Study On Species Diversity Of Endophytic Fungi In Leaf Of Camellia Oleifera And Its Antifungal Activity Against Colletotrichum Fructicola

Camellia oleifera is an important forest tree with high ecological and economic value in China.However,anthracnose disease caused by Colletotrichum fructicola is one of the most serious diseases affecting oil tea.Severe cases of anthracnose lead to flower and fruit drop,resulting in significant yield losses.Currently,there is little research on the diversity of endophytic fungi in oil tea leaves and their inhibition of anthracnose.This study aimed to isolate and classify endophytic fungi from oil tea leaves using tissue isolation methods.Classical morphology and molecular biology techniques were employed to identify the endophytic fungi at the species level.The isolated endophytic fungi were then tested for their inhibitory activity against C.fructicola.A highly active strain was initially screened,and further screening led to the identification of the most potent strain,which was analyzed for its active substances.The main findings of this study are as follows:(1)180 strains of endophytic fungi were isolated from oil tea leaves using the tissue isolation method.Based on morphology and molecular biology,these fungi were classified into 7 families,7 genera,and 16 species.Among them,two new species,Diaporthe qimensis and Diaporthe xianyushanennsis,were discovered,along with known species such as Arthrinium arundinis,Colletotrichum camelliae,C.fructicola,C.karstii,C.siamense,Diaporthe eres,D.hongkongensis,D.biguttulata,D.kadsurae,D.ovoicicola,Periconia byssoides,Pestalotiopsis lushanensis,Phyllosticta capitalensis,and Xylaria arbuscula.(2)The pathogenic fungus causing anthracnose in oil tea,C.fructicola,was successfully isolated and its pathogenicity was confirmed.Strain TJ1-1 was isolated from diseased oil tea leaves and identified as C.fructicola using molecular biology techniques.Inoculating healthy oil tea leaves with strain TJ1-1 resulted in disease symptoms appearing after 5 days.Strain TJ1-2,isolated from the infected leaves,was also identified as C.fructicola.These findings establish C.fructicola as one of the pathogens causing anthracnose in C.oleifera,and strain TJ1-1’s pathogenicity was confirmed.(3)Through the plate confrontation method,endophytic fungi with high inhibitory activity against C.fructicola were initially screened.Five-point confrontation tests were conducted to determine the antibacterial activity of endophytic fungi in oil tea leaves against strain TJ1-1.The results revealed that D.ere(YC202106-13,YC202106-12 and YC202107-105),D.kadsurae(YC202107-100 and YC202107-115)and D.biguttulata(YC202110-170)exhibited anti-fungal rates above 60%.D.eres(YC202107-105),D.kadsurae(YC202107-100 and YC202107-115)and D.biguttulata(YC202110-170)were selected for further screening based on their biological characteristics.(4)A plate containing crude extract was prepared to evaluate the anti-fungal activity of highly active strains against the TJ1-1 strain.Batch fermentation of D.eres(YC202107-105),D.kadsurae(YC202107-100 and YC202107-115)and D.biguttulata(YC202110-170)was ferment,and the fermentation broth and mycelium were extracted using ethyl acetate to obtain a plate containing crude extract.The results revealed that the mycelium extract of the D.kadsurae(YC202107-100)strain exhibited the highest anti-fungal rate of78.27±2.49%.(5)HPLC analysis was adopted to investigate the metabolites of the mycelium from the D.kadsurae(YC202107-100)strain.The analysis identified the presence of 10 compounds,including 5,6-Dihydro-6-(hydroxymethyl)-4-methoxy-2H-pyran-2-one(R)-form,Montagnetol,Phomaspyrone B,Neuropyrone A,Dothiorelone M,Phomadecalin B,Paxisterol,Anhydrochlioquinone A,Phomopsidin,and Dehydroxyintegration B.This study systematically examined the endophytic fungal diversity in oil tea and preliminarily explored the pathogenic fungi causing oil tea anthracnose and the antibacterial activity of endophytic fungi.The findings provide crucial foundational data and potential resources for the prevention and control of oil tea anthracnose.Moreover,the identification of as the causal agent of oil tea anthracnose significantly supports the pathogenicity research of this disease.The isolated endophytic fungi with high inhibitory activity against C.fructicola,particularly the mycelium extract of the D.kadsurae(YC202107-100)strain,hold substantial potential for various applications and can be considered as candidate agents for the development of anthracnose control measures.Furthermore,the HPLC analysis revealed the presence of multiple compounds,some of which may possess anti-fungal activity,offering significant implications for exploring the mechanisms of anthracnose prevention and developing natural antibacterial agents in oil tea.