Endophytic Fungi Isolated from Oil-Seed Crop Jatropha curcas Produces Oil and Exhibit Antifungal Activity

Jatropha curcas L., a perennial plant grown in tropics and subtropics is popularly known for its potential as biofuel. The plant is reported to survive under varying environmental conditions having tolerance to stress and an ability to manage pest and diseases. The plant was explored for its endophytic fungi for use in crop protection. Endophytic fungi were isolated from leaf of Jatropha curcas, collected from New Delhi, India. Four isolates were identified as Colletotrichum truncatum, and other isolates were identified as Nigrospora oryzae, Fusarium proliferatum, Guignardia cammillae, Alternaria destruens, and Chaetomium sp. Dual plate culture bioassays and bioactivity assays of solvent extracts of fungal mycelia showed that isolates of Colletotrichum truncatum were effective against plant pathogenic fungi Fusarium oxysporum and Sclerotinia sclerotiorum. Isolate EF13 had highest activity against S. sclerotiorum. Extracts of active endophytic fungi were prepared and tested against S. sclerotiorum. Ethyl acetate and methanol extract of C. truncatum EF10 showed 71.7% and 70% growth inhibition, respectively. Hexane extracts of C. truncatum isolates EF9, EF10, and EF13 yielded oil and the oil from EF10 was similar to oil of the host plant, i.e., J. curcas.


Introduction
Jatropha curcas L. (Family: Euphorbiaceae), a promising energy crop, is being extensively studied for developing bio-fuel technology as well as for other beneficial use, viz., antimicrobial and pesticidal activity. Leaf extract of Jatropha has been known to possess insecticidal activity against mosquito larvae [1]. Insecticidal property of the seed oil and plant extract has been reported against cotton bollworm, pests of pulses, potato and corn [2]. Diseases of J. curcas include anthracnose caused by Colletotrichum gloeosporioides [3], black rot caused by Botryosphaeria diplodea [4], root rot caused by Rhizoctonia bataticola [5] and root rot and collar rot caused by Lasiodiplodia theobromae [6]. J curcas is widely distributed in many parts of tropics and subtropics of the world and can be easily cultivated in low to high rainfall areas of saline and marshy lands [7]. Its adaptation to diverse agro-climatic condition is linked to its ecological fitness, which possibly could be in part, due to the presence of endophytic fungi [8]. Endophytic fungi live inside the plant without causing any overt negative effect on the host, rather they protect the host plant from pests and diseases [9]. The ability of endophytic fungi of grasses to provide protection from insect herbivore [10], [11], [12] drew the attention of researchers to exploit the endophytic microflora, especially fungi for better health of crop plants. Later, several workers reported endophytic fungi from plants and their bio-activity against wide range of pests and pathogens. Antifungal activity of endophytic fungi is well documented [13], [14], [15], [16], [17]. With the hypothesis that pesticidal property in Jatropha extracts and its seed oil is in part due to the presence of endophytic fungi, the present study was undertaken to assess the antifungal activity of endophytic fungi present in J. curcas against Rhizoctonia solani, Sclerotinia sclerotiorum, and Fusarium oxysporum phytopathogens. These phytopathogens have a wide host range and cause major losses in important food crops like rice, maize, wheat, and chickpea.

Isolation of endophytic fungi
Isolation of endophytic fungi was performed during June 2007 to August 2007. Nine endophytic fungi were isolated from 44 tissue segments of the leaf of J. curcas with isolation frequency of 20.5% and no endophytic fungus appeared out of 16 tissue segments of petiole kept for isolation up to 30 days. Pure cultures of the nine endophytic fungi EF8-EF16 were identified by rDNA sequencing of their ITS region [18]. Isolate EF8 was identified as Nigrospora oryzae, four isolates EF9, EF10, EF13, and EF 14 were identified as Colletotrichum truncatum, while EF11 was identified as Fusarium proliferatum, EF12 as Chaetomium sp., EF15 as Guignardia camelliae, and EF16 as Alternaria destruens. BLAST per cent similarity to sequences in the NCBI database from previously identified fungi ranged from 96% to 100% (Table 1).

Dual culture bioassay of endophytic fungi
Dual culture bioassay of the isolated endophytic fungi of J. curcas was carried out to assess their activity against plant pathogenic fungi R. solani, S. sclerotiorum, and F. oxysporum. All the isolates of the C. truncatum (EF9, EF10, EF13 and EF14) and Chaetomium isolate EF12 showed antagonistic activity against S. sclerotiorum and F. oxysporum (Table 1). While N. oryzae isolate EF8 was effective against F. oxysporum, and G. cammillae isolate EF15 was effective against S. sclerotiorum, none of the endophytic fungi were found effective against R. solani. Figure 1 shows the effect of endophytic fungi EF9 and EF10 against S. sclerotiorum. On the basis of these observations EF9, EF10, EF12 and EF13 were further taken up for batch culture fermentation and metabolite extraction. Batch culture fermentation was done by culturing each one of these endohytic fungi in one flask of 1 liter capacity containing 300 ml of the wickerham medium.

Extraction of metabolites from active endophytic fungi
Extraction of metabolites from endophytic fungi EF9, EF10, EF12, EF13, and EF15 was done with ethyl acetate partitioning. Endophytic fungi grown in the liquid culture for 4 weeks were ground after overnight soaking in ethyl acetate and filtered. The filtrate was partitioned with ethyl acetate and dried under vacuum evaporator. After extraction with ethyl acetate, residue was further extracted with butanol. The dried ethyl acetate extract was further partitioned between hexane and 90% methanol. Yield of the extracts in respective solvents is given in Table 2.
Hexane extract of C. truncatum Hexane extracts of C. truncatum isolates EF9, EF10, and EF13 yielded an oil and the oil from EF10 was found to be similar to oil of the host plant, i.e., J. curcas in fatty acid profile. Variation in the oil yield was recorded; EF10 produced the highest amount of oil, i.e., 99.3 mg/l of media while EF9 and EF13 produced 94.6 mg/l and 42 mg/l under un-optimized conditions. Chromatograms of the oils analyzed by gas chromatography are given in Figure 2 (A, B, C, D, E) and the fatty acid composition is given in Table 3. Fatty acid profile of hexane extract of EF10 was found similar to fatty acid profile of Jatropha seed oil. Jatropha seed oil is known to contain four major fatty acids namely; palmitic acid, stearic acid, oleic acid, and linoleic acid and these four fatty acids were also present in the hexane extract of endophytic fungus EF10, whereas EF12 has few more peaks which are in addition to the peaks of these four fatty acids. Chromatogram of EF9 and EF13 did not have the characteristics peaks of Jatropha seed oil, as they are having only one major peak with some smaller peaks.

Bioassay of the extracts
Among the four C. truncatum isolates, two isolates, viz., C. truncatum EF9 and C. truncatum EF10 were compared for their activity. EF10 recorded a better GI value of 71.8% compared to 58.4% GI with EF9 at concentration of 500 mg/ml on the 4 th day (Table 4). Therefore, for further sub-fractionation C. truncatum isolates EF10 and EF13 were selected along with Chaetomium sp EF12. Hexane, butanol, and methanol extracts of EF10, EF12, and EF13 were tested at 250 mg/ml and 500 mg/ml for their antifungal activity against S. sclerotiorum. Hexane extracts of these fungi were least effective and butanol extract of EF13 showed 66.6% GI on the 4 th day, while EF12 has a GI of 10% only (Table 5). Methanol extract of C. truncatum isolate EF13 exhibited the highest activity with 83.33% and 66.67% fungal growth inhibition at 500 mg/ml and 250 mg/ml, respectively. Statistically, no significant difference in the GI was observed. Methanol extract of C. truncatum isolate EF10 showed 70% GI at 500 mg/ml and 3.46% only at 250 mg/ml. While methanol extract of Chaetomium sp. isolate EF12 exhibited moderate activity with 43.33% of GI (Table 6). No growth inhibition was observed in hexane extracts of EF10, EF12, and EF13 at 250 mg/ml; hexane extract of EF10 at 500 mg/ml; butanol extracts of EF10, EF12 and EF13 at 250 mg/ ml; butanol extract of EF10 at 500 mg/ml; and methanol extract of EF12 at 250 mg/ml. Photographs of effect of extracts of endophytic fungi on S. sclerotiorum is given in Figure 3.

Discussion
Li et al. [19] isolated 57 strains of endophytic fungi from the roots and stems of Jatropha, among which two strains are antagonistic to Colletotrichum gloeosporioides. Later a group in India  isolated endophytic fungi from J. curcas and reported Leptosphaeria sp. as predominant fungus [20]. In our study, we isolated nine endophytic fungi from J. curcas leaves which represent six species, viz., C. truncatum (4) Colletotrichum truncatum has worldwide occurrence as a plant pathogen, causing diseases of several plants including soybean [21], broad bean, lentil [22], Stylosanthes sp., [23], cowpea [24], Pisum sativum [25], urdbean [26], christmas rose [27]. However, it has been reported as an endophyte from Artimisia sp. [28]. Despite the pathogenic nature of the fungus, it has been utilized for management of several weeds, including Sesbania exaltata a noxious weed of soybean [29]. In our study, we found this fungus as predominant endophyte of J. curcas. When tested against plant pathogenic fungi, the isolates of C. truncatum EF 9, EF10, and EF14 were found active against S. sclerotiorum and F. oxysporum. Ethyl acetate extract of EF10 isolate caused higher growth inhibition than EF9, although statistically non-significant. Maximum zone of inhibition was observed with EF10. The higher bioactivity of C. truncatum EF10 was also observed in its extracts. All the isolates of C. truncatum were found yielding oil, and fatty acid composition of oil isolated from EF10, was similar to Jatropha seed oil. C. truncatum isolate EF10 also recorded highest yield, while its methanol extract recorded very good antifungal activity.
Fusarium proliferatum has been isolated as an endophyte from the mangrove plant [39], pejibaye (Bactris gasipaes) [40], and inner bark tissue of Dysoxylum binectariferum [41]. It has also been shown to be a pathogen of crop plants causing head blight of oat [42], stem rot in vanilla [43], crown and root rot in wheat [44]. Endophytic fungi from pejibaye have the potential to be developed as biocontrol agent against plant pathogens. Beauvericin, an antibiotic against bacterial pathogens has been isolated from Fusarium proliferatum CECT 20569 grown on wheat; the technique of solid state fermentation [45]. In our study, F. proliferatum isolate EF11 did not show any activity, although the fungus was grown under different conditions.
Alternaria destruens is described from Cuscuta gronovii [57].  [64], and Gossypium sp. [65]. A. destruens has been reported to have herbicidal activity against Dodder, a parasitic weed and it works well in combination with other herbicides as well [66]. Our A. destruens isolate from Jatropha did not show any activity against R. solani, S. sclerotiorum, and F. oxysporum.
In the present study, the fungi we isolated from J. curcas leaves showed activity against S. sclerotiorum and F. oxysporum, however none exhibited activity against R. solani. Highest activity against S. sclerotiorum was recorded by C. truncatum isolate EF13 and its extract whereas G. camelliae isolate EF15 showed highest activity against F. oxysporum in dual culture bioassay. C. truncatum isolate EF13 and Guignardia camelliae isolate EF15 can further be explored for its biocontrol potential against S. sclerotiorum and F. oxysporum respectively. Their metabolites can also be explored for their potential as fungicide.
There are many reports of the production of metabolites by endophytic fungi, which are also produced by the host plant. For example taxol, a major anticancer drug being produced by Taxus sp. has been reported from the endophytic fungus of the plant, Taxomyces andreanae [76]. Camptothecin, a potent antineoplastic agent has been produced by endophytic fungi of the inner bark of Camptotheca acuminata, a known source of camptothecin [77]. In our experiment we found endophytic fungi producing an oil have a fatty acid profile similar to oil produced by its host J. curcas. Although, how this apparent co-metabolism is occurring is currently unknown; it may be due to the sharing of genetic material between these organisms.

Conclusion
The present study gives evidence that J. curcas harbors endophytic fungi of beneficial activity. Results indicate that these fungi may be helping the plants in protecting from pathogenic fungi. Endophytic fungi C. truncatum isolate EF13 found active in the study can be explored for its potential as a biocontrol agent against S. sclerotiorum and F. oxysporum, after studying its pathogenicity on crop plants and other non-target effects, while EF10 can be explored for its oil production capacity.

Sample collection
Leaf and petiole samples of Jatropha curcas were collected from plants grown at TERI, New Delhi, India, during June 2007 to August 2007. Immediately after the collection, plant parts were washed with tap water and processed for isolation of endophytic fungi.

Isolation of endophytic fungi
Endophytic fungi were isolated from the healthy plants as per the procedure of Kumar et al. [17]. The plant parts -leaves and petioles -were surface sterilized with 70% ethanol for 2 minutes followed by 1% sodium hypochlorite for 3 minutes. Surface sterilized plant parts were dried on sterile blotting sheet and then chopped and transferred to malt agar plates, after taking imprint of dried sterile plant part in a petri-plate containing media. These plates were incubated at 24uC for seven days. Hyphal tips of the developing fungal colonies were transferred to fresh malt agar plates.

Identification of endophytic fungi
Slide preparation. Fungal mycelium was stained with cotton blue and mounted in polyvinyl lactic acid glycerol (PVLG) by heating at 65uC for 2-3 days and observed under light microscope.
DNA Isolation and amplification. Fungal genomic DNA was isolated from fresh mycelia scrapped from potato dextrose agar (PDA) plates using the DNeasy plant minikit (Qiagen) according to manufacturers' protocol. DNA amplification by PCR was then performed according to the procedure described earlier by Kumar et al. [17].
Purification of the PCR product was done by MontageH PCR centrifugal filter devices as per the suppliers' protocol. Sequencing of the purified PCR product was carried out at LabIndia (Gurgaon, India) on an automated multicapillary DNA sequencer, ABI Prism 3130xl genetic analyzer (Applied Biosystems, USA).
To identify the isolates, sequences were subjected to the BLAST search with the NCBI database [78]. DNA sequences of representative isolates from this study have been submitted to NCBI GenBank database with accession no GQ176271 to GQ176279.

Bioassay of endophytic fungi against plant pathogenic fungi
Bioassay of endophytic fungi against plant pathogenic fungi was done by dual culture technique [79]. Potato dextrose agar medium

Extraction of metabolites of endophytic fungi
After 24 days of incubation, 250 ml of ethyl acetate was added to each flask, mixed, allowed to steep for 24 hrs, blended with a hand blender (INALSA Tech., India) for 15 minutes and filtered by whatman filter paper under vacuum [80]. The filtrate was collected and residual aqueous phase was partitioned thrice with ethyl acetate, followed by butanol (Qualigens, India) partitioning. The extracts were dried with vacuum rotary evaporator (Heidolph Inc, Germany). The ethyl acetate extract was further partitioned between 90% methanol (Qualigens, India) and n-hexane (Qualigens, India). The hexane, butanol, and methanol extracts after drying with vacuum rotary evaporator were subjected to further experimentation. Schematic diagram for the extraction of the metabolite has been given in Figure 4.

Bioassay of extracts of the endophytic fungi
Different extracts were tested against S. sclerotiorum by first dissolving 30 mg of dried extract in 800 ml of methanol. From this solution, 200 and 400 ml were added to 30 ml of molten PDA media, mixed and then poured into three 10 cm Petri plates to obtain 250 and 500 mg/ml extract concentrations, respectively. To obtain 1000 mg/ml concentration, 30 mg of dried extracts were dissolved in 400 ml of methanol and 400 ml were added to 30 ml of molten PDA. S. sclerotiorum was inoculated at the centre of the plate and radial growth was measured at intervals till the control plate attained the full growth. Control growth plates contained 400 ml of methanol. Per cent growth inhibitions of the extracts were calculated relative to the growth on the control plate.
Gas chromatography of hexane extract of C. truncatum isolates Gas chromatography (GC) was done to examine the fatty acid composition of the oil extracted from C. truncatum and J. curcas (host plant). GC was done on Nucon 5700 gas chromatograph equipped with Flame ionisation detector (FID) and capillary column of 30 m length and 0.25 mm ID. Nitrogen was used as carrier gas and hydrogen for flame. Sample preparation was done by mixing 200 ml of oil and 200 ml of methylating reagent (Ethanol:Benzene:Acetyl chloride at 20:4:1) followed by heating at 72uC for 1 hr and then partitioning with 200 ml of hexane. 2 ml of the hexane soluble part was injected to GC and allowed to run for 20 minute with starting temperature of 180uC and final temp of 232uC with increase of 4uC/minute.

Data analysis
Growth inhibition (GI) was calculated as per the following formula:

GI~f(A-B)=Ag|100
where A = radial diameter of fungus growing on the control plate B = radial diameter of fungus growing on the experimental plate All experiments were conducted in triplicate and GI for each replicate was calculated. Analysis of variance of the GI was performed by online statistical package (Web Agri Stat Package-WASP1) of ICAR-Goa regional centre, Goa, India. Least significant difference (LSD) and standard error were calculated and means were compared.