Establishment Of Molecular Diagnostic Method And Screening Of Biocontrol Agent For Major Fungal Diseases Of Medicinal Herbs In Zhejiang Province

Dendrobium officinale,Chrysanthemum morifolium Ramat,Ophiopogon japonicas and Fritillaria thunbergii are four traditional Chinese medicinal herbs in Zhejiang province and have huge commercial value.For tremendously strong market demand,nowadays,these herbs have been planted on a large scale.However,the non-standard planting technology and abuse of pesticides caused to the accumulation of pathogen,reduction of plant anti-disease ability,ultimately resulting at outbreak of plant fungal diseases.In this study,we isolated and identified the pathogenic fungi caused major fungal diseases of these four herbs,and established molecular detection method for each herb to dignose these diseases at the same time.In addition,the biological agents which can inhibit the growth of these pathogenic fungi were screened,and subsequently the field efficacy of selected biological agent was investigated.This study results provided theoretical basis and application foundation for dignose and biological control of major fungal diseases in four herbs.The results were as follows:1、 Field investigations were carried out on main fungal diseases of four herbs,and diseased tissues were collected.Pathogenic fungi were isolated,and identified by morphological characteristics and sequence analysis.The pathogenicity of pathogen was determined by Koch’s Rule.The results showed the pathogenic fungi that caused the five major fungal diseases of D.officinale(including sclerotium blight,anthracnose,dieback,root rot and leaf spot)were Sclerotium delphinii,Colletotrichum gloeosporioide,Fusarium chlamydosporum,Alternaria alternata and Cladosporium cladosporioides,respectively;Pathogenic fungi causing blight,leaf blight,black spot of C.morifolium Ramat were F.incarnatum,Chaetomium globosum and A.alternata,respectively;The pathogenic fungi causing anthrax and black spot of O.japonicas were C.liriopes and A.alternata respectively;The pathogeni c fungi causing the anthrax and dry rot of F.thunbergii were C.lineola and F.oxysporum,respectively.It is the first report that A.alternata can cause D.officinale root rot,F.incarnatum and A.alternata causes C.morifolium Ramat fusarium wilt and black spot disease,respectively,and C.lineola resuts in F.thunbergii anthracnose.2、 Specific primers were designed for the ITS sequences of above pathogenic fungi,and a multi-nested PCR system was constructed,which can be used to diagnose the field disease in a rapid stabilization system to each herbs,respectively.The multi-nested PCR system constructed for D.officinale could detected S.delphinii,,A.alternata and C.cladosporioides with a content of 10 fg/μL and detected C.gloeosporioide with a content of 100 pg/μL;The multi-nested PCR system constructed for C.morifolium Ramat could detected 10 pg/μL C.globosum,1 pg/μL F.incarnatum and 10 fg/μL Alternaria alternata;In multi-nested PCR system constructed for O.japonicas can detect at least 100 pg/μL C.liriopes and 10 fg/μL A.alternata DNA;For F.thunbergii the multi-nested PCR system constructed could detected C.lineola and F.oxysporum with a content of 100 pg/μL.Besides,the results of field sample testing also proved that these multi-nested PCR systems could diagnose corresponding disease rapidly and stably.3、 In order to screen biological agents that can effectively control these fungal diseases,several biological agents with different targets were studied by mycelia growth inhibition experiments.The results demonstrated that the metabolites of Streptomyces diastatochramogenes 1628 had a strong inhibitory effect on the growth of mycelia of these pathogenic fungi,and field control effects showed the control effect of S.diastatochramogenes 1628 metabolites significantly higher than carbendazim.