Endophytes are the microorganisms that persist within or between tissues of plants without causing any negative effect on plant’s growth and development. Endophytes are mutually co-evolved with host plant species and have been reported to produce secondary metabolites similar to host plants. In this study, we developed unique neem-based media to isolate neem endophytic fungi and bacteria. A total of 361 fungi and 80 bacterial endophytes were isolated from various parts of neem plant including leaf, flower, seed, bark, cortex, and root using neem-based media. This is the first of its kind to demonstrate culturing of endophytic fungi and bacteria on a selective neem-based media without using any external nutrients. Out of total 376 fungal endophytes and 80 bacterial endophytes, only 10 fungi and 3 bacteria were inhibiting the growth of pathogenic rice blast fungi, Magnaporthe oryzae which were further identified using ITS and 16S rRNA sequencing. Finally, two fungal isolates identified as Fusarium sp.,st.Ai.67A and Neocosmospora ramose Ai.51D and two bacterial isolates identified as Pantoea sp. Ai.A2 and Bacillus sp. Ai.C5 were further selected for secondary metabolite production. These microbes were found to produce three neem metabolites such as Epoxy/hydroxyazadiradione, nimbin, and salanin. These endophytes will have great potential applications in agriculture, medicine, and bio-energy.
KeywordsEndophytes Neem media 16S rRNA ITS Magnaporthe Metabolites
We would like to thank Mahesh H B, Indira B.S, Shirke M, Yenagi V and Janani S for their help in endophytes isolation and DNA fingerprinting.
- Debbab A, Aly HA, Edrada-Ebel RA, Müller WE et al (2009) Bioactive secondary metabolites from the endophytic fungus Chaetomium sp. isolated from Salvia officinalis growing in Morocco. Biotechnol Agron Soc Environ 13(2):229–234Google Scholar
- Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic acid techniques in bacterial systematics, pp 15–175Google Scholar
- Mahesh B, Tejasvi MV, Nalini MS, Prakash HS, Kini KR, Subbiah V, Shetty HS (2005) Endophytic mycoflora of inner bark of Azadirachta indica A Juss. Curr Sci 88(2):218–219Google Scholar
- Rajagopal R, Suryanarayanan TS (2000) Isolation of endophytic fungi from leaves of neem (Azadirachtaindica). Curr Sci 78:1375–1378Google Scholar
- Verma VC, Kharwar RN (2006) Efficacy of neem leaf extract against its own fungal endophyte Curvularia lunata. J Agr Tech 2:329–335Google Scholar
- White TJ, Bruns T, Lee SJWT, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications, vol 18, pp 315–322Google Scholar