Identification Of The Pathogen Of Bletilla Striata Anthracnose And Preliminary Study On Its Interaction Mechanism With Bletilla Striat

Bletilla striata is a traditional Chinese medicinal herb that is widely cultivated in China.In recent years,as the demand for B.striata has increased,the scale of artificial cultivation of B.striata has continued to expand,and the types,distribution,and degree of damage caused by B.striata diseases have been worsening year by year,causing significant economic losses to the B.striata industry.Among them,anthracnose is one of the main diseases,which occurs widely and severely in the main B.striata producing areas in Guizhou.However,there is a lack of systematic research on the anthracnose of B.striata in Guizhou Province,which has affected the effective control of the disease.This study systematically investigated the occurrence and harm of anthracnose in the main B.striata producing areas of Guizhou Province,conducted a systematic survey of the pathogenic fungi,collected diseased samples,isolated and identified the pathogenic fungi,and observed the infection process of the dominant pathogenic fungus C.spaethanium,as well as conducted transcriptome analysis on different infected B.striata leaves.The main research findings are as follows:1)The pathogenic fungi of B.striata anthracnose have been identified.From 2020 to2022,60 samples with anthracnose symptoms were collected from the main B.striata planting areas in Guizhou Province,and 51 Colletotrichum spp.fungi were obtained using tissue isolation and single-spore isolation methods.Based on the combination of phylogenetic analysis of multiple genes(ITS,ACT,GAPDH,TUB2,CHS-1,HIS3)and morphological observation,they were identified as belonging to C.spaethanium,C.liriopes,C.fructicola,C.plurivorum,and C.boninense,with isolation rates of 41.1%,21.5%,19.6%,11.7%,and 5.8%,respectively.Pathogenicity tests were conducted on representative strains of each group,and the results showed that all selected strains were pathogenic.According to the speed of leaf disease development and the size of the lesion,it was found that C.spaethanium,C.liriopes,and C.fructicola were more pathogenic,while C.plurivorum and C.boninense were less pathogenic and had slower disease development after inoculation.The results showed that there were differences in pathogenicity among different species of B.striata anthracnose pathogens.2)The infection process of the dominant pathogenic fungus SB7(C.spaethanium)on B.striata leaves was observed,and cell biology studies were conducted.B.striata leaves were inoculated using a spray method,and he infection process of C.spaethanium conidia on the leaves of B.striata was systematically described by using optical microscope,scanning electron microscope and paraffin section.C.spaethanium produces infection pegs through appressorium to penetrate the host cuticle and invade the host,and spore germination began 4~8 h later.After 12~24 h of inoculation,attachment cells and infection pegs were produced and attached to the surface of leaf cells,causing local shrinkage of the leaf epidermal cells.After 48 h of inoculation,the infected area appeared sunken.After 72 h of inoculation,the primary hyphae began to invade and expand into the adjacent intercellular space and cells.The epidermal cells of the inoculated leaves were damaged,and the internal tissue cells were partially digested.Plant-related defense enzyme activities(SOD,PAL,CAT,chitinase)were measured in B.striata leaves at 0 h,12 h,24 h,48 h,and 72 h after inoculation,The activities of SOD,PAL and chitinase reached the maximum at 72 h after inoculation,and the activity of CAT reached the maximum at 48 h after inoculation.,indicating that SOD,PAL,CAT,and chitinase activities were positively correlated with the resistance of B.striata to disease.3)Transcriptome analysis was conducted using high-throughput sequencing technology on infected B.striata and leaf samples of C.spaethanium at 12 h,24 h,48 h,and72 h as the experimental groups,and sterile water sprayed at 0 h as the control group.A total of 18,996 DEGs were found,and GO and KEGG analyses were performed.KEGG pathway enrichment analysis was performed on the differentially expressed genes at 12 h,24 h,48,and 72 h after C.spaethanium infection in B.striata samples,and peroxisomes were found to be enriched in CK-vs-T1,T3,and T4,with the expression levels of CAT and PAL defense enzyme genes reaching the highest after 12 h of pathogen infection.The expression levels of SOD and chitinase-related genes reached the highest after 72 h of pathogen infection.Phenylpropanoid biosynthesis was enriched in CK-vs-T2.Phenylalanine,tyrosine,and tryptophan biosynthesis were enriched in CK-vs-T1 and T4,flavonoid biosynthesis was enriched in CK-vs-T1,T2,and T4,and phenylalanine metabolism was enriched in CK-vs-T3.The expression levels of genes such as cinnamyl alcohol dehydrogenase(CAD),chalcone isomerase(CHI),dihydroflavanol 4-reductase(DFR),flavonoid 3’-monooxygenase(CYP75B1),and flavonol synthase(FLS)in the phenylpropanoid metabolic pathway increased with the time of C.spaethanium infection in B.striata samples.