Endophytic Fungi Isolated from Oil- Seed Crop Jatropha curcas Produces Oil and Exhibit Antifungal Activity Susheel Kumar,Nutan Kaushik February 2013,Volume 8,Issue 2,e56202

Intruduction

Jatropha curcas

Energy crop – developing biofuel technology Antimicrobial pesticidal activity – Leaf extract against mosquito larvae – seed oil and plant extract against cotton bollworm, pests of pulses, potato and corn

Adaptation to diverse agro-climatic condition widely distributed in tropics and subtropics Easily cultivated in – low to high rainfall areas of saline – marshy lands

Ecological fitness because… the presence of endophytic fungi

Endophytic fungi live inside the plant without causing any overt negative effect on the host protect the host plant from pests and diseases endophytic fungi of grasses to provide protection from insect herbivore

Colletotrichum truncatum Disease can occur widely, especially during warm, rainy conditions It can reduce yields, decrease stands, and reduce seed quality

Other endophytic Fungi Nigrospora oryzae Colletotrichum truncatum Fusarium proliferatum Chaetomium sp. Guignardia camelliae Alternaria destruens

hypothesis due to the presence of endophytic fungi – pesticidal property in Jatropha extracts – seed oil is in part

Diseases of J. curcas Anthracnose caused by Colletotrichum gloeosporioides Black rot caused by Botryosphaeria diplodea Root rot caused by Rhizoctonia bataticola Root rot and collar rot caused by Lasiodiplodia theobromae

Aim

Undertaken to assess antifungal activity of endophytic fungi present in J. curcas against the phytopathogens – Rhizoctonia solani – Sclerotinia sclerotiorum – Fusarium oxysporum have a wide host range and cause major losses in important food crops like rice, maize, wheat, and chickpea

Materials, Methods and Result

Sample collection Leaf and petiole samples of Jatropha curcas were collected from plants washed with tap water

Isolation of endophytic fungi leaves and petioles surface sterilized with 70% ethanol 2min 1% sodium hypochlorite 3min dried on sterile blotting sheet

chopped Transferred leafe to malt agar plates, petioles in petri-plate containing media incubated at 24 ℃ for seven days Hyphal tips of the developing fungal colonies transferr to fresh malt agar plates Nine endophytic fungi were isolated from leaves(EF8-EF16), no one from petioles

Slide preparation Fungal mycelium stain with cotton blue and mounted in polyvinyl lactic acid glycerol (PVLG) heating at 65 ℃ for 2–3 days observed under light microscope

DNA Isolation and amplification scrap fresh mycelia from potato dextrose agar (PDA) plates using the DNeasy plant minikit (Qiagen) according to manufacturers’ protocol PCR identified by rDNA sequencing

DNeasy plant minikit (Qiagen)

Sequencing

Nine endophytic fungi were isolated － (EF8-EF16)

Bioassay of endophytic fungi against plant pathogenic fungi dual culture technique

Endophytic fungi showing antagonistic property against plant pathogenic fungi inhibition zone

Bioassay of endophytic fungi against plant pathogenic fungi Plant pathogenic fungi and endophytic fungi inoculate on PDA plate opposite to each other After seven days at 24 ℃ presence of inhibition zone at the point of interaction → antagonism dual culture technique

antagonistic property inhibition zone

Nine endophytic fungi were isolated － (EF8-EF16) C. Truncatum 、 Chaetomium and G. cammillae against S. sclerotiorum N. Oryzae 、 C. Truncatum and Chaetomium against F. oxysporum none of the endophytic fungi against R. solani

Batch culture fermentation of endophytic fungi and extraction of their metabolites incubated at 24 ℃ for 24 days under static culture condition Endophytic fungi inoculated in Wickerham medium

Extraction of metabolites of endophytic fungi

Bioassay of extracts of the endophytic fungi Different extracts test against S. sclerotiorum dissolving 30 mg of dried extract in methanol Add different concentration extract to molten PDA media, mixed poured into three Petri plates

S. sclerotiorum Different extracts with the concentration of 250, 500 and 1000ug/ml PDA Control growth plates contained 400 ml of methanol V.S.

effect of extracts of endophytic fungi on S. sclerotiorum control

Data analysis Growth inhibitions(GI) of the extracts calculate relative to the growth on the control plate GI=[(A-B)/A]*100% Radial diameter of fungus: A → control plate B → experimental plate The better GI value, the better antagonistic property against plant pathogenic fungi

Ethyl acetate extracts against S. sclerotiorum C. truncatum EF10 show a better GI value compared to GI with EF9

Butanol extracts against S. sclerotiorum Chaetomium sp No effect highest activity fungal growth inhibition effect

Methanol extracts against S. sclerotiorum EF13 is the highest activity fungal growth inhibition No effect E13 show the higher growth inhibition than E10 effect E13 is the highest of all

Hexane extracts against S. sclerotiorum No growth inhibition was observed in hexane extracts

Gas chromatography of hexane extract of C. truncatum isolates examine the fatty acid composition of the oil extracted from C. truncatum and J. curcas

Gas chromatography He sample Heat detect

Sample preparation was done by mixing 200 ml of oil and 200 ml of methylating reagent heating at 72 ℃ for 1 hr partitioning with 200 ml of hexane 2 ml of the hexane soluble part inject to GC

run for 20 minute with starting temperature of 180 ℃ increase of 4 ℃ /minute final temp of 232 ℃

EF10 was similar to fatty acid profile of Jatropha seed oil EF12 has more peaks, addition to the peaks of these four fatty acids EF9 and EF13 are having only one major peak with some smaller peaks

four major fatty acids of Jatropha seed oil EF9EF10EF13 oil yield 94.6 mg/l of media 99.3 mg/l of media 42 mg/l of media EF10 produced the highest amount of oil similiar EF10 has oil production capacity

Conclusion

Endophytic fungi of beneficial activity : Protecting from pathogenic fungi C. truncatum of EF13 agent against S. sclerotiorum and F. oxysporum oil production capacity C. truncatum of EF10 was similar to fatty acid profile of Jatropha seed oil

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