Production of Volatile Organic Compounds of Fusarium oxysporum f. sp . lycopersici on Coinoculation with the Metabolites of Chaetomium globosum

The present study focused on divulging the effect of 15 and 30 days old culture filtrates of Chaetomium globosum on the mycelial growth and toxin production of Fusarium oxysporum f. sp. lycopersici . The cell-free culture (CFC) filtrate of C. globosum was inoculated in the liquid medium of F. oxysporum f. sp. lycopersici . The results revealed that mycelial growth was reduced in the CFC filtrate of C. globosum inoculated medium compared to control (pathogen alone). The mycelial dry weight of the F. oxysporum f. sp. lycopersici was 0.864g in fifteen days old CFC filtrate followed by 30 days old CFC filtrate of C. globosum (1.374g) amended medium. Metabolites from the CFC filtrate and control were extracted separately using chloroform followed by ethyl acetate and the extract was subjected to GC-MS analysis. GC-MS analyses showed that methyl ester group was present in 15 days old culture filtrate condensate compared to 30 days old CFC filtrate condensate. The compounds are methyl tetradecanoate, octadecanoic acid, methyl ester, and DL-Proline, 5-oxo-, methyl ester. Thirty days old culture filtrate condensate of C. globosum contained more phenol groups like phenol 2,4-bis (1,1-dimethylethyl) at 17.62 RT followed by diisooctyl phthalate, 2-methylene-, and Metabolites of F. o. f. sp. lycopersici were also c haracterized by GC-MS, which showed very few volatile compounds like methyl tetradecanone, oleic acid, eicosyl ester, methyl stearate, and bis(2-ethylhexyl)phthalate. These compounds were not detected in co-inoculation of 15 and 30 days old CFC filtrate of C. globosum and F. o . f. sp. lycopersici . The difference in the volatile profile may be due to the effect of the metabolites of C. globosum on F. o . f.sp. lycopersici .


INTRODUCTION
Fusarium causing wilt disease in tomato persists in soil for long period and produces survival structures such as chlamydospores (Goyal et al., 1973 andShuxian et al., 1998). Complete control of Fusarium is very difficult with a conventional method. Even though fungicides are used for the management of many pathogens, continuous use of such chemicals are reported to induce the resurgence of existing pathogens and the emergence of new races, posing a greater threat. At this juncture, biomolecules from the secondary metabolites have multiple advantages to manage plant pathogens and induce plant growth. Microbes producing secondary metabolites are called mycotoxin, present in the growing medium as well as in the plant tissue. Secondary metabolites of several fungi are known to possess an inhibitory effect on plant pathogenic fungi, bacteria and viruses (Janssens et al., 1992;Breheret et al., 1997 andMarumoto et al., 1997). The involvement of secondary metabolites in plant disease and particularly in the interaction between beneficial and pathogenic fungi, has been studied by several scientists (Fravel, 1988). Every microorganism produces different nature of secondary metabolites with low molecular weight compounds. Trichoderma spp is known to produce several secondary metabolites that are toxic for plant pathogenic fungi. Secondary metabolite compounds from Trichoderma spp are pyrone 6-pentyl-2H-pyran-2-one; koninginins; viridians; harzianopyridone; azaphilones; butenolides ; hydroxyl -lactones; isocyano metabolites; diketopiperazines; gliotoxin; gliovirin and peptaibols. A pyrone 6-pentyl-2Hpyran-2-one is a metabolite purified from the culture filtrate of Trichoderma spp, responsible for coconut aroma and has shown antifungal activity against several plant pathogens (Scarselletti and Faull., 1994;Worasatit et al., 1994). Likewise, Chaetomium spp also produces several biologically active compounds such as chaetoglobosin A, C, D, E, G, R, chaetoviridins, chetomin, chaetocin, chaetochromins, cochliodinol, mollicellin, oosporein, ergosterol, allantoin, uracil, and sterigmatocystin (Brewer et al., 1968;Cole and Cox, 1981;Park et al., 2005;Fogle et al., 2007;Aggrawal, 2015). Biswas et al. (2000 and2012) studied the characterization of antifungal metabolites from C. globosum and their antagonism against fungal plant pathogens. Soytong (1992) and Soytong et al. (2001) showed that a specific isolate of C. cupreum produced secondary metabolites that significantly suppressed tomato wilt caused by F. oxysporum f. sp. lycopersici and later found that this isolate of C. cupreum produced rotiorinols A to C and rotiorin, which also exhibited antifungal activity against Candida albicans (Kanokmedhakul et al., 2006 andPhong et al., 2016). C. globosum produces the antibiotic chaetoglobosin C, which suppressed the growth of many plant pathogens such as Colletotrichum gloeosporioides, C. dematium, Fusarium oxysporum, Phytophthora palmivora, P. parasitica, P. cactorum, Pyricularia oryzae, and Sclerotium rolfsii (Soytong et al., 2001). Some compounds have been reported to possess significant biological activities, such as cytotoxic, enzyme inhibitory, and antibiotic (Gunatilaka, 2006;Scherlach et al., 2010). The antimicrobial activity of chaetoglobosin-C, which is produced by C. elatum ChE01, and C. lucknowense CLT01 could be involved in the disease control mechanism of these antagonistic fungi against tomato wilt fungus F. oxysporum f. sp. lycopersici (Thohinung et al., 2010). With this background, the current study on the reduction of secondary metabolite secretion of F. oxysporum. f. sp. lycopersici in the culture medium was tested by adding beneficial microbe of C. globosum under in vitro conditions.

MATERIAL AND METHODS
Isolation and identification of C. globosum and F. oxysporum f. sp. lycopersici Fusarium sp. was isolated from the infected plant tissue of the tomato. The same field bioagent of Chaetomium spp was also isolated and both pathogens and bioagents were identified based on the morphological and molecular level, and the molecular sequence of C. globosum (MN069626.1) and F. oxysporum f. sp. lycopersici (MN071397.1) were submitted into NCBI.

Collection of cell free culture (CFC) filtrates
The conical flasks containing sterilized PDA broth were inoculated with mycelial discs of C. globosum, measuring 6 mm diameter (5 numbers in each flask). The flasks were placed in an incubator cum shaker maintained at 25°C and agitated at 120 rpm. The culture filtrates were collected at 15 and 30 days after inoculation under aseptic conditions, filtered through a membrane filter (0.2 µm). The CFC filtrates were used to arrest the mycelial growth and metabolite production of F. oxysporum f. sp. lycopersici. Cell-free culture filtrates (15 and 30 days old) of C. globosum were aseptically mixed with sterilized liquid medium separately in each flask at the rate of 1 mL in 100 mL broth. Then, a 5 mm mycelial disc of the test pathogen was inoculated in the same flask, placed in an incubator, and maintained at 25°C, and agitated at 120 rpm. The ingredients of the liquid medium contained 0.5 g malt extract; one gram of peptone; one gram of KH 2 PO 4 , 0.3g MgSO 4 .7H 2 O; 0.3g KCL; one mL of CuSO 4 .5H 2 O solution (0.005g dissolved in one lit.) and one mL of ZnSO 4 .7H 2 O solution (0.01g dissolved in one lit) per lit. The pathogen alone in the liquid medium without cell-free culture filtrates served as a control. Three replications were maintained in each treatment. Ten days after incubation, the mycelial mat and the culture filtrates were separated by filtration through Whatman No.40 filter paper. The mycelial dry weight of the pathogen was taken separately in each treatment to know the biomass production.

Extraction of Toxin
After separating the mycelial mat, the ten days old cell free culture filtrates of F. o. f. sp. lycopersici were taken, which were grown separately in a liquid medium containing one percent of 15 and 30 days old cell free culture filtrates of C. globosum. The pathogen alone was grown in a liquid medium as a control used for toxin extraction. Then the filtrate was further centrifuged at 10,000 rpm for 15 min. The cell-free culture filtrate was extracted with organic solvents like chloroform and ethyl acetate. Liquidliquid extraction was carried out using chloroform, and the compound-containing layer was collected separately. Subsequently, the remaining aqueous layer was mixed equally with ethyl acetate. Then, the compound containing layer was separated. This was repeated three times with an equal volume of solvents. The two organic solvent layers were pooled together and it was evaporated under reduced pressure using a rotary evaporator to obtain the compound residues. The condensate of the solvent layer was dried and dissolved in methanol (1mg/mL), filtered through a membrane filter (0.2 μm), and stored at 4°C for GC-MS analysis. The compounds were also extracted from the 15 and 30 th day old culture filtrate of C. globosum and liquid medium alone without fungus by using a method as described above. The extracted compounds were further characterized by GC-MS for compound identification.

Characterization of toxin through GC-MS
Characterization of CFC filtrate condensate of F. oxysporum f. sp. lycopersici was done by GC-MS analysis. The column Elite-5MS (100 per cent Dimethyl poly siloxane), 30 x 0.25 mm x 0.25 μm df equipped with GC Clarus 500 Perkin Elmer and turbo mass -gold -Perkin -Elmer detector was used. The carrier gas (helium) flow rate was one mL per min, split 10:1 and injected volumes were two μL. The column temperature was maintained initially at 110°C at the rate of 10°C /min followed by increasing up to 280°C at the rate of 5° C /min and hold time 9 min. The injector temperature was 250°C and this temperature was held constant for 36 min. The electron impact energy was 70 e V, the Julet line temperature was set at 2000°C and the source temperature was set at 200°C. Electron impact (EI) mass scan (m /z) was recorded in the 45-450 aMU range. Using computer searches on the NIST Ver. 2011 MS data library and comparing the spectrum obtained through GC -MS the compounds present in the sample were identified.

RESULTS AND DISCUSSION
Growth of Fusarium oxysporum f. sp. lycopersici in metabolite amended medium F. oxysporum f. sp. lycopersici caused wilt disease in tomatoes leading to yield losses reducing production and productivity of tomato (Hussain et al., 2016). The result of the current study is revealed that the fungus, F. oxysporum f. sp. lycopersici was able to grow in fifteen and thirty days old CFC filtrate of C. globosum amended medium at one per cent concentration. Nevertheless, the mycelia growth of F. oxysporum f. sp. lycopersici was less in these two treatments when compared with control. lycopersici was recorded only 0.864g in fifteen days old CFC filtrate followed by thirty days old CFC filtrate of C. globosum (1.374g) amended medium ( Figure  1). Rashmi et al. (2011) reported that antagonistic fungal mycelial growth was suppressed when the other fungus was grown in a toxin amended medium.  Figure 2). Soytong (1992) and Soytong et al. (2001) also reported that an isolate of Chaetomium cupreum producing metabolites significantly suppressed the mycelial growth of F. oxysporum f. sp. lycopersici causing wilt disease of tomato.  Figure 3). Biswas et al. (2000 and2012) studied the identification and characterization of antifungal compounds like chaetoglobosin and chaetomin produced by C. globosum. These two compounds significantly suppressed the redial growth of the plant pathogens such as Bipolaris sorokiniana, Macrophomina phaseolina, Rhizoctonia solani and Phytium ultimum. They also reported that chaetoglobosin at the rate of 1000ppm and 500ppm mixed medium showed only 13mm and 17mm mycelial growth of Bipolaris sorokiniana compared with control (69mm).

CONCLUSION
The current investigation revealed the reduction of mycelial growth and toxin production of F. oxysporum f. sp. lycopersici under different days old cell-free culture (CFC) filtrate of Chaetomium globosum amended medium. The mycelial dry weight of F. oxysporum f. sp. lycopersici was recorded to be only fifty per cent in fifteen days old cell-free culture filtrate amended medium. The volatile organic compounds were characterized through GC-MS. Fatty acid, phenol, and terpenoids compounds were present in cell-free culture filtrate of C. globosum and this might be responsible for the reduction of mycelial dry weight. However, the additional compounds from the culture filtrate of F. oxysporum f. sp. lycopersici have antimicrobial nature for their self-survivability. Further study is needed to test compound compatibility and explore a mode of action against plant pathogens.