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Open Access

Peer-reviewed

Research Article

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

Abstract

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.

Citation: Kumar S, Kaushik N (2013) Endophytic Fungi Isolated from Oil-Seed Crop Jatropha curcas Produces Oil and Exhibit Antifungal Activity. PLoS ONE 8(2): e56202. https://doi.org/10.1371/journal.pone.0056202

Editor: Scott E. Baker, Pacific Northwest National Laboratory, United States of America

Received: May 7, 2012; Accepted: January 10, 2013; Published: February 8, 2013

Copyright: © 2013 Kumar, Kaushik. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The study was funded by the host institution, i.e., The Energy and Resources Institute (TERI), New Delhi, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

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.

Results

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).

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Table 1. Identification of endophytic fungi isolated from Jatropha curcas, their per cent similarity in BLAST and their bio-efficacy against plant pathogens.

https://doi.org/10.1371/journal.pone.0056202.t001

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.

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Figure 1. Dual culture bioassay of Jatropha endophytic fungi against Sclerotinia (S).

https://doi.org/10.1371/journal.pone.0056202.g001

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.

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Table 2. Yield of the different extracts obtained from endophytic fungi isolated from J. curcas.

https://doi.org/10.1371/journal.pone.0056202.t002

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.

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Figure 2. Gas chromatograms of fatty acid composition of hexane extract of endophytic fungi EF 9 (A), EF 10 (B), EF 12 (C) and EF 13 (D) isolated from J. curcas and jatropha seed oil (E).

https://doi.org/10.1371/journal.pone.0056202.g002

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Table 3. Fatty acid profile of oil isolated from endophytic fungi and Jatropha seed oil.

https://doi.org/10.1371/journal.pone.0056202.t003

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 µg/ml on the 4th 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 µg/ml and 500 µg/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 4th 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 µg/ml and 250 µg/ml, respectively. Statistically, no significant difference in the GI was observed. Methanol extract of C. truncatum isolate EF10 showed 70% GI at 500 µg/ml and 3.46% only at 250 µg/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 µg/ml; hexane extract of EF10 at 500 µg/ml; butanol extracts of EF10, EF12 and EF13 at 250 µg/ml; butanol extract of EF10 at 500 µg/ml; and methanol extract of EF12 at 250 µg/ml. Photographs of effect of extracts of endophytic fungi on S. sclerotiorum is given in Figure 3.

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Figure 3. Antifungal activity of solvent extracts of J. curcas endophytic fungi against Sclerotinia sclerotirum.

A- EF9 EtoAc 500 µg/ml, B- EF9 EtoAc 1000 µg/ml, C- EF10 EtoAc 500 µg/ml, D- EF10 EtoAc 1000 µg/ml, E- EF13 EtoAc 500 µg/ml, F- EF13 EtoAc 1000 µg/ml, G- check plate of S. sclerotiorum.

https://doi.org/10.1371/journal.pone.0056202.g003

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Table 4. Effect of ethyl acetate extracts of endophytic fungi of Jatropha curcas on growth of Sclerotinia sclerotiorum.

https://doi.org/10.1371/journal.pone.0056202.t004

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Table 5. Effect of butanol extracts of endophytic fungi of Jatropha curcas on growth of Sclerotinia sclerotiorum.

https://doi.org/10.1371/journal.pone.0056202.t005

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Table 6. Effect of methanol extracts of endophytic fungi of Jatropha curcas on growth of Sclerotinia sclerotiorum.

https://doi.org/10.1371/journal.pone.0056202.t006

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), N. oryzae (1), F. proliferatum (1), G. cammillae (1), A. destruens (1), and Chaetomium sp. (1) with C. truncatum as predominant species. No endophytic fungi emerged from J. curcas petiole.

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.

Endophytic association of N. oryzae is well documented and reported from crop plants, including maize [30], ornamental plant, Rosa hybrida [31], fruit plant, banana [32], weed, Parthenium hysterophorus [33], Eucalyptus citriodora Hook. [34], medicinal plant Tylophora indica [17], Nyctanthes arbor-tristis [35], and Crataeva magna [36]. Extracts from an isolate of N. arbor-tristis have been shown to have strong antifungal and antibacterial activity also. It has also been shown to be a pathogen of crop plants causing diseases of rice [37], wheat, sorghum, barley [38]. In present study our isolate of N. oryzae was found active against F. oxysporum.

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.

Guignardia sp. has been isolated as an endophyte from several host plant including Kandelia candel, a mangrove plant [46], Centella asiatica [47], Garcinia [48], Heterosmilex japonica [49], sugarcane [50], Coffea arabica [51], Tryptergium wilfordii [52], Spondias mombin [53], Musa acuminata [54], leaves of ericaceous plant [55] and Undaria pinnatifida [56]. Our isolate Guignardia camelliae EF15 from J. curcus showed activity against S. sclerotiorum, which is the first report of antifungal activity of the fungus. Ethyl acetate extract of this endophytic isolate also showed moderate level of antifungal activity against Sclerotinia disease of chickpea.

Alternaria destruens is described from Cuscuta gronovii [57]. Alternaria sp. have been isolated as an endophyte from several host plants including medicinal plants from the western ghats of India [19], Deschampsia antarctica [58], Bletilla ochracea [59], Rosa damascaena [60], Azadirachta indica [61], Catheranthus roseus [62], Polygonum senegalense [63], mangrove plants [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.

Chaetomium was isolated as an endophyte from many of the plants including Cinnamomum camphora [67], Cucumis sativus [68], and Huperzia serrata [69]. Chaetomium sp. from cucumber showed nematicidal activity when applied as seed treatment. C. globosum has been the most common species of all Chaetomium sp. and has been reported as an endophyte from several host plants including Canvalia maritime [70], Ipopmea pes-caprae, Launea sarmentosa and Polycarpaea corymbosa [71], Oryza sativa [72], and Ginkgo biloba [73]. Endophytic association of C. globosum has also been reported from medicinal plants, viz., Terminalia arjuna, Crataeva magna, Azadirachta indica, and Holarrhena antidysentrica [74]. Antagonistic activity of C. globosum has been identified against major pathogens of cotton, viz., Macrophomina phaseolina, Fusarium solani, and Rhizoctonia solani, [75]. As an endophyte from Tylophora indica, it showed antifungal activity against S. sclerotiorum and F. oxysporum [17].

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.

Materials and Methods

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 24°C 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 65°C 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 Montage® 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 (Himedia) was selected for dual culture as it favours growth of plant pathogenic fungi — Rhizoctonia solani, Sclerotinia sclerotiorum, and Fusarium oxysporum. The cultures were obtained from Indian Type Culture Collection, Indian Agricultural Research Institute, New Delhi. Plant pathogenic fungi and endophytic fungi were inoculated on PDA plate at periphery, opposite to each other. After incubation at 24°C for seven days plates were observed and antagonism was expressed by presence of inhibition zone at the point of interaction.

Batch culture fermentation of endophytic fungi and extraction of their metabolites

Endophytic fungi showing antagonistic property against plant pathogenic fungi, in dual culture bioassay, were inoculated in 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] (300 ml in 1 litre conical flask) and incubated at 24°C for 24 days under static culture condition. One flask of medium without any inoculam served as a control.

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.

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Figure 4. Schematic diagram of extraction procedure for obtaining crude fungal extracts.

https://doi.org/10.1371/journal.pone.0056202.g004

Bioassay of extracts of the endophytic fungi

Different extracts were tested against S. sclerotiorum by first dissolving 30 mg of dried extract in 800 µl of methanol. From this solution, 200 and 400 µl were added to 30 ml of molten PDA media, mixed and then poured into three 10 cm Petri plates to obtain 250 and 500 µg/ml extract concentrations, respectively. To obtain 1000 µg/ml concentration, 30 mg of dried extracts were dissolved in 400 µl of methanol and 400 µl 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 µl 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 µl of oil and 200 µl of methylating reagent (Ethanol∶Benzene∶Acetyl chloride at 20∶4∶1) followed by heating at 72°C for 1 hr and then partitioning with 200 µl of hexane. 2 µl of the hexane soluble part was injected to GC and allowed to run for 20 minute with starting temperature of 180°C and final temp of 232°C with increase of 4°C/minute.

Data analysis

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

Acknowledgments

We are grateful to Late Dr KG Mukherji, for identification of the fungus. Susheel Kumar is grateful to University Grant Commission, New Delhi for the research fellowship.

Author Contributions

Conceived and designed the experiments: NK SK. Performed the experiments: SK. Analyzed the data: SK. Contributed reagents/materials/analysis tools: NK. Wrote the paper: SK NK.

References

  1. 1. Karmegam N, Sakthivadivel M, Anuradha V, Daniel T (1997) Indigenous-plant extracts as larvicidal agents against Culex quinquefasciatus Say. Bioresour Technol 59: 137–140.
  2. 2. Kumar A, Sharma S (2008) An evaluation of multipurpose oil seed crop for industrial uses (Jatropha curcas L.): A review. Ind Crops Prod 28: 1–10.
  3. 3. Terren M, Saverys S, De Haveskercke PJ, Toussaint A, Baudoin P, et al. (2012) Study of agronomic constraints to the dissemination of the cultivation of Jatropha curcas L. in Senegal. Commun Agric Appl Biol Sci 77: 245–249.
  4. 4. Rao CS, Kumari MP, Wani SP, Marimuthu S (2011) Occurrence of black rot in Jatropha curcas L. plantations in India caused by Botryosphaeria dothidea. Curr Sci 100: 1547–1549.
  5. 5. Kumar S, Sharma S, Pathak DV, Beniwal J (2011) Integrated management of jatropha root rot caused by Rhizoctonia bataticola. J Trop Forest Sci 23: 35–41.
  6. 6. Latha P, Prakasam V, Kamalakannan A, Gopalakrishnan C, Raguchander T, et al. (2009) First report of Lasiodiplodia theobromae (Pat.) Griffon Maubl causing root rot and collar rot disease of physic nut (Jatropha curcas L.) in India. Aus Plant Dis Notes 4: 19–20.
  7. 7. Openshaw K (2000) A review of Jatropha curcas: An oil plant of unfulfilled promise. Biomass Bioenergy 19: 1–15.
  8. 8. Rodriguez RJ, White JF Jr, Arnold AE, Redman RS (2009) Fungal endophytes: diversity and functional roles. New Phytol 182: 314–330.
  9. 9. Saikkonen K, Wali P, Helander M, Faeth SH (2004) Evolution of endophyte–plant symbioses. Trend Plant Sci 9: 275–280.
  10. 10. Clay K, Hardy TN, Hammond AM Jr (1985) Fungal endophytes of grasses and their effects on an insect herbivore. Oecologia 66: 1–5.
  11. 11. Clay K (1988) Fungal endophytes of grasses: Defensive mutualism between plants and fungi. Ecology 69: 10–16.
  12. 12. Breen JP (1994) Acremonium endophyte interactions with enhanced plant resistance to insects. Annu Rev Entomol 39: 401–423.
  13. 13. Narisawa K, Kawamata H, Currah RS, Hashiba T (2002) Suppression of Verticillium wilt in eggplant by some fungal root endophyte. Eur J Plant Pathol 108: 103–109.
  14. 14. Clarke BB, White JF Jr, Hurley RH, Torres MS, Sun S, et al. (2006) Endophyte-mediated suppression of dollar spot disease in fine fescues. Plant Dis 90: 994–998.
  15. 15. Vaz ABM, Mota RC, Bomfim MRQ, Vieira MLA, Zani CL, et al. (2009) Antimicrobial activity of endophytic fungi associated with Orchidaceae in Brazil. Can J Microbiol 55: 1381–1391.
  16. 16. Li HY, Zhao CA, Liu CJ, Xu XF (2010) Endophytic fungi diversity of aquatic/riparian plants and their antifungal activity in vitro. J Microbiol 48: 1–6.
  17. 17. Kumar S, Kaushik N, Edrada-Ebel RA, Ebel R, Proksch P (2011) Isolation, characterization, and bioactivity of endophytic fungi of Tylophora indica. World J Mircobiol Biotechnol 27: 571–577.
  18. 18. Nilsson RH, Kristiansson E, Ryberg M, Hallenberg N, Larsson K-H (2008) Intraspecific ITS variability in the kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identification. Evol Bioinform online 4: 193–201.
  19. 19. Li HY, Wang L, Zhao ZW (2006) Study on endophytic fungi of Jatropha curcas and their antifungal activity. Nat Prod Res Develop 18: 78–80.
  20. 20. Shankar Naik B, Shashikala J, Krishnamurthy YL (2008) Diversity of fungal endophytes in shrubby medicinal plants of Malnad region, Western Ghats, Southern India. Fungal Ecol 1: 89–93.
  21. 21. Begum MM, Sariah M, Puteh AB, Abidin MAZ (2008) Pathogenicity of Colletotrichum truncatum and its influence on soybean seed quality. Int J Agric Biol 10: 393–398.
  22. 22. Latunde-Dada AO, Lucas JA (2007) Localized hemibiotrophy in Colletotrichum: Cytological and molecular taxonomic similarities among C. destructivum, C. linicola and C. truncatum. Plant Pathol 56: 437–447.
  23. 23. Nan ZB, Hanson J (1998) Detection of seedborne fungi in Stylosanthes guianensis, S. hamata, and S. scabra. Seed Sci Technol 26: 333–345.
  24. 24. Bankole SA, Adebanjo A (1996) Biocontrol of brown blotch of cowpea caused by Colletotrichum truncatum with Trichoderma viride. Crop Prot 15: 633–636.
  25. 25. O'Connell RJ, Uronu AB, Waksman G, Nash C, Keon JPR, et al. (1993) Hemibiotrophic infection of Pisum sativum by Colletotrichum truncatum. Plant Pathol 42: 774–783.
  26. 26. Kaushal RP, Singh BM (1988) Genetics of disease resistance in urdbean (Vigna mungo (L.) Hepper) to the leaf spots caused by Colletotrichum truncatum (Schw.) Andrus & Moore. Euphytica 37: 279–281.
  27. 27. Sugawara K, Matsudate A, Ito Y, Namai T (2009) Anthracnose of christmas rose caused by Colletotrichum sp. J Gen Plant Pathol 75: 163–166.
  28. 28. Huang WY, Cai YZ, Survesvaran S, Hyde KD, Corke H, et al. (2009) Molecular phylogenitic identification of endophytic fungi isolated from three Artemisia spp. Fungal Divers 36: 69–88.
  29. 29. Boyette CD, Jackson MA, Bryson CT, Hoagland RE, Connick WJ Jr, et al. (2007) Sesbania exaltata biocontrol with Colletotrichum truncatum microsclerotia formulated in ‘Pesta’ granules. BioControl 52: 413–426.
  30. 30. Saunders M, Kohn LM (2008) Host-synthesized secondary compounds influence the in vitro interactions between fungal endophytes of maize. Appl Environ Microbiol 74: 136–142.
  31. 31. Salgado Salazar C, Cepero De García MC (2005) Endophytic fungi in rose (Rosa hybrida) in Bogota, Colombia. Rev Iberoam Micol 22: 99–101.
  32. 32. Brown KB, Hyde KD, Guest DI (1998) Preliminary studies on endophytic fungal communities of Musa acuminata species complex in Hong Kong and Australia. Fungal Divers 1: 27–51.
  33. 33. Romero A, Carrión G, Rico-Gray V (2001) Fungal latent pathogens and endophytes from leaves of Parthenium hysterophorus (Asteraceae). Fungal Divers 7: 81–87.
  34. 34. Kharwar RN, Gond SK, Kumar A, Mishra A (2010) A comparative study of endophytic and epiphytic fungal association with leaf of Eucalyptus citriodora Hook., and their antimicrobial activity. World J Microbiol Biotechnol 26: 1941–1948.
  35. 35. Gond SK, Mishra A, Sharma VK, Verma SK, Kumar J, et al. (2012) Diversity and antimicrobial activity of endophytic fungi isolated from Nyctanthes arbor-tristis, a well-known medicinal plant of India. Mycoscience 53: 113–121.
  36. 36. Nalini MS, Mahesh B, Tejesvi MV, Prakash HS, Subbaiah V, et al. (2005) Fungal endophytes from the three-leaved caper, Crataeva magna (Lour.) DC. (Capparidaceae). Mycopathologia 159: 245–249.
  37. 37. Ahmad I, Iram S (2008) Effect of zero and conventional tillage on the soil mycoflora of rice-wheat cropping system. Arch Phytopathol Plant Prot 41: 67–74.
  38. 38. Fakhrunnisa , Hashmi MH, Ghaffar A (2006) Seed-borne mycoflora of wheat, sorghum and barley. Pak J Bot 38: 185–192.
  39. 39. Cheng ZS, Tang WC, Su ZJ, Cai Y, Sun SF, et al. (2008) Identification of mangrove endophytic fungus 1403 (Fusarium proliferatum) based on morphological and molecular evidence. J Forest Res 19: 219–224.
  40. 40. De Almeida CV, Yara R, De Almeida M (2005) Endophytic fungi in shoot tip of the pejibaye cultivated in vivo and in vitro, Pesqui. Agrop Bras 40: 467–470.
  41. 41. Mohana Kumara P, Zuehlke S, Priti V, Ramesha BT, Shweta S, et al. (2012) Fusarium proliferatum, an endophytic fungus from Dysoxylum binectariferum Hook.f, produces rohitukine, a chromane alkaloid possessing anti-cancer activity. Antonie van Leeuwenhoek 101: 323–329.
  42. 42. Stenglein SA, Dinolfo MI, Moreno MV, Galizio R, Salerno G (2010) Fusarium proliferatum, a new pathogen causing head blight on oat in Argentina. Plant Dis 94: 783.
  43. 43. Pinaria AG, Liew ECY, Burgess LW (2010) Fusarium species associated with vanilla stem rot in Indonesia. Australasian Plant Pathol 39: 176–183.
  44. 44. Hajieghrari B (2009) Wheat crown and root rotting fungi in Moghan area, Northwest of Iran. African J Biotechnol 8: 6214–6219.
  45. 45. Meca G, Soriano JM, Gaspari A, Ritieni A, Moretti A, et al. (2010) Antifungal effects of the bioactive compounds enniatins A, A1, B, B1. Toxicon 56: 480–485.
  46. 46. Pang KL, Vrijmoed LLP, Goh TK, Plaingam N, Jones EBG (2008) Fungal endophytes associated with Kandelia candel (Rhizophoraceae) in Mai Po nature reserve, Hong Kong. Bot Mar 51: 171–178.
  47. 47. Rakotoniriana EF, Munaut F, Decock C, Randriamampionona D, Andriambololoniaina M, et al. (2008) Endophytic fungi from leaves of Centella asiatica: Occurrence and potential interactions within leaves. Antonie van Leeuwenhoek 93: 27–36.
  48. 48. Phongpaichit S, Nikom J, Rungjindamai N, Sakayaroj J, Hutadilok-Towatana N, et al. (2007) Biological activities of extracts from endophytic fungi isolated from Garcinia plants. FEMS Immunol Med Microbiol 51: 517–525.
  49. 49. Gao XX, Zhou H, Xu DY, Yu CH, Chen YQ, et al. (2005) High diversity of endophytic fungi from the pharmaceutical plant, Heterosmilax japonica Kunth revealed by cultivation-independent approach. FEMS Microbiol Lett 249: 255–266.
  50. 50. Stuart RM, Romão AS, Pizzirani-Kleiner AA, Azevedo JL, Araújo WL (2010) Culturable endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane and its non-transgenic isolines. Arch Microbiol 192: 307–313.
  51. 51. Santamaría J, Bayman P (2005) Fungal epiphytes and endophytes of coffee leaves (Coffea arabica). Microb Ecol 50: 1–8.
  52. 52. Kumar DSS, Hyde KD (2004) Biodiversity and tissue-recurrence of endophytic fungi in Tripterygium wilfordii. Fungal Divers 17: 69–90.
  53. 53. Rodrigues KF, Samuels GJ (1999) Fungal endophytes of Spondias mombin leaves in Brazil. J Basic Microbiol 39: 131–135.
  54. 54. Photita W, Lumyong S, Lumyong P, Hyde KD (2001) Endophytic fungi of wild banana (Musa acuminata) at Doi Suthep Pui National Park, Thailand. Mycol Res 105: 1508–1513.
  55. 55. Okane I, Nakagiri A, Ito T (1998) Endophytic fungi in leaves of ericaceous plants. Can J Bot 76: 657–663.
  56. 56. Wang FW (2012) Bioactive metabolites from Guignardia sp., an endophytic fungus residing in Undaria pinnatifida. Chin J Nat Med 10: 72–76.
  57. 57. Simmons EG (1998) Alternaria themes and variations (224–225). Mycotaxon 68: 417–427.
  58. 58. Rosa LH, Vaz ABM, Caligiorne RB, Campolina S, Rosa CA (2009) Endophytic fungi associated with the Antarctic grass Deschampsia antarctica Desv. (Poaceae). Polar Biol 32: 161–167.
  59. 59. Tao G, Liu ZY, Hyde KD, Liu XZ, Yu ZN (2008) Whole rDNA analysis reveals novel and endophytic fungi in Bletilla ochracea (Orchidaceae). Fungal Divers 33: 101–122.
  60. 60. Kaul S, Wani M, Dhar KL, Dhar MK (2008) Production and GC-MS trace analysis of methyl eugenol from endophytic isolate of Alternaria from Rose. Ann Microbiol 58: 443–445.
  61. 61. Verma VC, Gond SK, Mishra A, Kumar A, Kharwar RN (2008) Selection of natural strains of fungal endophytes from Azadirachta indica A. Juss, with anti-microbial activity against dermatophytes. Curr Bioact Comp 4: 36–40.
  62. 62. Kharwar RN, Verma VC, Strobel G, Ezra D (2008) The endophytic fungal complex of Catharanthus roseus (L.) G. Don. Curr Sci 95: 228–233.
  63. 63. Aly AH, Edrada-Ebel R, Indriani ID, Wray V, Müller WEG, et al. (2008) Cytotoxic metabolites from the fungal endophyte Alternaria sp. and their subsequent detection in its host plant Polygonum senegalense. J Nat Prod 71: 972–980.
  64. 64. Liu AR, Wu XP, Xu T (2007) Research advances in endophytic fungi of mangrove. Ying Yong Sheng Tai Xue Bao 18: 912–918.
  65. 65. Wang B, Priest MJ, Davidson A, Brubaker CL, Woods MJ, et al. (2007) Fungal endophytes of native Gossypium species in Australia. Mycol Res 111: 347–354.
  66. 66. Cook JC, Charudattan R, Zimmerman TW, Rosskopf EN, Stall WM, et al. (2009) Effects of Alternaria destruens, glyphosate, and ammonium sulfate individually and integrated for control of dodder (Cuscuta pentagona). Weed Technol 23: 550–555.
  67. 67. He X, Han G, Lin Y, Tian X, Xiang C, et al. (2012) Diversity and decomposition potential of endophytes in leaves of a Cinnamomum camphora plantation in China. Ecol Res 27: 273–284.
  68. 68. Yan XN, Sikora RA, Zheng JW (2011) Potential use of cucumber (Cucumis sativus L.) endophytic fungi as seed treatment agents against root-knot nematode Meloidogyne incognita. J Zhejiang Uni Sci B 12: 219–225.
  69. 69. Chen XY, Qi YD, Wei JH, Zhang Z, Wang DL, et al. (2011) Molecular identification of endophytic fungi from medicinal plant Huperzia serrata based on rDNA ITS analysis. World J Microbiol Biotechnol 27: 495–503.
  70. 70. Seena S, Sridhar KR (2004) Endophytic fungal diversity of 2 sand dune wild legumes from the southwest coast of India. Can J Microbiol 50: 1015–1021.
  71. 71. Beena KR, Ananda K, Sridhar KR (2000) Fungal endophytes of three sand dune plant species of west coast of India. Sydowia 52: 1–9.
  72. 72. Shankarnaik BS, Shashikala J, Krishnamurthy YL (2009) Study on the diversity of endophytic communities from rice (Oryza sativa L.) and their antagonistic activities in vitro. Microbiol Res 164: 290–296.
  73. 73. Qin JC, Zhang YM, Gao JM, Bai MS, Yang SX, et al. (2009) Bioactive metabolites produced by Chaetomium globosum, an endophytic fungus isolated from Ginkgo biloba. Bioorg Med Chem Lett 19: 1572–1574.
  74. 74. Tejesvi MV, Mahesh B, Nalini MS, Prakash HS, Kini KR, et al. (2006) Fungal endophyte assemblages from ethanopharmaceutically important medicinal trees. Can J Microbiol 52: 427–435.
  75. 75. Asran-Amal A, Moustafa-Mahmoud SM, Sabet KK, El Banna OH (2010) In vitro antagonism of cotton seedlings fungi and characterization of chitinase isozyme activities in Trichoderma harzianum. Saudi J Biological Sci 17: 153–157.
  76. 76. Stierle A, Strobel G, Stierle D (1993) Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew. Science 260: 214–216.
  77. 77. Kusari S, Zühlke S, Spiteller M (2009) An endophytic fungus from Camptotheca acuminata that produces camptothecin and analogues. J Nat Prod 72: 2–7.
  78. 78. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410.
  79. 79. Oldenburg KR, Vo KT, Ruhland B, Schatz PJ, Yuan Z (1996) A dual culture assay for detection of antimicrobial activity. J Biomol Screen 1: 123–130.
  80. 80. Wicklow DT, Joshi BK, Gamble WR, Gloer JB, Dowd PF (1998) Antifungal metabolites (monorden, monocillin IV, and cerebrosides) from Humicola fuscoatra Traaen NRRL 22980, a mycoparasite of Aspergillus flavus sclerotia. App Environ Microbiol 64: 4482–4484.