Screening Of Antifungal Strains And The Study On The Secondary Metabolites From Three Strains Endophytes

Fungal infection is one of the common clinical diseases,which has currently challenged to the therapy with the development of drug resistance fungi.Therefore,the therapy of fungal infection has become the hot point and it is exigent to find sensitive,broad-spectrum and safe novel antifungal drugs. Marine fungi and plant endophytes could product secondary metabolites with unique structures and novel skeletons,and become important resources for the development of novel antifungal drugs because of their specific habitats.In this study,we screened marine fungi and plant endophytes having antifungal activity and studied the secondary metabolites of strong antifungal strains for discovering potential antifungal strains to develop novel antifungal drugs.Using two strains of Candida albicans as indicator organisms,we detected 228 extract samples of marine fungi and plant endophytes by Oxford plate assay for primary antifungal active screening.The result showed 63 antifungal strains,28%of test strains.Microdilution bioassay and germ tube inhibition assay were adopted for secondary screening.The results displayed15 strains of strong antifungal activity(MIC₅₀≤25μg/mL) and 22 strains highly inhibiting germ tube(IC₅₀≤25μg/mL).The secondary metabolites of three antifungal plant endophytes including one endophytic fungi strain AF274,one Marine-derived fungi strain AF044 and one endophytic actinomycete strain AF349 were studied.Totally,9 compounds (DZ-3,DZ-6,DZ-7,DZ-9,DZ-17,DZ-20,DZ-22,DZ-23,DZ-24) were isolated and identified from the three strains,including 3 new compounds(DZ-6,DZ-7, DZ-23),2 compounds(DZ-17,DZ-24) as nature product for the first time.The bioactivities of partial compounds were studied.The results showed that compound DZ-3 exhibited significant antifungal activities.The MIC was 8μg/mL against both strains of Candida albicans.The IC₅₀ was 2.21μg/mL and MTC was 4μg/mL against Candida albicans ATCC 10231,and the IC₅₀ was 5.03μg/mL and MTC was 8μg/mL against Candida albicans AS 2.538. Further more,it displayed strong activities against plant pathogens (Trichoderma sp.,Rhizoctonia solani Kuhn,Verticillium dahliae Kleb, Colletotrichum lagenarium(Passerini) Ellis et Halsted,Phytophthora parasitica Destur,Pellicularia gramineum Ikata et Matsumura).The flow cytometry analysis showed that compound DZ-3 could affect membranes integrity and damage membranes,with DZ-3(8μg/mL) treated 48h,the ratio of membrane damage were 64%and 80%against Candida albicans ATCC 10231 and AS 2.538.Endophytic fungi strain AF274,marine-derived fungi strain AF044 and endophytic actinomycete strain AF349 were identified.The result indicated that the strain AF274 belonged to Fusarium sp.and strain AF044 belonged to Aspergillus sp.according to their morphological characteristics,strain AF349 belonged to Streptomyces parvus according to its morphological characteristic and 16SrDNA sequence analysis.In conclusion,our study indicated that marine fungi and plant endophytes contained abundant strains that could product antifungal active compounds, which as valuable resources for metabolites with antifungal activity.