The Diversity Of Endophyte Isolated From Ocimum Basilicum L. And Biological Function

There are rich diversity of microbial taxa in plants,which have an indispensable positive effect on the growth of plants.In order to investigate biodiversity and physiological function of endophytic bacteria,the plant Ocimum basilicum L.was chosen as experiment materials.The endophytic bacteria were isolated and identified,and the endophytes were tested for various functions for growth promoting and disease resistance.The pure culture of endophyte was obtained by spread plate and streak plate methods from surface disinfectant plants,utilizing different culture medium.Genetic diversity of isolates was studied by insertion sequence-based PCR(IS-PCR)finger-printing at molecular levels.The 16 S r RNA gene phylogenetic analysis and physiological and biochemical tests were used to identify the representative strains of all clusters.The species were identified and described by the methods of 16 S r RNA gene complete sequence similarity comparison,phylogenetic analysis,phenotypic and physiological characterization.In addition,functional diversity of identified endophytic bacteria was studied by nitrogenase activity test,secretion of IAA and siderophore production test,dissolving phosphorus and potassium test,and antagonistic plant pathogenic fungi test.The fermentation broth products of endophytic strains with strong antagonistic plant pathogens were analyzed.The target disease-resistant lipopeptides were detected by Agilent High Performance Liquid Chromatography Mass Spectrometer under positive and negative ion modes,and the strains with strong disease resistance were carried out for rice seed germination and potted growth test.The main results were as follows:(1)48 endophytic bacteria were isolated and purified from the host plant Ocimum basilicum L.They were clustered into 13 groups by IS-PCR insertion sequence fingerprint.According to the identification results based on molecular technology,representative strains of various groups belong to 13 species of 10 genera,showing abundant endophytic bacterial diversity in the plant Ocimum basilicum L.(2)The endophytic bacteria isolated and purified in this experiment have functional diversity.Among the 13 representative strains of various groups,9 strains have nitrogenase activity and Nif H gene was amplified by PCR,10 strains have potassium-dissolving ability,8 strains have the ability to dissolve phosphorus,12 strains could secrete auxin,5 strains have protease-producing activity,6 strains have the ability to secrete iron carriers.(3)Two biocontrol strains H1401 and H4309 were obtained,which had good antagonistic effects against pathogenic fungus Fusarium oxysporum and Rhizoctonia solani.At the same time,they had functions of potassium release,auxin secretion,protease production and iron carrier.Four disease-resistant genes Itu A,Itu B,Itu C and Itu D were obtained by PCR amplification.Further liquid chromatography-mass spectrometry analysis showed that their fermentation broth contained three main lipid peptide compounds,Surfactin,Iturin and Fengycin,which were related to disease resistance.(4)The results of rice seed germination and growth test showed that H1401 and H4309 could promote rice seed germination and growth.Therefore,H1401 and H4309 can be exploited as potential engineering strains.