) who reported that 25-30OC
temperatures was optimum for the colony growth and sporulation of lasiodiplodia theobromae.
However,
fungi grow on diverse habitants in nature and ark cosmopolitan in distribution
requiring several specifics element for growth and reproduction. A wide range
of media are used for isolation of different fungi and colony morphology (Kuhn
and Ghannoun 2003; Kumara and Rawal,
2008). Observed that culture media influenced the growth and sporulation of
some indian isolates of Colletotrichum gloeosporides.
Similarly, Kuhn and Ghonnoum (2003) reported that a
wide range of media are used for isolation of different groups of fungi that
influence the vegetative growth and colony, morphology, pigmentation and
sporulation depending upon the composition of specific culture medium. Ray (2004) showed that lactose and glucose
had similar effect on growth of L. theobromae. Jash et al., (2003) also, observed that
sucrose was the best carbon source for growth of Altemaria zinnia followed by starch and maltose, mannitol
produced the least growth.
The
results of the effects of light and darkness on fungal growth (Table 3) revealed
that there was an increase in growth of L.
theobromae in both light and darkness. Rewal and Grewal (1989) studied
the effect of light on conidial germination of three strains of Botrytis cinerea infecting
chickpea, and found that conidia of B. theobromae
germinated best under continuous light and strain B2, B. theobromae of germinated well under light and darkness
treatment. From the study, it implied that light and darkness are necessary for
growth and sporulation of test fungi.
This is in agreement with Ahmed (1985) who observed that light promoted
the growth and sporulation of Collectotrichum gloeosporoides.
Similarly, Marshi et al., (1959) reported that fungi
exhibited varying response to light depending on the light intensity, quality
and duration of exposure. Prota (1992), Oladiran and Iwu (1993) and Pihet et al., (2009) reported that ultra
violet (UV) radiation or sunlight affected the survival of fungal spores, sclerotia and pycndia. However,
some fungi need light to sporulate whereas other
fungi sporulate better in darkness. In their investigation, Aspergillus ornatus produced abundant conidia when
grown in continuous light and virtually none when grown in dark while cleistothecia and ascospores are
produced in the dark whereas neither is produced in continuous light (Schwemmin, 1960).
Hill
(1976) further explained that light inhibits glucose uptake and phosphorylation
which caused starvation and retards fungi growth and conidia formation.
Conversely the growth of Mycospharella pinodes, Aspergillus niger
increased when exposed to darkness.
On
the contrary, Alam et al., (2001) reported that light is not necessary for growth and
sporulation of B. theobromae, but it enhances the growth and the number of conidia formation
which is in partial agreement with the observation of Rewal
and Grewal (1989). However, the increasing glucose in
medium, may have cause the fungus to utilize it in a
certain level and grow properly, and after that level, the fungal physiology
does not permit the utilization of glucose for the growth of the pathogen. The fungus might utilize the glucose by
different ways instead of growth and formed more pigmentation using more
glucose (Teyegaga and Clerk 1972). According to Cochrane (1958), temperature
range permitting reproduction is usually narrower than that permitting
growth. Earlier, Leach (1979) had
reported variations in optimum temperature requirements within the same species
for light induced sporulation at continuous light and continuous darkness. Alam et al.,
(2001) obtained more growth of L. theobromae under continuous light and less in
continuous darkness. These findings agreed with the present research work where
L. theobromae
test fungi had a good growth performance for both light and darkness. However, Teygaga and Clerk (1972) earlier demonstrated the
relationship between Cercospora
canescens
conidia longevity and storage humidity, and observed that in the dark there was
longest survival of conidia at low humidity than those under light. Generally the spores stored in the darkness
appeared to be more viable than those in light.
This may be due to metabolic disruption by light or that light inhibited
the spores of test fungi thus reducing their percentage conidial germination.
CONCLUSION AND
RECOMMENDATIONS
Conclusion
Our findings revealed
that culture media differently influenced the growth, colony character and sporulation
of the test fungi in the two test media employed in the present study, potato
dextrose agar stem exudate (PDASE) was found to be most suitable for mycelial growth while potato dextrose agar PDA produced
most visible colony morphology. It is concluded that instead of using single
culture medium, a combination of two or more media will be more appropriate for
routine cultural and morphological characterization.
Recommendations
Based on the present
findings the following recommendations are made;
1.
From the study potato dextrose agar stem
exudates was found to be good medium of growth that supported the growth of L. theobromae.
2.
It is revealed from the study that
temperature, light and darkness significantly (P≤0.05) affected the mycelial growth of L.
theobromae.
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