Abstract: Medicinal plants have been used by marginal communities to treat various ailments. However, the potential of endophytes within these bio-prospective medicinal plants remains unknown. The present study elucidates the endophytic diversity of medicinal plants (Caralluma acutangula, Rhazya stricta, and Moringa peregrina) and the endophyte role in seed growth and oxidative stress. Various organs of medicinal plants yielded ten endophytes, which were identified as Phoma sp. (6 isolates), Alternaria sp. (2), Bipolaris sp. (1), and Cladosporium sp. (1) based on 18S rDNA sequencing and phylogenetic analysis. The culture filtrates (CFs; 25%, 50%, and 100% concentrations) from these endophytes were tested against the growth of normal and dwarf mutant rice lines. Endophytic CF exhibited dose-dependent growth stimulation and suppression effects. CF (100%) of Phoma sp. significantly increased rice seed germination and growth compared to controls and other endophytes. This growth-promoting effect was due to the presence of indole acetic acid in endophytic CF. The gas chromatography/mass spectrometry (GC/MS) analysis showed the highest indole acetic acid content ((54.31±0.21) µmol/L) in Bipolaris sp. In addition, the isolate of Bipolaris sp. exhibited significantly higher radical scavenging and anti-lipid peroxidation activity than the other isolates. Bipolaris sp. and Phoma sp. also exhibited significantly higher flavonoid and phenolic contents. The medicinal plants exhibited the presence of bio-prospective endophytic strains, which could be used for the improvement of crop growth and the mitigation of oxidative stresses.

药用植物内生菌对作物生长及氧化应激的作用

[1]Ansari, M.W., Trivedi, D.K., Sahoo, R.K., et al., 2013. A critical review on fungi mediated plant responses with special emphasis to Piriformospora indica on improved production and protection of crops. Plant Physiol. Biochem., 70:403-410.

[2]Arnold, A.E., Lutzoni, F., 2007. Diversity and host range of foliar fungal endophytes: are tropical leaves biodiversity hotspots? Ecology, 88(3):541-549.

[3]Bajpai, V.K., Agrawal, P., Park, Y.H., 2014, Phytochemicals, antioxidant and anti-lipid peroxidation activities of ethanolic extract of a medicinal plant, Andrographis paniculata. J. Food Biochem., 38(6):584-591.

[4]Chen, J., Hu, K.X., Hou, X.Q., et al., 2011. Endophytic fungi assemblages from 10 Dendrobium medicinal plants (Orchidaceae). World J. Microbiol. Biotechnol., 27(5):1009-1016.

[5]Chutima, R., Lumyong, S., 2012. Production of indole-3-acetic acid by Thai native orchid-associated fungi. Symbiosis, 56(1):35-44.

[6]de Hoog, G.S., Horre, R., 2002. Molecular taxonomy of the Alternaria and Ulocladium species from humans and their identification in the routine laboratory. Mycoses, 45(7-8):259-276.

[7]Ding, X., Liu, K., Deng, B., et al., 2013. Isolation and characterization of endophytic fungi from Camptotheca acuminata. World J. Microbiol. Biotechnol., 29(10):1831-1838.

[8]Gao, J.M., Xiao, J., Zhang, Q., et al., 2014. Secondary metabolites from the endophytic Botryosphaeria dothidea of Melia azedarach and their antifungal, antibacterial, antioxidant, and cytotoxic activities. J. Agric. Food Chem., 62(16):3584-3590.

[9]Garcia, A., Rhoden, S.A., Rubin-Filho, C.J., et al., 2012. Diversity of foliar endophytic fungi from the medicinal plant Sapindus saponaria L. and their localization by scanning electron microscopy. Biol. Res., 45(2):139-148.

[10]Ghimire, S.R., Charlton, N.D., Bell, J.D., et al., 2011. Biodiversity of fungal endophyte communities inhabiting switchgrass (Panicum virgatum L.) growing in the native tall grass prairie of northern Oklahoma. Fungal Div., 47(1):19-27.

[11]Ghosh, S., Derle, A., Ahire, M., et al., 2013. Phytochemical analysis and free radical scavenging activity of medicinal plants Gnidia glauca and Dioscorea bulbifera. PLoS ONE, 8(12):e82529.

[12]Gubiani, J.R., Zeraik, M.L., Oliveira, C.M., et al., 2014. Biologically active eremophilane-type sesquiterpenes from Camarops sp., an endophytic fungus isolated from Alibertia macrophylla. J. Nat. Prod., 77(3):668-672.

[13]Gulati, V., Harding, I.H., Palombo, E.A., 2012. Enzyme inhibitory and antioxidant activities of traditional medicinal plants: potential application in the management of hyperglycemia. BMC Comp. Alter. Med., 12:77.

[14]Hilbert, M., Voll, L.M., Ding, Y., et al., 2012. Indole derivative production by the root endophyte Piriformospora indica is not required for growth promotion but for biotrophic colonization of barley roots. New Phytol., 196(2):520-534.

[15]Huang, W.Y., Cai, Y.Z., Xing, J., et al., 2007. A potential antioxidant resource: endophytic fungi from medicinal plants. Econ. Bot., 61(1):14-30.

[16]Keerthi, M., Jumpponen, A., 2014. Unraveling the Dark Septate Endophyte Functions: Insights from the Arabidopsis Model. Advances in Endophytic Research. Springer India, p.115-141.

[17]Kende, H., 2001. Hormone response mutants: a plethora of surprises. Plant Physiol., 125(1):81-84.

[18]Khan, A.L., Hamayun, M., Kim, Y.H., et al., 2011. Ameliorative symbiosis of endophyte (Penicillium funiculosum LHL06) under salt stress elevated plant growth of Glycine max L. Plant Physiol. Biochem., 49(8):852-862.

[19]Khan, A.L., Hussain, J., Al-Harrasi, A., et al., 2013. Endophytic fungi: a source of gibberellins and crop resistance to abiotic stress. Crit. Rev. Biotech., 35(1):1-13.

[20]Kipkore, W., Wanjohi, B., Rono, H., et al., 2014. A study of the medicinal plants used by the Marakwet Community in Kenya. J. Ethnobiol. Ethnomed., 10(1):24.

[21]Kumar, D.S.S., Hyde, K.D., 2004. Biodiversity and tissue-recurrence of endophytic fungi in Tripterygium wilfordii. Fungal Divers., 17:69-90.

[22]Kusari, S., Singh, S., Jayabaskaran, C., 2014. Biotechnological potential of plant-associated endophytic fungi: hope versus hype. Trends Biotechnol., 32(6):297-303.

[23]Liu, X., Dong, M., Chen, X., et al., 2007. Antioxidant activity and phenolics of an endophytic Xylaria sp. from Ginkgo biloba. Food Chem., 105(2):548-554.

[24]Mandyam, K.G., Roe, J., Jumpponen, A., 2013. Arabidopsis thaliana model system reveals a continuum of responses to root endophyte colonization. Fungal Biol., 117(4):250-260.

[25]Murphy, B.R., Doohan, F.M., Hodkinson, T.R., 2014. Yield increase induced by the fungal root endophyte Piriformospora indica in barley grown at low temperature is nutrient limited. Symbiosis, 62(1):29-39.

[26]Nalini, M.S., Sunayana, N., Prakash, H.S., 2014. Endophytic fungal diversity in medicinal plants of Western Ghats, India. Int. J. Biodiv., 2014:1-9.

[27]Nath, A., Chattopadhyay, A., Joshi, S.R., 2015. Biological activity of endophytic fungi of Rauwolfia serpentine Benth: an ethnomedicinal plant used in folk medicines in northeast India. PNAS, 85(1):233-240.

[28]Nishijima, T., Koshioka, M., Yamazaki, H., 1994. Use of several gibberellin biosynthesis inhibitors in sensitized rice seedling bioassays. Biosci. Biotechnol. Biochem., 58(3):572-573.

[29]Orlandelli, R.C., Alberto, R.N., Rubin Filho, C.J., et al., 2012. Diversity of endophytic fungal community associated with Piper hispidum (Piperaceae) leaves. Genet. Mol. Res., 11(2):1575-1585.

[30]Rai, M.R., Rathod, D., Agarkar, G., et al., 2014. Fungal growth promotor endophytes: a pragmatic approach towards sustainable food and agriculture. Symbiosis, 62(2):63-79.

[31]Redman, R.S., Kim, Y.O., Woodward, C.J.D.A., et al., 2011. Increased fitness of rice plants to abiotic stress via habitat adapted symbiosis: a strategy for mitigating impacts of climate change. PLoS ONE, 6(7):e14823.

[32]Reinhardt, D., Mandel, T., Kuhlemeier, C., 2000. Auxin regulates the initiation and radial position of plant lateral organs. Plant Cell, 12(4):507-518.

[33]Saeed, N., Khan, M.R., Shabbir, M., 2012. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Comp. Alter. Med., 12:221.

[34]Sakayaroj, Y., Preedanon, S., Supaphon, O., et al., 2010. Phylogenetic diversity of endophyte assemblages associated with the tropical seagrass Enhalus acoroides in Thailand. Fungal Div., 42(1):27-45.

[35]Schulz, B., Boyle, C., 2005. The endophytic continuum. Mycol. Res., 109(6):661-686.

[36]Slinkard, K., Singleton, L., 1977. Total phenol analyses: automation and comparison with manual methods. Am. J. Enol. Vitic., 28:49-55.

[37]Strobel, G., Daisy, B., Castillo, U., et al., 2004. Natural products from endophytic microorganisms. J. Nat. Prod., 67(2):257-268.

[38]Tamura, K., Peterson, D., Peterson, N., et al., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol., 28(10):2731-2739.

[39]Tang, A.M.C., Jeewon, R., Hyde, K.D., 2009. A re-evaluation of the evolutionary relationships within the Xylariaceae based on ribosomal and protein-coding gene sequences. Fungal Div., 34:127-155.

[40]Thakur, A., Kaur, S., Kaur, A., et al., 2013. Enhanced resistance to Spodoptera litura in endophyte infected cauliflower plants. Environ. Entomol., 42(2):240-246.

[41]Torres, M.S., White, J.F.Jr., Zhang, X., et al., 2012. Endophyte-mediated adjustments in host morphology and physiology and effects on host fitness traits in grasses. Fungal Ecol., 5(3):322-330.

[42]Ullah, I., Khan, A.R., Park, G.S., et al., 2013. Analysis of phytohormones and phosphate solubilization in Photorhabdus spp. Food Sci. Biotechnol., 22(S1):25-31.

[43]Vadassery, V., Ritter, C., Venus, Y., et al., 2008. The role of auxins and cytokinins in the mutualistic interaction between Arabidopsis and Piriformospora indica. MPMI, 21(10):1371-1383.

[44]Waller, F., Achatz, B., Baltruschat, H., et al., 2005. The endophytic fungus Piriformis indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. PNAS, 102(38):13386-13391.

[45]Wang, L.W., Xu, B.G., Wang, J.Y., et al., 2012. Bioactive metabolites from Phoma species, an endophytic fungus from the Chinese medicinal plant Arisaema erubescens. Appl. Microbiol. Biotechnol., 93(3):1231-1239.

[46]Waqas, M., Khan, A.L., Lee, I.J., 2014. Bioactive chemical constituents produced by endophytes and effects on rice plant growth. J. Plant Interact., 9(1):478-487.

[47]White, J.F.Jr., Torres, M.S., 2010. Is plant endophyte mediated defensive mutualism the result of oxidative stress protection? Physiol. Plant., 138(4):440-446.

[48]Xiao, J., Zhang, Q., Gao, Y.Q., et al., 2014. Secondary metabolites from the endophytic Botryosphaeria dothidea of Melia azedarach and their antifungal, antibacterial, antioxidant, and cytotoxic activities. J. Agric. Food Chem., 62(16):3584-3590.

[49]Zhang, H., Xiong, H., Zhao, H., et al., 2013. An antimicrobial compound from the endophytic fungus Phoma sp. isolated from the medicinal plant Taraxacum mongolicum. J. Taiwan Inst. Chem. Eng., 44(2):177-181.

[50]Zhang, Y., Crous, P.W., Schoch, C.L., et al., 2011. Pleosporales. Fungal Div., 53(1):1-221.

[51]Zhao, J.T., Ma, D.H., Luo, M., et al., 2014. In vitro antioxidant activities and antioxidant enzyme activities in HepG2 cells and main active compounds of endophytic fungus from pigeon pea [Cajanus cajan (L.) Millsp.]. Food Res. Int., 56:243-251.

[52]List of electronic supplementary materials

[53]Fig. S1 Individual phylogenetic analysis of the 10 fungal endophytes isolated from medicinal plants

[54]Table S1 Location and length of 18S, 28S, 5.8S and ITS1, ITS2 sequences of various fungal strains

[55]Table S2 Estimates of evolutionary divergence between sequences

[56]Table S3 Sequence homology matrix of fungal strains