Identification Of The Pathogen Of Tobacco Leaf Spot And Screening Of Control Agent

Tobacco belongs to dicotyledonous plants,Tuber,Solanaceae,Nicotiana,is one of the important economic crops in China.Leaf spot caused by Corynespora cassiicola is one of the factors causing the decline of tobacco quality and yield in recent years.In order to control tobacco Corynespora leaf spot more scientifically,in this study,pathogen isolation and identification,mitochondrial genome analysis,biological characteristics,fungicides and biocontrol bacteria screening of tobacco Corynespora leaf spot were studied.The main results were as follows:1.The pathogenic bacteria were isolated and purified from tobacco leaves collected in Guizhou Province by conventional tissue isolation method.According to Koch’s law,two representative strains YC1002 and YC1104 were obtained.They were identified as Corynespora cassiicola by morphological characteristics,ITS sequence analysis and phylogenetic analysis.Illumina and Nanopore sequencing platforms were used to sequence the mitochondrial genome of Corynespora cassiicola.The results showed that the mitochondrial genome of annular fungi was43963 bp,consisting of 15 protein-coding genes,27 t RNA genes and 2 r RNA genes.Phylogenetic analysis showed that YC1104 was closely related to Corynespora cassiicola.2.In the study of biological characteristics of tobacco Corynespora leaf spot pathogen in Guizhou,it was concluded that the pathogen grew fastest on carrot agar medium（CA）,The highest sporulation was observed on potato sucrose agar（PSA）on the 11th day.The optimum growth temperature was 30°C.Pathogens have a wide range of p H adaptation and can grow in the range of p H 4～11.,and the lethal temperature of mycelium was 52°C for 10 min.The light had little effect on the growth of mycelium.The optimal carbon sources were maltose and soluble starch,and the optimal nitrogen sources were glycine and threonine.3.Studies on the effects of different temperatures,p H,sporulation modes and carbon and nitrogen sources on the sporulation of pathogens showed that the carbon and nitrogen sources with the largest sporulation of pathogens were fructose and sodium nitrate,respectively.On potato sucrose agar（PSA）medium,the temperature with the highest spore yield of the pathogen was30°C,and the p H value was 7.The sporulation mode was as follows:healthy tobacco leaves were cut off,cleaned,and cut into squares of 2×2 cm in length.After high pressure sterilization at 121°C for 20 min,they were cooled,placed on the surface of inoculated cakes,and subjected to12 h near-UV irradiation/12 h dark alternating culture.On potato dextrose agar（PDA）medium,the temperature with the highest spore yield of the pathogen was 28°C,and the p H value was 6.The spore induction mode was as follows:healthy tobacco leaves were cut down,cleaned,and cut into squares of 2×2 cm length.After high-pressure sterilization at 121°C for 20 min,they were cooled and placed on the surface of inoculated bacterial cakes.After 12 h of near-UV irradiation/12 h of dark alternating culture,and 12 h of near-UV irradiation/12 h of dark alternating culture.4.The sensitivity of pathogenic bacteria to eight fungicides was determined by mycelium growth rate method,and the biocontrol strains were screened.The results showed that the EC₅₀values of prochloraz and fluazinam were the smallest in the indoor toxicity test,which were0.2092μg/m L and 0.2510μg/m L,respectively.Seven strains were screened out from 85biocontrol strains.Among them,two strains YC2140 and YC2148 had the best inhibitory effect on target pathogens,with inhibition rates of 67.3%and 63.52%.The two strains were identified as Pseudomonas fluorescens and Chryseobacterium indologenes,respectively.The pot and field control experiments showed that the control effects of fluazinam and prochloraz were both above70%and 40%,with good control effects.