Diversity And Applied Potential Of Endophytic Actinomycetes Isolated From Melia Toosendan Sieb.et Zucc.

In this study,culture-independent and culture-dependent approaches were employed to assessed the structure of endophytic actinobacteria associated to Melia toosendan Sieb.et Zucc,evaluating the bioactivities against pathogenetic microorganisms,and screening efficiently plant growth-promoting actinobacteria in vitro.2 isolates display hight plant growth promoting activity to tobacco.A total of 387 endophytic actinobacterial isolates were obtained from different tissues of Melia toosendan Sieb.et Zucc.,which collected from 7 districts of Sichuan province.Based on morphological characteristics,129 representative isolates were selected to analyze the diversity and evaluate antimicrobial and PGP activity of endophytic actinobacteria associated with Melia toosendan Sieb.et Zucc.The 16S rRNA sequence analysis showed that most of the isolates belong to the genera Streptomyces genus and other non-Streptomyces strains distributed into Micromonospora,Tsukamurella,Actinokineospora,Nocardia,Actinobispora,Microbispora,Pseudonocardia,Streptosporangium,Cellulosimicrobium,Actinomadura,Rhodococcus,Nocardiopsis,Nornomuraea,Actinoplanes,Verrucosispora.16S rRNA PCR-DGGE analysis revealed 17 genera of endophytic actinobacteria,which belong to Strentomyces,Nakamurella,Blaslococcus,Cryobacterium,Microbacterium,Leifsonia,Nocardioides,A eromicrobium,Rhodococcus,Corynebacterium,Mycobacterium,A rthrobacter,Nocardiopsis,Kulzneria,Gordonia,Kitasatospora,Planobispora,from Melia toosendan Sseb.et Zucc.Antimicrobial activity analysis combined with the results of amplifying genes coding for polyketide synthetase(PKSI,PKSII),nonribosomal peptide synthetase(NRPS)and Halo showed that the representative endophytic actinomycetes isolated from M Toosendan had broad-spectrum antimicrobial activity and potential natural product diversity,which further proved that endophytic actinomycetes are valuable reservoirsof novel bioactive compounds.Moreover,out of 77 isolates produce indole-3-acetic acid(IAA),Siderophore production was positive in 44 strains,77 strains produced cellulases.Strains P29 and P60 were tested in vivo and evaluated a range of growth parameters in tobacco under greenhouse conditions.The content of TN,TP and TK of tobacco and the content of TN,TP,TK and organic matter of rhizospheric and non-rhizospheric soils treated with strains P29＋60 were higher than the negative control.However,there were no significant differences on rhizospheric soil.Compared to control plants,the leaf length and width were increased by 19.35%,19.36%and 17.6%,11.7%,repectively,under individual treatment strain P29 and P60,whereas inoculation with strain P29+60 increased all biometric parameters of tobacco,including length,dry weight of shoots and roots and photosynthetic rate of tobacco,which exhibited significant promoting effects on plant growth.A 16S rRNA gene PCR-DGGE analysis was performed to explore the structure of the microbial communities associated to tobacco,which inoculate strains P29,P60,and P29+60,repectively.The result showed the diversity and abumdance of microbe in leaf,root,stem,rhizosphere and non rhizosphere of tobacco were changed,the bacterial diversity of leaf,root,rhizospheric and non-rhizospheric soil were increased except stem.The actinobacterial diversity of leaf and non-rhizospheric soil and the fungi diversity of leaf,stem and rhizospheric soil were increased.Whereas the abundance of actinobacteria in root,stem and rhizospheric and fungi in root and non-rhizospheric soil were decreased,repectively.In addition,the microbial counts in leaf,root,stem,rhizosphere and non rhizosphere of tobacco were significant different under treatment of P29,P60 and P29+P60,respectively.In a word,the endophytic actinobacterial isolates showed abundant diversity and antimicrobial and PGP activity.In addition,the genes encoding polyketide synthases,nonribosomal peptide synthetase and FADH2-dependent halogenase were amplified Based on the PGP screening results,isolates P29 and P60 were selected to evaluate their effects on growth of tobacco,which provide scientific basis for exploiting and utilization the endophytic actinobacteria of medicinal plants.