SYMPTOMATOLOGY OF FUNGAL COMPETITORS ON OYSTER MUSHROOM’S SPAWN PACKETS AND IN VITRO EVALUATION USING PHYTOEXTRACTS AND A FUNGICIDE

An experiment was conducted to find out the fungal competitors and symptom studies in damaged Oyster Mushroom spawn packets at National Mushroom Development and Extension Center, Savar, Dhaka, Bangladesh. A total of nine fungal competitors of oyster mushroom were isolated and identified namelyTrichoderma harzianum Rifai, T. viride Pers. (Green strain), T. viride Pers. (Yellow strain), T. koningii Oudem, Mucor hiemalis Wehmer, Papulaspora byssina Hotson, Neurospora sp. Shear and B.O. Dodge., Aspergillus flavus Link., and Botryodiplodia theobromae Pat. on the basis of microscopic, morphological and cultural characteristics. To produce oyster mushroom in an eco-friendly manner and to find out their antifungal potency, 23 plant species belonging to 19 families were screened out against isolated nine fungal competitors of oyster mushroom. Among 23 extracts, the maximum (44%) mycelial inhibition of T. harzianum was found due to Aegle marmelos whereas Eclipta alba showed the highest mycelial inhibition (62%) of T. viride (Green strain); in case of T. viride (Yellow strain), Cassia tora exhibited the highest mycelial inhibition (39%); Diospyros cordifolia showed the maximum mycelial inhibition (48%) of T. koningii; Curcuma longa (rhizome) gave the maximum mycelial inhibition (90%) of Neurospora sp. There were no significant effects found to control of P. byssina, B. theobromae, M. hiemalis and A. flavus due to 23 different types of botanicals tested. Trichoderma harzianum, T. viride (Green strain), T. viride (Yellow strain), T. koningii, A. flavus, Neurospora sp. and P. byssina was successfully inhibited by 30, 50 and 70 ppm of fungicide-Bavistin 50 WP but B. theobromae and M. hiemalis were not affected by Bavistin at mentioned concentration.

Kumm.] is one of the popular and widely cultivated mushroom varieties in Bangladesh and cheaply available named as PO-2 at National Mushroom Development and Extension Centre (NAMDEC) and cultivated all the year round. A number of competitor moulds have been reported to occur in the substratum, which used for oyster mushroom production. Variations in the types of moulds are mainly due to the use of a diversity of substrates, different methods of substrate preparation and the conditions and containers used for cultivation. Different saprophytic and plant pathogenic fungi occurring in the substrate and competing with mushroom mycelium for space and nutrition are Aspergillus niger, A. flavus, Alternaria alternata, Drechslera bicolor, Fusarium moniliforme, Mucor sp., Penicillium sp., Rhizopus spp., Rhizpus stolonifer, Sclerotium rolfsii,, Trichoderma viride (Sharma et al., 2007;Sharma and Kumar, 2011). There might be an interaction between Trichoderma sp. and the mushroom due to the enzymatic action on substrate by mushroom that favors green mold fungal growth (Colavolpe et al., 2015). Antifungal activity of different plant extracts have been reported earlier by several investigators against a number of plant pathogens (Ashrafuzzaman et al., 1990). Present study was undertaken with the aim to investigate the symptoms produced due to fungal competitors of mushroom during mushroom production; isolation, identification of competitor fungi of Oyster mushrooms, to evaluate in vitro antifungal potency of several phytoextracts and fungicide against the fungal competitors.

Symptomatological study of damaged spawn packets, isolation and identification of fungal competitors
On the basis of visual observation range of different symptoms were noticed in spawn packets where the mycelium of mushroom were damaged or dominated by the competitors. The symptoms and signs were closely and carefully observed. A total of ten infected spawn packets were taken randomly to isolate the mushroom competitors responsible for damaging as methods given by Dhingra and Sinclair (1985). Individual isolates were identified following Commonwealth Mycological Institute description as described by Barnett (1960) for imperfect fungi, Alexopolus et al. (1996) for perfect fungi.

In vitro evalulation of phytoextracts and fungicide-Bavistin 50 WP
A total of twenty three plant species belonging to 19 families were collected from different locations of Jahangirnagar University campus namely-Aegle marmelos (L.) Correa., Axonopus compressus (Sw) P. Beauv., Blumea lacera (Burm. f) DC., Bougainvillea glabra Choisy, Calamus viminalis Willd., Cassia tora L., Catharanthus roseus L., Curcuma longa (leaf) L., Curcuma longa (rhizome) L., Diospyros cordifolia Roxb., Eclipta alba L., Hemidesmus indicus Br., Hollarhena antidysenterica (Linn) Wall., Ixora coccinea L., Lantana camara L., Melastoma malabathicum L., Mesua nagesarium Kost., Mucuna pruriens L., Pimenta acris Wt., Pteris sp. L., Rungia pectinata (L.) Nees, Catunaregam spinosa (Thunb) Tirveng., Zingiber offinale Rosc. Ethanol was used to extract the active constituent of plant materials. Filtration of extract through Membrane filters was carried out as described by Cappuccino and Sherman (1998). The extracts were tested by introducing 0.5 ml of filtrate spreaded onto 20 ml PDA media containing Petri plate and incubated at 30°C for five days. In a sterile Petri plate, 20 ml of PDA was poured and 2 wells of 5 mm were dug at two sides. 100 µl of each botanical were poured into these wells using sterile micropipette. Fungal discs (5 mm) were punched from 5 days old cultures of the test fungus and placed at the centre of the Petri plates to evaluate the efficacy of the extracts. Petri dish containing PDA medium with each fungal inoculums alone served as control. The plates were incubated at room temperature (28±2°C) for 7 days. The mean radial growth of the fungal colony was recorded after 7 days. The efficacy of extract was determined by comparing the radial growth in treatment (T) with the control (C). The inhibition percentage (I) was calculated using the formula given by Vincent (1947): Mycelial inhibition (%) = Three different concentrations (30, 50, 70 ppm) of a recommended fungicide-Bavistin 50 WP (Carbendazim) were used in the experiment. PDA medium served with requisite amount of distilled water and poured in sterile petri plate and inoculated with test fungus served as control. Each treatment was replicated thrice and kept at room temperature (28±2°C) for 7 days. The inhibition percentages (I) of fungicides were calculated using the formula given by Vincent (1947). The data obtained from different treatments related to phytoextracts and fungicide were analyzed statistically to find out the variation resulting from experimental treatments using SPSS-18 programme.

Symptomatological of fungal competitor in oyster spawn packets
The symptoms appeared in the spawn packets and observed damaging mushroom mycelium were distinctly different from each other depending on different causal competitors. The different symptoms appeared have been described in Table 1. A total of nine fungal competitors were identified in oyster spawn packets namely Trichoderma viride (green strain), Trichoderma viride (yellow strain), T. harzianum, T. koningii, Papulaspora byssina, Mucor hiemalis, Botrydiplodia theobromae, Aspergillus flavus, Neurospora sp. Table 1. Different symptoms appeared in Oyster spawn packets due to different competitors.

Causal organisms Symptoms Trichoderma harzianum
Appeared white in color and compete mushroom mycelium, distinctly showed the green sporulation and ceased the growth of mushroom.

Trichoderma viride (Green strain)
Deep green and compact sporulation found growing over the mushroom mycelium and covered the whole packet.

Trichoderma viride (Yellow strain)
Creamy white or yellowish, light green sporulation appeared over the spawn packet.

Trichoderma koningii
Green sporulation found spreaded over spawn packet.

Mucor hiemalis
Pinheaded mold became mature vigorously and run over mushroom for space and nutrition.

Botrydiplodia theobromae
Destroyed the spawn packet substrate and black acervuli appeared.
Pink colored vigorously growing mycelium observed covered mushroom.

Papulaspora byssina
Brown powdery substance recorded which completely covered the spawn packet space.
Int. J. Agril. Res. Innov. & Tech. 6(2): 24-30, December, 2016 The symptoms appeared and time of expression varied with the different species. Trichoderma spp. initially found to produce the denser compact mycelia compared to Pleurotus, which gradually turned green in color due to heavy sporulation, within two to three days, a characteristic symptom of green mold disease (Table 1). Trichoderma spp. having a green, green-yellow, or white color on the mushroom compost, compete with other mushrooms for nutrients, cause parasitic damage and no fruit bodies observed in infected spawn packets. The occurrence of different species of Trichoderma on Pleurotus spawn packets, the incidence of T. harzianum was the highest at low temperature. The incidence of T. harzianum became lower while temperature raised but incidence of other Trichoderma spp. increased. The findings of the present study are in agreement with those described by Choi et al. (2003). Mushrooms infected with T. harzianum developed larger, light brown spots (Dano, 2000); T. koningii developed reddish spots (Fletcher et al., 1989), T. viride developed dark brown spots (Rinker and Wuest, 1994), which are not similar to present findings. During present study, it was observed that T. harzianum and T. viride (green) caused maximum damage in mushroom production. Dano (2000) also reported the similar findings and cited that T. harzianum and T. viride are more severe than T. koningii. The present findings are in agreement with the results of Sharma and Kumar (2011)

In vitro evaluation of botanicals against mushroom competitors
The present investigation revealed the antifungal activity of some botanicals against the isolated fungal competitors of oyster mushroom. Among 23 botanical extracts, Aegle marmelos showed the highest mycelia growth inhibition (44%) of T. harzianum, followed by Zingiber offinale (12%) while rest of 21 botanical extract did not show any inhibitory effect on green mould-T. harzianum (

In-vitro effect of fungicide on vegetative growth of test fungus
A fungicide-Bavistin 50 WP (Carbendazim) was applied against the selected competitors to observe the antifungal efficacy (Table 7).  Carbendazim was also found to be efficiently inhibiting the mycelial growth green mould isolates (T. harzianum) at very low concentrations (0.63 μg mL -1 to 5 μg mL -1 ) and did not influence the growth of Oyster mushroom (Pleurotus ostreatus and button mushroom (Hatvani et al., 2012;Woo et al., 2004). Parvez et al. (2009) found that the combination of formalin and Carbendazim (500 mL+ 75 ppm) was the best in inhibiting the mycelial radial growth of all the identified microflora of oyster mushroom substrate. Maurya et al. (2013) reported that Carbendazim (0.05%) exhibited strong antifungal properties which inhibited more than 80% mycelial growth of the T. harzianum and P. byssina but mycelial growth of mushroom  Muhammad et al. (2009) who reported that Carbendazim showed complete inhibition of Botryodiplodia theobromae over at both 50 and 100 ppm doses.
In conclusion, fungicide-Bavistin was found to be effective to control a range of microflora associated with oyster mushroom substrate.