IN VITRO EVALUATION OF BIOCONTROL AGENTS AND FUNGICIDES ON WOOD DECAY FUNGI-GANODERMA ASSOCIATED WITH MORTALITY OF TREE LEGUMES

An experiment was conducted to isolate a number of biocontrol agentTrichoderma spp. from infected spawn packets of oyster mushroom at National Mushroom Development and Extension Centre, Savar, Dhaka, Bangladesh. These bio-control agents were used as antagonist against four wild wood decay fungi of Ganoderma, viz., G. lucidum-1, G. lucidum-2, G. lucidum-3, G. applanatum and two cultivated G. lucidum-4, G.lucidum-6 under in vitro condition. An in vitro trial of Trichoderma spp. against Ganoderma were performed by dual culture, by treating with volatile, non-volatile and naturally untreated metabolites of bio-control agents. In dual culture, all the Trichoderma species showed 70100% mycelia inhibition of G. lucidum-1 and G. lucidum-2, 55.6-100% inhibition of G. lucidum-3, 20-66.7% of G. applanatum, 100% of G. lucidum-5, 75-100% of G. lucidum-6. Effects of heat killed extracts of Trichoderma spp. on growth of G. lucidum-2 (wild) and G. lucidum-6 (cultivated) were also evaluated. Fungicides Bavistin and Dithane M-45 were also used to investigate the mycelial growth inhibition of Ganoderma spp.


Introduction
Tree legumes are important throughout the tropics as sources of forage, firewood, charcoal, green manure and timber (Hughes and Styles, 1989).Ganoderma spp.are important wooddecaying fungi, occurring on conifers and hardwoods across the world.They are known as white-rot fungi which able to decay lignin as well as cellulose (Adaskaveg and Gilbertson, 1994).Ganoderma species caused the root and stem rot diseases result in losses of crops and trees in worldwide (Miller et al., 1994).Seven year-old trees had 10-15% mortality at moist sites due to Ganoderma lucidum (Pathak, 1986).Stressed and damaged Canary Island date palms often become inflicted by Ganoderma applanatum.Large numbers of trees have been known to kill in ten-year-old plantations due to Ganoderma spp. in Peninsular Malaysia (Lee, 2000).Tree mortality generally increases with time in areas where the Ganoderma disease is already present.Control of root rot diseases is difficult as the pathogens survive on woody material in the soil.Green mould disease caused by Trichoderma spp.one of the serious problem of oyster mushroom and white button mushroom.It causes large economic losses to the mushroom growers (Hatvani et al., 2007).Present investigation was carried out to evaluate the potential of fungi as biological control agents (BCA) and fungicides against pathogenic Ganoderma to tree legumes.
The cultural and microscopic characteristics of Ganoderma lucidum was determined as according to Schwarze and Ferner (2003) and Fernando (2008).The efficacy of Trichoderma isolates were evaluated against Ganoderma (4 wild, 2 cultivated) by dual culture technique as described by Dennis and Webster (1971).The pathogens inoculated the precolonized agar plate method as described by Forley and Deacon (1985).The effect of released volatile metabolites of Trichoderma isolates on the mycelial growth of the Ganoderma spp.were evaluated as method described by Dennis and Webster (1971).The effect of non-volatile metabolites on tested fungi were evaluated as according to Kaur et al. (2006).Effects of natural untreated metabolites by dipping culture disc method was performed as mentioned by Ashrafuzzaman and Aminur (1992).There are different concentrations (30, 50 and 70 ppm) of fungicides, namely Bavistin and Dithane M-45 were used to see the mycelial growth inhibition of Ganoderma spp. on PDA medium using food poison technique.All of the inoculated and noninoculated plates were incubated at 28±2ºC and percent of mycelia inhibition was calculated as the formula given by Kaur et al. (2006).

Mycelial inhibition (%) =
Where, C=Radial growth of control plates T = Radial growth of treated plates

Inhibition of Ganoderma spp. by biocontrol agents
In vitro dual culture tests against wild Ganoderma spp.revealed that percent of inhibition range of Ganoderma lucidum-1, 2, 3 and G. applanatum were: 85-100%, 70-100%, 55.6-100%, 55-67.7%due to T. harzianum, T. koningii, T. viride (green strain), T. viride (yellow strain), respectively (Table 1).During present study, Trichoderma showed the overgrowth on pathogens in some case that indicates the mycoparasitic nature of Trichoderma spp.Trichoderma viride effectively inhibited the growth of G. lucidum under in vitro condition.Cultivated G. lucidum-4 and 6 were inhibited 75-100% by Trichoderma spp. at 7 days after incubation during present study (Table 1).In our study, Trichoderma showed overgrowth on pathogens, which indicates the mycoparasitic nature of Trichoderma spp.Similarly, in dual culture technique, the maximum suppression of Ganoderma applanatum (72%) and G. lucidum (75%) over control was noted with Trichoderma harzianum (Srinivasulu and Raghava, 2009).Idris et al. (2008) also recognized Trichoderma spp. as well-known antagonists to many plant pathogenic Ganoderma spp. in oil palm.Trichoderma viride effectively inhibited the growth of G. lucidum under in vitro condition (Lingan et al., 2007).Trichoderma atroviride was also consistently and highly competitive against most wood decay fungi (Schubert et al., 2008).Red root disease of rubber (Ganoderma psuedoferreum) was inhibited by Trichoderma spp.(Ogbebor et al., 2010).The mycelial growth of G. lucidum was inhibited successfully by T. viride, T. harzianum and T. virens with 66.55%, 63.99% and 62.12%, respectively after 96 hrs of incubation (Chakrabarty et al., 2013).It has been revealed that Trichoderma spp.coiled round the hyphae of Ganoderma spp.both sparsely and intensely which was followed by penetration of Trichoderma spp.into the hyphae of Ganoderma spp., finally, lysis of the host mycelium was noticed (Srinivasulu and Raghava, 2009).

Effect of volatile, non-volatile and natural untreated metabolites
The current study confirmed that the volatile metabolites had a fungistatic rather than a fungicidal effect.Volatile metabolites secreted by Trichoderma spp.showed significant effect in controlling Ganoderma spp.The range of inhibition of Ganoderma lucidum-1, 2, 3 and G.
(Table 2).Volatile metabolites of T. viride showed the maximum inhibition than other isolates.In the present study, the average inhibition was recorded as 0-33.3% in Ganoderma spp.by nonvolatile compound and T. viride was found more effective than others (Table 2).Present results are supported by earlier workers.Trichoderma viride, T. hamatum and T. harzianum were reported to be very effective in producing volatile and non-volatile metabolites against Ganoderma lucidum and G. applanatum (Srinivasulu and Raghava, 2009).Bruce et al. (2000) cited that volatile metabolites of T. viride having significant effect on wood decay fungi.Idris et al. (2008) reported 318 isolates of Trichoderma and tested against pathogenic Ganoderma.Effect of natural untreated metabolites of Trichoderma spp.
showed variable inhibitory effects on studied organisms.T. viride (green strain) showed the maximum inhibition in test fungus except G. applanatum (Table 2).There is lack of information regarding the effect of natural untreated metabolites on Ganoderma spp.
There is no literature available in this regard by previous workers.During present investigation, the aggressiveness of Trichoderma spp.studied varied more or less to previous mentioned workers.This might be due to difference in site of isolation.In literature, Trichoderma spp.were collected from soil rhizosphere but in the present study isolates were collected from spent mushroom compost.

Effect of fungicides on Ganoderma spp.
In vitro fungicidal effects on studied organisms found very significant.Bavistin showed complete mycelial inhibition in case of all selected organisms at 30, 50 and 70 ppm concentrations (Table 4) while Dithane M-45 was not satisfactory as compared to Bavistin.Present results are in conformity with the previous findings.Data recorded after 7 of incubation; Data represent as mean value of three replications; Column having the same letters do not differ significantly at 5% level of significance; G1 = Ganoderma lucidum-1, G2 = G. lucidum-2, G3 = G. lucidum-3, G4 = G. applanatum.
It can be concluded that both biocontrol agent-Trichoderma and Bavistin were found to be effective to control Ganoderma infection.Therefore, either utilization of Trichoderma or Bavistin is preferable to control stem and root rot of higher plant like tree legume.

Table 1 .
An in vitro mycelial growth inhibition (%) of Ganoderma spp.by four Trichoderma spp. in dual culture technique at 32±2ºC temperature.

Table 2 .
An in vitro mycelial growth inhibition (%) of Ganoderma spp.by four Trichoderma spp. at 28±2ºC temperature due to volatile, non-volatile and naturally untreated metabolites.

Table 3 .
Effects of heat killed extracts of Trichoderma spp. on mycelia growth of G.lucidum-2 (wild) and G. lucidum-6 (cultivated) at three different temperatures.