Evaluation of Some Botanicals in Controlling Green Mold (Trichoderma harzianum) Disease in Oyster Mushroom Cultivation
Successful methods to control the damaging weed mold, Trichoderma harzianum (green mold) in mushroom beds are not presently known. The aim of the present study was to determine antifungal potentials of extracts of some selected botanicals against the infection of Green mold (Trichoderma harzianum) associated with the Pleurotus sp. cultivation. Eight botanicals namely Azadiracta indica, Allium sativum, Artemesia indica, Urtica dioeca, Lycopersicon esculentum, Datura stramonium, Mentha spicata and Juglans regia were evaluated in the laboratory for their efficacy against both Pleurotus mycelium and pathogen (Trichoderma harzianum), causing Green mold of mushroom. The efficiency of botanicals against Trichoderma was examined by poison food technique. The percent inhibition produced by botanicals against Trichoderma recorded in vitro, was: Azadiracta indica (34.1%), Allium sativum (28.4%), Artemesia indica (21.8%), Urtica dioeca (22.2%), Lycopersicon esculentum (17.4%), Datura stramonium (17.6%), Mentha spicata (20.8%) and Juglans regia (51.9%). Botanicals showing maximum efficacy against the pathogen and minimum inhibition against the mushroom were further evaluated against Trichoderma infection in mushroom house (in vivo test). In in vivo test, the polybags which receive Azadiracta indica show maximum mean increase in yield (32.8%) over control and exhibited minimum mean disease incidence (27.7%) than control. Ecofriendly management practice, i.e., use of botanicals was studied both in vitro and in vivo which gave better results and these practices can be economical, long lasting and free from residual side effects.
The cultivation of edible mushrooms is regarded as a biotechnological process
for conservation of various lignocellulosic, agricultural, industrial, forestry
and horticultural wastes or their by-products into proteins, especially in developing
countries. Mushroom cultivation is a viable alternative venture for minimizing
the ever increasing protein malnutrition gap and multitude of allied problems
in these countries (Eswaran and Ramabadran, 2000). Mushrooms
are saprophytic in nature and are included in a separate group of organisms
called fungi, belong to the sub-division Basidiomycotina. They lack the green
pigment chlorophyll, generally present in plants. Mushrooms which are in commercial
or semi-commercial cultivation in India, include Agaricus bisporus (white
button mushroom) Volvariella spp. (paddy straw mushroom) and Pleurotus
spp. (oyster mushroom) (Munshi and Ghani, 2003). Pleurotus
commonly called as oyster mushroom is one of the most widely eaten mushrooms.
Pleurotus sp. is appreciated for its culinary properties and can grow
under varied agro-climatic conditions (Baysal et al.,
2003). Oyster mushroom cultivation can help in managing organic wastes whose
disposal is very complicating and time consuming (Das and
Oyster mushrooms provide good nutritional and medicinal value. Oyster mushrooms
are rich in proteins, vitamins and minerals (Mattila et
al., 2000). Oyster mushrooms contain about 85-95% water, 3% protein,
4% carbohydrates, 0.1% fats, 1% minerals and vitamins (Wasser,
2002). They contain minerals like potassium, phosphorous, copper and iron
but little amount of calcium (Vetter, 2007).
After Agaricus spp. (button mushrooms), Pleurotus species are
the second most popular mushrooms growing in India. Three species of Pleurotus
are commonly available in Jammu and Kashmir State-P. fossulatus,
P. flabellatus and P. mambranacens. In Kashmir, commercial cultivation
of Pleurotus specie was introduced by number of organizations since
long and a good amount of reasonable work has been done on various aspects of
this mushroom in SKUAST-K. The cultivation of Oyster mushroom has increased
in the Kashmir valley due to its easy cultivation process. But the major constraint
in the speedy popularisation of this crop is diseases and pests. The diseases
and pests happen to be devastating and perpetuate easily from one season to
another. Trichoderma is responsible for causing a serious disease called
as Green mold which is a major fungal problem for mushroom growers. The pathogen
inhibits the growth of mushrooms and hinders the production of fruiting bodies.
It is a common contaminant, badly hindering the production of mushrooms in the
Kashmir valley. Some workers have recommended fungicides for the treatment of
green mold. But growers hardly use the fungicides for the treatment of this
havoc disease. They often found fungicidal treatment as non-economical (Shah
and Nasreen, 2011). The present study was carried out to develop economically
viable and eco-friendly management of this devastating disease through the application
MATERIALS AND METHODS
The study was conducted during 2007. The pathogen was collected from Mushroom Research and Training Center (M.R.T.C), SKAUST-K. The in vitro study was carried out in Division of Environmental Science, SKAUST-K and in vivo trial was laid in mushroom house of M.R.T.C, SKAUST-K, Srinagar.
Isolation of pathogen: Samples of pathogen were collected from green
mold infected substrate poly bags and spawn bottles in Mushroom Research and
Training Center (M.R.T.C), SKAUST-K, Srinagar, during the period August 2007
to November 2007. The sampling included paddy straw bags with or without visual
growth of Pleurotus sajor-caju mycelium and also spawn bottles of Pleurotus
sajor-caju. Pieces of mycelium taken from affected areas of infected paddy
straw and infected spawn grains were placed on Potato Dextrose Agar (PDA) using
a sterilized inoculating needle. The samples from infected bottles were obtained
by dilution and plating of sampled material on rose benegal agar medium. The
plates were incubated at room temperature (27°C) until fungal growth was
visible. The fungi were then sub-cultured on fresh PDA medium for identification.
The fungus causing Green Mold was identified as Trichoderma harzianum.
The fungus so obtained was stored in refrigerator at 4°C and was maintained
by periodic sub-culturing in PDA slants after every 15 days (Aneja,
In vitro evaluation: In this study, ethanol extracts of 8 botanicals
were evaluated in the laboratory for their efficacy against both Pleurotus
mycelium and pathogen Trichoderma harzianum. The plant extracts were
evaluated in vitro through Poison food technique (Nene
and Thapliyal,. 2000). Test concentrations of 5 and 10% obtained by adding
appropriate amount of sterile distilled water to the standard solution (100%).
Two milliliter of each test extract (5 and 10%) was dispensed in petriplates
(9 cm) and then 20 mL of molten PDA was poured gently in petri plates containing
the extract solution. After solidification, inoculations done with 5 mm dia
mycelial cut from 6 days old cultures of both Trichoderma harzianum and
Pleurotus sajor-caju separately. The media without the botanical extract
served as check. The plates were incubated at 27±1°C till the complete
growth was observed in control plates. Percent inhibition in growth was calculated
in relation to growth in control using the following formula of Vincent
In vivo evaluation: In this study, the botanicals which displayed
least adverse effects on the growth of Pleurotus sajor-caju were evaluated
to determine their inhibitory effect against the pathogen (Trichoderma harzianum)
in the cultivation trial during 2009 in M.R.T.C, SKAUST-K. The cultivation trails
were laid under natural temperate climatic conditions of Kashmir during June-July.
The spawn of Pleurotus sajor-caju was procured from Mushroom Research
and Training Centre, Division of Plant Pathology, SKUAST-K. Paddy straw was
used as substrate for cultivation. The chopping of paddy straw was done manually
into bits of 3-5 cm in length and were cleaned thoroughly 2-3 times with tap
water and then soaked in water for 12 h. The paddy straw bits were then drained
off. The dried powder at the rate of 1, 2 and 3% (w/w) of selected plant materials
was incorporated separately in the paddy straw and then filled in polythene
bags at the rate of 1 kg dry substrate. The untreated bags (devoid of botanicals)
were kept as control. All the treatments including control were replicated six
times in CRBD (Complete Randomized Block Design). Spawn of P. sajor-caju
was added at the rate of 1% on dry weight basis of substrate. The bags were
then incubated inside the cropping room, where temperature (28°C max. and
25°C min.) and relative humidity (80%) was maintained. Room having spawn
running bags was kept in dark for 10-15 days till complete colonization of the
compost with fungal mycelium (El-Kattan and El-Hadded, 1998).
The polythene bags were cut open when the substrate was completely colonized with mycelium. The blocks were then inoculated with 3 mL ascospore suspension of Trichoderma harzianum with a spore load of (1x103 spores mL-1) in the middle, with the help of syringe. The untreated bags (devoid of botanicals) with the same inoculums load were kept as control. All the treatments including control were replicated six times in CRBD (Complete Randomized Block Design).
While carrying the above experiment in vivo, the observations on days for spawn run, days for pin head formation, percent increase in yield over control and disease incidence were recorded. Percent increase in yield in treated bags over control (un-treated) was calculated using the formula:
Statistical analysis: In the in vitro experiments, R.B.D (Randomized
Block Design) was applied. In in vivo trial, C.R.B.D (Complete Randomized
Block Design) was applied. All the experiments were analyzed statistically by
the Analysis of Variance (ANOVA). The analyses of variance technique was applied
for drawing conclusions from the data. The calculated value was compared with
tabulated value at 0.05% level of probability for the appropriate degree of
The results obtained on inhibition of mycelial growth of Trichoderma harzianum
in food poisoning plate technique were presented in Table 1.
It could be seen from the Table 1, that all eight botanicals
more or less, significantly inhibited mycelial growth of Trichoderma harzianum
at both test concentrations (5 and 10%). The results revealed that, out
of 8 botanical extracts tested, maximum inhibition of mycelial growth of Trichoderma
harzianum (51.9%) was observed in the Juglans regia hull extract.
It was followed by Azadiracta indica (34.1%), Allium sativum (28.4%),
Urtica dioeca (22.2%), Artemesia indica (21.8%) and Mentha
spicata (20.8%). The least inhibition was exhibited by Lycopersicon esculentum
(17.4%) followed by statistically identical Datura stramonium (17.6%).
As maximum inhibition of pathogen was recorded by J. regia, it was found
that this botanical showed the toxicity to Pleurotus sajor-caju, inhibiting
the mycelial growth of mushroom by (61.4%) (Table 2). It was
further observed that the effect of two concentrations (5 and 10%) on mycelial
inhibition of T. harzianum varied significantly i.e., with the
increase in concentration from 5 to 10%, there was an increase in the inhibition
of mycelial growth of pathogen (T. harzianum) as well. However,
P. sajor-caju also showed the same results except in the treatments which
contain Artemesia indica foliage extract and Lycopersicon esculentum
leaf extract, inhibiting mushroom mycelium by 33.7 and 58.6% at the rate
of 5% but exhibit stimulatory effect on mushroom, showing the inhibition of
22.8 and 56.2% at the rate of 10%. The least toxicity to P. sajor-caju
was shown by M. spicata, inhibiting mushroom by (16.8%) (Table
2). But this botanical exhibit only (20.8%) mycelial inhibition of pathogen,
T. harzianum (Table 1). It was further observed that
the interaction between the treatment (botanical) and concentration was recorded
as significant, both in P. sajor caju and T. harzianum.
In this bioassay, the botanicals which displayed the maximum efficacy against
T. harzianum and least adverse effects on the growth of P. sajor-caju
were further evaluated in vivo.
||In vitro efficacy of ethanol extract of various botanicals
on inhibition of mycelial growth of Trichoderma harzianum
|*Mean of three replications. **Figures in parenthesis are
Arc sine transformed value
||In vitro efficacy of ethanol extract of various botanicals
on inhibition of mycelial growth of Pleurotus sajor-caju (Dhingri
|**Mean of three replications. **Figures in parenthesis are
Arc sine transformed values
||Influence of various botanicals on time taken for complete
colonization by mycelium of Pleurotus sajor-caju
|Control = 18 days
||Influence of various botanicals on time taken for pin formation
by Pleurotus sajor-caju
|Control = 7.6 days, N.S= Non significant
The botanicals selected for in vivo trail were: Azadiracta indica,
Artemesia indica and Urtica dioeca.
The data pertaining to in vivo evaluation of botanicals against Pleurotus sajor-caju and Trichoderma harzianum is presented under the following heads:
Time taken for complete colonization/Spawn-run: It is evident from the (Table 3) that there was significant difference between the influences of botanicals on time taken for complete colonization by mycelium of P. sajor-caju. The average number of the days required for spawn run in P. sajor-caju was significantly less (15.3 days) in Azadiracta indica. It was followed by Artemesia indica and Urtica dioeca (15.4 days) which were statistically identical to Azadiracta indica. The average number of the days for spawn-run was significantly more (18 days) in control, devoid of botanical.
Days taken for pin head formation: The data recorded with regard to influence of botanicals on time taken for pin head formation by P. sajor-caju presented in (Table 4) revealed that there was no significant difference between the effect of botanicals and their concentrations on time taken for pinhead formation by P. sajor-caju. All the botanicals slightly reduced the days (6.3-6.4 days) for pin formation as compared to control (7.6 days).
Percent increase in yield over control: It was revealed that there was a significant difference between the influences of the botanicals on the effect of total yield of P. sajor-caju (Table 5). Maximum increase in yield (32.8%) over control was recorded in treatment which received Azadiracta indica as botanical, followed by Artemesia indica (31.0%) which were found statistically identical with each other. Urtica dioeca gives the least performance of increase in yield (1.6%) over control. It was further observed that the mushroom yield increased on increasing the concentrations (1, 2 and 3%) of botanicals. Maximum increase in yield (35.3%) over control was recorded in treatment which received Azadiracta indica as botanical at a concentration of 3% (34.3%) at 2% concentration, followed by Artemesia indica (32.9%) (32.4%) at 3, 2%, followed by Azadiracta indica (29.0%) at 1% concentration which were found statistically identical with each other. Minimum increase in yield (0.5%) over control was obtained incase of Urtica dioeca at 1% concentration, followed by (1.7%) at 2% and (2.7%) at 3% concentration.
Percent disease incidence: It is clear from the (Table 6) that all the three botanicals at all concentrations of 1, 2 and 3% were more or less significantly effective in reducing the incidence of Green mold of P. sajor caju as compared to the control. However, Azadiracta indica was most effective where the incidence was reduced to (27.7%). It was followed by Artemesia indica (40.6%) and then by Urtica dioeca (70.3%). The disease incidence (83.3%) was recorded in control which was devoid of botanicals. Furthermore, it was found with increase in concentrations of botanicals, the disease incidence was reduced. Minimum disease incidence (22.2%) was recorded in Azadiracta indica at the highest concentration of 3% (27.7%) at the concentration of 2 and (33.3%) at lowest concentration of 1%. It was followed by (38.8%) in Artemesia indica at 3 and 2% and (44.4%) at lowest concentration of 1%. Maximum disease incidence (77.7%) was recorded in Urtica dioeca at the lowest concentration of 1%, followed by (66.6%) at 2 and 3% concentrations.
In vitro evaluation of Botanicals against both Trichoderma harzianum
and Pleurotus sajor-caju revealed that all the botanicals more or less
suppress the growth of T. harzianum. Among the eight botanical extracts
tested, Juglans regia expressed the strongest antifungal activity against
T. harzianum. But this botanical showed the strong inhibitory effect
against Pleurotus mycelium also. The best botanical that inhibits T.
harzianum was Azadiracta indica (Neem). It was followed by Allium
sativum, Urtica dioeca, Artemesia indica and Mentha
|| Influence of botanicals on yield of Pleurotus sajor-caju
during one month cropping period
|*Mean of three flushes
||Effect of botanicals on disease incidence of Trichoderma
harzianum in Pleurotus sajor-caju (Dhinghri mushroom) cultivation
|*Mean of six replications. **Figures in parenthesis are Transformed
These plant species may contain chemical compounds having antifungal properties.
Leaf extracts of Lycopersicon esculentum and Datura stramonium were
less effective. Out of eight botanicals tested, only three botanicals, viz.,
Azadiracta indica, Artemesia indica and Urtica dioeca were further
evaluated against T. harzianum under mushroom house conditions. Sharma
and Jarial (2000) evaluated Neem leaves and Neem cake against False Truffle
(Diehliomyces microsporus) disease of Agaricus spp. and recorded
good results in controlling this disease in vitro which supports the
present investigation. Sharma and Rajesh (2005) observed
that 10 percent neem leaf extract was effective in inhibiting the mycelial growth
of Sepedonium chrysospermum, responsible for causing Yellow mold in button
mushrooms which supports the present investigation. Mishra
(2009) also reported similar results with the use of Neem leaf extract,
Neem cake solution and Neem saw dust against Trichoderma viride. Narzari
et al. (2007) reported that complete inhibition of T. harzianum
was obtained by 0.4% Allium sativum (garlic) extract while 0.2% extract
of consumable and non-consumable garlic inhibited the green mold by 42.2 and
12.2%, respectively, over the control. Arora et al.
(2003) reported the antifungal activity of leaf extracts of Mentha longifolia
and Urtica dioeca against mushroom fungal pathogens, Mycogone
perniciosa and Verticillum which again supports the present work.
In vivo evaluation of selected botanicals, viz., Azadiracta indica,
Artemesia indica and Urtica dioeca against Trichoderma harzianum
was carried out in mushroom house. All the botanicals reduced the time taken
for complete spawn colonization as compared to control. A minimum mean average
of (15.3 days) were taken by the treatment that receive Azadiracta indica.
It was followed by Artemesia indica and U. dioeca (15.4 days).
Pin formation was observed (6.3-6.4 days) after complete spawn-run in all the
treatments which received different botanicals. The botanicals slightly reduced
the time taken for pin head formation as compared to control (7.6 days) (Table
4). Similarly the maximum mean increase in yield (32.8%) over control was
shown by the treatment which received Azadiracta indica, followed by
Artemesia indica (31.0%) (Table 5). Minimum mean increase
in yield (1.6%) over control was recorded in the treatment which was amended
with Urtica dioeca (Table 5). It was further recorded
that with the increase in concentration, the percent increase in yield over
control also increased. Efficacy of botanicals against percent disease incidence
of T. harzianum was also recorded; it was observed that all the botanicals
were effective in reducing the disease incidence when compared to control. Azadiracta
indica was most superior in reducing the incidence rate to (27.7%). It was
followed by Artemesia indica (40.6%) and Urtica dioeca (70.3%)
(Table 6). It was further observed that increase in concentrations
of botanicals decreased the disease incidence of T. harzianum. Antifungal
activity of leaf extracts of Mentha longifolia and Urtica dioeca against
mushroom fungal pathogens, Mycogone perniciosa and Verticillum fungicola
was reported by Govindachari et al. (1998). In
addition, the products of Neem plant have very low toxic effects to mammals
(Kleeberg, 1992) and are relatively safe to non-target
organisms (Schmutterer, 1995). Grewal
and Grewal (1988) reported that incorporation of dried leaves of Azadiracta
indica and Eucaylptus in mushroom compost eliminated Fusarium
and Spendonium sp. which supports the present investigation. Sharma
and Jandiak (1994) reported that leaves from Azadirachta indica, Eucalyptus
tereticornis and Eichhornia crassipes and Allium sativum (cloves)
when incorporated in compost inoculated with various weed fungi, increased mushroom
yield which is in support with the present work. Sharma
and Jarial (2000) evaluated Neem cake, Neem leaves and water hyacinth against
false truffle disease of Agaricus spp. These botanicals when incorporated
with compost resulted in drastic reduction of false truffle and significant
increase in yield of Agaricus spp., again supporting the present investigation.
Sharma and Rajesh (2005) reported that addition of bhang
(2%) at spawning and before casing gave maximum yield followed by Neem leaves
as compared to untreated control which is in agreement with the present investigation.
Narzari et al. (2007) reported that spraying
of 0.3, 0.4 and 0.5% garlic extract on the pasteurized straw before spawning
resulted in 69.6, 77.5 and 78.3% increase in oyster mushroom yield and while
spraying after opening the bags contributed 28.3, 34.3 and 37.9% increase in
yield, respectively, over the control which supports the present work. Inam-ul-Haq
et al. (2010) investigated that certain active components of Eucalyptus
camaldulensis, Azadirachta indica, Citrus lemon and Cymbopogon
marginatus were capable of increasing mushroom yield and controls pathogenic
microbes in oyster mushroom cultivation which again supports the present investigation.
It was found that Neem-cake and Citrus lemon enhances the yield and reduces
the incidence of pathogens. It supports the present investigation that neem
increases the yield and suppresses the infection by T. harzianum.
Currently, the use of inhibitory botanicals is one of the most possible methods for controlling some plant diseases. It is reasonable to collect and screen for more plants with high activity to suppress the green mold infection. This study has found two most promising botanicals, Azadiracta indica and Artemesia indica that were able to minimize the infection of green mold both in in vitro and in vivo treatments. In in vivo trial, incorporation of these botanicals into the compost reduces the disease intensity and enhances the yield when compared to the check (without botanical). However, there are still several further studies to be carried out. Moreover, breeding for mushroom that can resist Trichoderma sp. is one of the interesting aspects, that will make mushroom cultivation a success.
The authors would like to thank my mom and dad who helped me in collecting botanicals. We also thank Dr. Parvez for his valuable advice for mushroom cultivation.
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