Antifungal Bioactive Compounds from Chinese Caterpillar Fungus ( Ophiocordyceps sinensis (Berk.) G. H. Sung et al .) against Plant Pathogens

Studies were conducted to extract and evaluate the intracellular bioactive molecules of Ophiocordyceps sinensis against plant pathogens. Methanolic fraction of mycelium borne bioactive molecules at 200  L concentration showed the maximum mycelial inhibition of Fusarium oxysporum f. sp. cubense (48.67 per cent) and F. o. f. sp. lycopersici (46.47 per cent), which cause wilt disease in banana and tomato, respectively. GC-MS analysis of methanolic fraction of mycelial mat extract indicated the presence of 14 different compounds hydroxylamine; glycerin; oxime-,methoxy-phenyl; adipamide; 1,2,3-Benzenetriol; cyclododecane; imidazole-5-carboxylic acid; 1,3,5-Benzenetriol; 1,3-Cyclohexanedione, 2-propyl; 2-Propenoic acid, tridecyl ester; n-Decanoic acid; 5-Undecene, 9-methyl; L-Ornithine, N5-(aminocarbonyl) and pyrrolo [1,2-a]pyrazine-1,4-dione, hexahydro. Among these, the compound hydroxylamine identified at 1.817 RT exhibited the maximum peak area of 35.00 per cent. This compound is also known to possess important pharmacological and antimicrobial properties.

Ophiocordyceps sinensis (Berk) G. H. Sung et al., is an entomopathogenic fungus belonging to Ascomycota, Sordariomycetidae, Hypocreales and Ophiocordycepitaceae (Sung et al., 2007).Sporophores of this fungus are highly valued medicinal mushrooms around the world (Borchers et al., 2004 andZaidman et al., 2005).The name Cordyceps comes from the two Latin words cord and ceps, meaning club and head.The fungus is known to parasitize the larvae of ghost moth, Hepialus armoricanus (Pegler et al., 1994;Wang, 1995 andYao, 2004).The erstwhile known anamorphic state(s) of the genus Cordyceps viz., Beauveria, Metarhizium and Paecilomyces have been reported to kill nematodes and insect pests by direct parasitism or lysis (St. Leger et al., 1992;Gillespie et al., 1998;De-faria and Wraight, 2007).Cordyceps species are parasitic rather specifically on insects, nematodes, sclerotia of Claviceps, or hypogeous ascocarps (Jeffries and young, 1994).Ophiocordyceps spp are known to produce several novel bioactive compounds like cordycepin, cordymin, adenosine, cordycepic acid, amino acid, ergosterol, sterol, superoxide dismutase, myriocin and multivitamins (Isaka et al., 2000;Li et al., 2010;Varshney et al., 2011;Kumar and Spandana, 2013).In the present study, bioactive compounds from mycelial mat of O. sinensis extracted with methanol were characterized by TLC and GC-MS.The combined action of these compounds has also been tested for their efficacy against the root rot, wilt and sheath blight pathogens.

Materials and Methods
O. sinensis isolate No.1220 was obtained from Forest Research Institute (FRI), Dehradun, India.This fungus was grown on Mushroom Complete Medium (MCM) in 90 mm diameter Petri dishes and incubated at 25°C.

Extraction of bio-active compounds
Four mycelial discs measuring 6 mm diameter each, cut from the margin of a 5 days old colony of O. sinensis were inoculated in 250 mL conical flasks containing 100 mL of sterilized MC broth (pH 5.5).The flasks were placed on a rotary shaker maintained at 120 rpm and incubated at 25°C in diffused day light (600 -800 lux) for 15 days.After incubation, the culture filtrate and the mycelial mat were separated by filtration through Whatman No.40 filter paper.Five gram of mycelial mat was powdered with liquid nitrogen and extracted three times with equal volume of methanol.The extracted sample taken in the Eppendorf tubes was centrifuged at 10,000 rpm for 15 min in a bench centrifuge (at 4°C) and the supernatants were evaporated to dryness, dissolved in methanol and stored at 4°C.

Detection of bioactive compounds Thin layer chromatography (TLC)
Bioactive compounds of mycelial mat extract were spotted on to silica gel 60 TLC plates (60 F254, 0.12 mm thick, 5×20 cm, Merck, Germany) at the rate of 5 μL /spot.After drying, chromatographs were developed using the solvent system, butanol: water (86:14).The developed TLC plates were air dried overnight to remove the remaining solvents and viewed under UV light at 250 nm.The retention factor (Rf) values of various compounds resolved on TLC plates were calculated using the formula given by Sadasivam and Manickam (1992).

Distance travelled by solute Rf =
Distance travelled by solvent

GC -MS analysis
Characterization of biomolecules of methanolic fraction of mycelial mat extract was done by GC -MS analysis.The column Elite-5MS (100 per cent

Bioassay of mycelium borne bioactive compounds
The concoction of mycelium borne bioactive compounds of O. sinensis was evaluated against F. o. f. sp.cubense and F. o. f. sp.lycopersici causing wilt disease in banana and tomato, respectively; Macrophomina phaseolina causing root rot in red gram and Rhizoctonia solani causing sheath blight in rice by agar well diffusion method (Stoke and Ridgway, 1980).After solidification of the sterile PDA medium in Petri plates, wells of 10 mm diameter were made on each of the plate using sterile cork borer on all four sides, giving equal distance; and also by leaving one cm space from the periphery of Petri plates.Secondary metabolites composite in methanolic fraction was poured separately, at the rate of 50, 100, 150 and 200 L per well using a micro pipette.Actively growing five d old mycelial discs of pathogenic fungi measuring 6 mm in diameter were inoculated separately, at the centre of each of the Petri plate and incubated at 28 ± 2ÚC for seven days.The radial growth of mycelium (mm) and zone of inhibition (mm) were recorded after 7 days of incubation.Based on the observation, per cent inhibition over control was calculated.Each dose was replicated three times.Sterile water served as control.

Results and Discussion
The results showed that the methanolic fraction of mycelial mat of O. sinensis was highly inhibitory to F. o. f. sp.lycopersici and F. o. f. sp.cubense at all concentrations tested, when compared to control (Table 2).At high concentration (200 μL), F. o. f. sp.cubense, F. o. f. sp.lycopersici and R. solani exhibited

Detection and characterization of bioactive molecules
Bioactive molecules extracted from mycelial mat of O. sinensis were identified by TLC and GC-MS.TLC results exhibited four different compounds (Rf -0.12, 0.35, 0.39 and 0.76) as shown in Fig. 1.GC-MS analysis indicated the presence of 14 different compounds (Table 1 and Fig maximum peak area of 35.00 per cent.Several heterocyclic compounds (diazepines, imidazoles and thiadiazole), alpha substituted hydroxlamine derivatives and related compounds are known to possess important pharmacological properties and antimicrobial properties (Baregama et al., 2004).Presence of glycerine (10.01 per cent expressed at 11.09 RT) in the CFC filtrate and mycelial preparations of O. sinensis would hypothetically indicate the antibacterial nature of the fungus as rightly pointed out by Kern (1971), which could be used as a form of preservative in bio-extract preparations.Another compound namely, 1, 2, 3benzenetriol and its derivatives 1, 3, 5-benzenetriol pyrogallol compound (8.51 and 3.00 per cent, respectively) detected at 18.18 and 22.94 RT (Table 1) are aromatic alcoholic compounds known for fungicidal, insecticidal, antioxidant and antiseptic activities (Euginamala and Jeyaraj, 2014).In addition, Sangeetha et al. (2015a and b) reported that the bioactive molecules present cell free culture (CFC) filtrates and mycelium of Ophiocordyceps sinensis could be potentially explored for the management of root knot nematode, Meloidogyne incognita and Fusarium spp.

Conclusion
The results of the current investigation conclusively indicate that the bioactive compounds of O. sinensis could be potentially explored to contain Fusarium spp.which cause wilt disease in crop plants.The results also pave way to explore hitherto unutilized biomolecules of O. sinensis in a more potential way against plant pathogens.Further systematic insight into individual molecules and developing step up protocols to enhance their level of production and innovative formulation will have a greater stake hold in agrochemical industry.
47 and 22.24 per cent inhibition, respectively.But, the methanolic fraction is ineffective in reducing the mycelial growth of Macrophomina phaseolina.The overall results of bioassay experiment revealed that the wilt pathogens (Fusarium spp) are more sensitive when compared to root rot and sheath blight pathogens (Plate 1).Sekaran et al. (2011) had evaluated the antifungal activity of methanolic and aqueous extract of Ganoderma lucidum and reported inhibition of mycelial growth of five fungal pathogens including Aspergillus fumigatus and Penicillium sp.Ragul (2013) reported that methanolic extract of Calocybe indica and Pleurotus florida sporophores had effectively inhibited soil borne plant pathogens at a concentration of 150 L, when tested by agar well diffusion technique.

Table 2 . Bioassay of intracellular compounds of O. sinensis against plant pathogens
PI -Per cent Inhibition ; Values are mean of five replications ; Means followed by a common letter is not significantly different by one way ANOVA48.67,46.