The Diversity And Biocontrol Effects On Bacterial Will Of Endophytic Fungi From Patchouli

Objective Patchouli is the "top ten traditional south Chinese medicinal",which has efficacy on removing dampness by means of aromatics,harmonizing the stomach,stopping vomiting and renoving summer-heat to relieve superficies.At present,there is a great demand for patchouli at home and abroad.The bacterial wilt caused by the infection of Ralstonia solanacearum is the most serious systemic disease in the growing of patchouli,which seriously threatens the survival and development of the patchouli industry.Therefore,it is of great significance to search for a new safe and effective method for the control of bacterial wilt in order to promote the cultivation of patchouli,solve the shortage of patchouli resources and develop the patchouli industry.There are rich active material resource in endophytic fungi from plants,which can provides important resources for screening new compounds and drugs.In addition,endophytic fungi have established close relationship with host plants in the long evolutionary process,and it can improve the resistance of plant disease through a variety of mechanisms,thus it shows a good application prospect in biological control of plant disease.At present,there is a lack of comprehensive understanding about the biodiversity of endophytic fungi in patchouli,and its potential role in the prevention of patchouli bacterial wilt has not yet covered.The aim of this paper is to systematically analyze the taxonomic structure and biodiversity of the endophytic fungi in patchouli and screen the endophytic fungi possessing the potential for antagonizing patchouli bacterial wilt,which will provide the basis for the comprehensive exploitation of the medicinal resources of endophytic fungi and the biocontrol system of endophytic “fungus-patchouli”.Method1.The diversity of cultivated endophytic fungi from patchouli was analysed by traditional culture method The endophytic fungi were isolated from the roots,stems,and leaves of the healthy patchouli using the tissue isolation method.The taxonomic structure and biodiversity of the endophytic fungi from patchouli were explored based on the morphological traits,and fungal ITS region sequence analysis.2.The diversity of endophytic fungi(including uncultured and cultured microbes)from patchouli was analysed by High-throughput sequencing The total DNA was extracted from roots,stems and leaves of patchouli,and ITS5-1737 F,ITS2-2043 R primers were used to amplify the ITS region sequence of fungi.Illumina Miseq high-throughput sequencing was used to analyze the distribution and diversity of endophytic fungi in different parts of patchouli.3.Screening of endophytic fungi possessing the potential for against patchouli bacterial wilt.The antagonistic activity of patchouli endophytic fungi to Pseudomonas solanacearum in vitro was analyzed by double plate antagonistic method and the biocontrol effect of active strains on patchouli wilt was verified by pot experiments.Results1.In the results,313 strains belonging to 30 genera of endophytic fungi were isolated from patchouli.The dominant groups of the endophytic fungi were Alternaria(28.75%),Phompsis(23.00%)and Colletorichum(11.82%),respectively.In addition,there were some rare fungal endophytes taxa such as Biscogniauxia,Eutypella,Gibellulopsis ?Neosartorya,Neofusicoccum,Talaromyces were also isolated.The colonization rates and the isolation rates of endophytic fungi in patchouli stems and leaves were higher than those in roots.However,the diversity index of fungal endophytes in roots(2.64)was higher than those in stems(2.00)and leaves(1.97).2.Through high-throughput sequencing,41188 valid sequences and 1305 OTUs were obtained and 2 kinds of phylums named Ascomycota and Basidiomycota were tested from3 parts of patchouli.The dominant groups of the endophytic fungi in roots were Athelia(11.13 %)and Myrothecium(10.19%),while the dominant groups of the endophytic fungi in stems and leaves were Aspergillus,accounted for 5.58% and 0.64% respectively.The species abundance and total diversity of endophytic fungi in different parts of patchouli showed the trend of stem> root> leaf.3.16 stains of endophytic fungi showed the antagonistic activities to Ralstonia solanacearum(Smith)Yabuuchi.Among the 16 strains,GHXR07,GHXR27 and GHXR29 showed the best antagonistic activities,and they were identified as Talaromyces sp.,Myrothecium roridum and Talaromyces wortmannii,respectively.Pot experiments showed that the relative control effects of the strains GHXR07 and GHXR29 on patchouli bacterial wilt were 57.47% and 69.36%,respectively.Conclussion The endophytic fungi in patchouli were very diverse and their distribution showed the tissue specificity,and the antagonistic strains against Ralstonia solanacearum(Smith)Yabuuchi were also demonstrated.The strains GHXR07 and GHXR29 showed better biocontrol for patchouli bacterial wilt.Therefore,the biocontrol system of “endophytic fungi-patchouli” is expected to provide a new method for the control of patchouli bacterial wilt.