Identification of antifungal principle in the solvent extract of an endophytic fungus Chaetomium globosum from Withania somnifera

Extracts of Chaetomium globosum EF18, isolated as endophytic fungus from Withania somnifera, were found effective against Sclerotinia sclerotiorum. Ethyl acetate and methanol extracts were more effective than hexane extract showing >80% growth inhibition. Bioactive compound (antibiotic Sch 210971, m/z 445 and λmax 290) having antifungal activity against S. sclerotiorum has been isolated in pure form from the ethyl acetate extract following bioassay guided fractionation. Apart from this compound other fractions of polar to medium polarity were also found effective. Fraction no. VIII from VLC (Vacuum liquid chromatography) column of ethyl acetate extract was most active having IC50 value 35.4 μg/ml.


Introduction
The need for new and useful compounds to provide protection and relief to crop plants from pests and thereby sustenance of food production for human consumption is ever growing. Plant diseases have been causing devastating effects on crop plants and human life since the human civilization evolved (Agrios 2005). To combat such diseases, safer and greener chemicals have to be developed because conventional chemicals are posing greater threat to ecology and biodiversity and also causing ill effects to human health. Microbes and their compounds are emerging as alternative strategies for pest control (Montesinos 2003).
Many endophytic fungi and their metabolites have been reported to have insecticidal and fungicidal activity (Kumar et al. 2008). In our effort to isolate bioactive endophytic fungi, we isolated various endophytic fungi from Withania somnifera, among which Chaetomium globosum EF18 showed good activity against Sclerotinia sclerotiorum, Fusarium oxysporum and Rhizoctonia solani (Kumar et al., communicated paper). S. sclerotiorum is non-specific and omnivorous pathogen causing diseases to plants belonging to 75 families, 278 genera, and 408 species (Boland and Hall 1994). Some of the major crops affected by S. sclerotiorum are brassicas, potato, chickpea, pea, sunflower, beans, carrot, lettuce, soybean, kiwifruit and grapes. Although the most common disease caused by S. sclerotiorum is white mould, yet cottony rot, watery soft rot, stem rot, drop, crown rot, blossom blight are also of common occurrence in crop plants causing yield loss up to 100% (Purdy 1979). 1.5 million Tones of yield reduction due to Sclerotinia infection in soybean have been reported in US during 2004 (Wrather and Koenning 2006). This paper describes the isolation of active metabolites of endophytic fungus through bioassay mediated fractionation using VLC, Column chromatography and preparative HPLC. Bioactivity was tested against S. sclerotiorum during the experimentation.

Results
Ethyl acetate, methanol (a portion of EtoAc extract was partitioned between methanol and hexane and then assayed to see if polarity has major effect on antifungal activity) and butanol extracts of endophytic fungus C. globosum were tested at 250 μg/ml and 500 μg/ml against S. sclerotiorum. Methanol extract showed 76.58% mycelial growth inhibition (GI) at 500 μg/ml on 4 th day, which further increased to 80.83% on 5 th day. Ethyl acetate extract was effective at 500 μg/ml having 75.68% growth inhibition on 4 th day. No significant difference was observed in the growth inhibition caused by ethyl acetate extract and methanol extract on 4 th day. Butanol extract was least effective showing 33.32% growth inhibition at 500 μg/ml on 4 th day. Graphical representation of GI over no. of days is given in Figure 1.
Ethyl acetate extract was further fractionated by VLC on silica gel. On the basis of HPLC and LC-MS analysis it was observed that all the VLC fractions were mixtures of many compounds. When tested for their bioactivity, fraction WSL-2E_VIII exhibited maximum mycelial growth inhibition of 95.9%, 83.7% and 75.6% at 500 μg/ml, 200 μg/ml and 100 μg/ml respectively (Table 1). Fraction WSL-2E_VI, WSL-2E_VII, WSL-2E_IX and WSL-2E_X were statistically at par and caused more than 80% growth inhibition at 500 μg/ml. Fraction IV and V exhibited least growth inhibition,~70% at 500 μg/ml, among all the fractions. Figure 2 shows growth inhibition caused by these fractions at different concentrations. IC 50 values were calculated for these fractions and are provided in Table 2, which shows that lowest IC 50 of 40.57 μg/ml and 35.4 μg/ml on 4 th and 5 th day respectively was observed in fraction no. VIII. Chi-square values and regression equation have also been provided in the Table 2. Fraction no. IX stands next to fraction VIII showing IC 50 value of 80.14 μg/ml on 5 th day of observation.
The VLC fractions VI-X showing activity against S. sclerotiorum were having a common peak at RT 42.3-42.4 minutes (from Waters HPLC) ( Figure 3). Correlation of chemical analysis and bioassay results, it is apparent that the compound eluting at RT 42.5 is responsible for the antifungal activity. As depicted in the Table 3, increased concentration-corresponds to increased area of the peak-of this compound in the fraction was prime cause of increased bioactivity of the fraction. Table 4 shows the regression model and correlation between increasing concentration of active compound in the fraction and bioactivity of fraction. Correlation coefficient (r) of 0.86 was calculated between the % area of the compound in the HPLC chromatogram and biological activity at 100 μg/ml, which says that statistically 86% of the activity in these fractions is due to the compound and rest 14% is due to the unexplained reason. LC-MS analysis suggested the molecular weight of the compound associated with peak (RT 33.78 in LC-MS) as 445 (m/z + 446 and m/z -444) and UV maxima 226 and 288 nm (as presented in    Figure 7 shows the plate photograph of growth inhibition in Sclerotinia sclerotiorum due to the toxicant present in VLC fractions VIII, IX and X. WSL-2E_VI _I_S1 fraction was subjected to preparative HPLC to obtain pure compound ' A' , which was further analyzed by NMR spectroscopy. Comparative analysis of NMR and MASS revealed that compound ' A' is similar to Antibiotic Sch 210971 (m/z 445 and λ max 290) (Figure 8), which has previously been isolated from C. globosum by Yang et al. (2006). In the finding by Yang et al. molecular weight of the isolated compound was 445 Da and showed the protonated molecular ion at m/z + 446 similar to the compound isolated by us. Also UV absorption of Sch 210971 at 220 and 295 was near to that of our compound i.e. 226 and 288.

Discussion
Hexane extract of C. globosum has been reported as antifungal against S. sclerotiorum and Botrytis cineria (Nakashina et al. 1991   viz. methanol and ethyl acetate were more active than hexane extract against S. sclerotiorum. Antibiotic Sch 210971 has been previously reported as chemokine receptor CCR-5 inhibitor (Yang et al. 2006); however this is the first report of antifungal activity of this compound. HPLC has been utilized for the first time in our study in finding out the principle component of C. globosum responsible for antifungal activity. Previously chaetomugilin D, together with three known metabolites, chaetomugilin A, chaetoglobosins A and C have been isolated by a bioassay-guided fractionation from the EtOAc extract of the cultures of C. globosum. Chaetomugilin D also reported to have antifungal activity against Mucor miehei (Qin et al. 2009). C. globosum an endophyte to a medicinal plant Curcuma wenyujin has yielded chaetoglobosin X, which showed broad antifungal activity (Wang et al. 2012  chaetoglobosins A and C from C. globosum and they found that chaetoglobosin A is having antifungal activity against Setosphaeria turcica, causal agent of northern corn leaf blight. Two other antifungal substances viz. chaetoviridins A and B have been purified from culture broth of C. globosum isolated from barnyard grass. Chaetoviridin A exhibited higher antifungal activity with 80% reduction in disease development at 62.5 μg/ml concentration against rice blast and wheat leaf rust and 50% control of late blight of tomato at 125 μg/ml (Park et al. 2005), however purified fraction isolated in our experiment has IC 50 value of 35.4 μg/ml. In present study we have found that Antibiotic Sch 210971 can be used as marker for antifungal activity against S. sclerotiorum. Role of C. globosum in biological control has been well documented and commercial formulation has also been developed (Soytong et al. 2001). Culture filtrate of C. globosum has been reported to successfully inhibit the mycelial growth of Pythium ultimum in In vitro and pot culture experiments. Chaetomin has been the principle compound responsible for this antifungal activity (Di-Pietro et al. 1992). Cell wall degradation caused by beta-glucanases and carboxymethyl cellulases is one of the possible modes of action of C. globosum against P. ultimum (Inglis and Kawchuk 2002). Mode of action of Antibiotic Sch 210971 needs to be studied further. Antibiotic Sch 210971 enriched fraction can be formulated as antifungal biopesticide.

Batch culture fermentation of C. globosum EF18 and its extraction
Fungus was multiplied in 11.7 litres of wickerham medium [Malt extract (3 g/l); Yeast extract (3 g/l); Peptone (5 g/l); Glucose (Qualigens)-10 g/l; pH-7.2-7.4] at 24°C for 3-4 weeks. Media chemicals were purchased from Himedia, India. One flask of medium without the inoculum was kept as control. Metabolite extraction was done as per the protocol of Wicklow et al. (1998). Extraction procedure has been depicted in Figure 9 as flow diagram.

Isolation of antifungal compound
A schematic diagram of procedure for separation and purification is presented in Figure 10. Extract was subjected to VLC and eluted with dichloromethane: methanol in the following order: 100% DCM, 95:5 DCM: MeOH,90:10,80:20,70:30,60:40,50:50,30:50,25:75 and 100% MeOH. These fractions (WSL 2E I-X; Figure 10) were dried in rotary under vacuum and investigated by TLC and HPLC. Based on the HPLC and LC-MS profiles of the fractions, fraction no. WSL2E VI was further purified by sephadex LH 20 (Merck) column with dichloromethane and methanol 50:50. Based of TLC pattern, fractions were grouped in to 5 groups-WSL2EVI_I-V. These 5 fractions were analyzed by HPLC and LC-MS. Sub-fraction 1 (WSL2EVI_I) was further purified by sephadex column with 100% methanol followed by preparative HPLC (Figure 10). Preparative HPLC was done with the following gradient 0-5 min 50% methanol and 5-35 min increase from 50-100% of methanol and from 35-40 100% methanol. One major compound was obtained (compound ' A' in Figure 10).

Identification of antifungal Sch 210971
The Identification was done by MASS and NMR.  Table 5.
Bioassay of extracts/fractions of C. globosum EF18 against Sclerotinia sclerotiorum Bioassay of crude extract was done as per the procedure described by Kumar and Kaushik (2013). Whereas, for bioassay of fractions 30 mg of dried extract was dissolved in 600 μl of methanol and out of this 60, 120 and 300 μl were mixed to 30 ml of media for 100, 200 and 500 μg/ml concentrations. There were 3 replications for each treatment.

Data analysis
Percent growth inhibition (GI) and analysis of variance of the GI was performed as described earlier (Kumar and Kaushik 2013). Concentration at which 50% growth inhibition occurs i.e. IC 50 was calculated by probit analysis (Finney 1971) Figure 9 Schematic diagram of extraction procedure for obtaining crude fungal extracts of C. globosum EF18 (WSL2).  Figure 10 Schematic diagram of procedure of separation/ purification of ethyl acetate extracts of C. globosum isolate EF18 (WSL2E).