| In recent years,tobacco leaf spot disease has been on the rise in the northeast tobacco area.It is an important disease in the late stage of tobacco growth,and has a serious impact on the yield and quality of tobacco leaves.In this paper,pathogenic bacteria were isolated and purified from tobacco leaves with symptoms of leaf spot disease in Fuxin district.Pathogens were identified by culture traits,morphological observation,pathogenicity determination,physiological and biochemical properties,and were identified as Pseudomonas syringae pv.tabaci FX.The genomic sequence analysis of the FX strain was performed.In addition,biological fungicide screening and control application in field were conducted.The main results obtained in this study are as follows:1.In the Fuxin district of Liaoning Province,the susceptible leaves with obvious symptoms of tobacco leaf spot disease were collected,and the strain FX was isolated and purified by tissue separation and compared with the standard strains of Pseudomonas syringae pv.tabaci 216 and Pseudomonas syringae pv.tabaci DN.The isolated strain FX was back-to-back by Koch’s law.The lesions were irregular polygons,and the edges were limited by the veins.The symptoms were the same as those produced by the control strain 216 after inoculation.This was a typical tobacco leaf spot disease.Pathogens were observed by electron microscopy.The size of the bacteria was about 0.5~0.7×1.5~2.0 μm,and 1 flagellum was produced.The result of physiological and biochemical tests showed that glucose,lactose,sucrose,nitrate reduction,arginine decarboxylase,starch,and gelatin were shown.The results of the reaction between the tested strain and the control strain 216 were the same.DNA extraction and concentration determination of strain FX was performed.A pair of universal primers 1612JF/2591 JR and specific primers 2F2/2R2 were used to perform PCR amplification of the obtained DNA according to the relevant literature.The results showed that the strain FX was Pseudomonas syringae pv.tabaci2.Whole genome sequencing of Pseudomonas syringae pv.tabaci FX was performed and analysis revealed that: The total number of high-quality reads was 152,166 for the second-generation sequencing,and the total number of high-quality reads was 1,892,123,741 bp.After assembled through three generations of sequencing,the length of the assembled genomic sequence is 4,756,717 bp,and the percentage of GC content is 55.14%;the predicted coding gene is 4328,the length of the sequence is 4,106,799 bp;the length of the repeat sequence is 0.26% of the total gene sequence length;non-coding RNA TRNA and r RNA are divided into two major categories;CRISPR 12 genes were found in the nuclear genome of this strain;a total of 444 predicted protein sequences and transporters;and 912 pathogen-host interaction factors in this prediction;4 antibiotic resistance genes;proteins containing signal peptides removed proteins containing transmembrane helices,and the remaining secreted proteins were shared 331.The predicted gene sequence was used to annotate 3475 COG functional genes,3200 GO genes,2738 KEGG pathways,and 3619 Swiss-Pro gene information.In this study,the bacterial complete map of the strain Pseudomonas syringae pv.tabaci FX was constructed and the genome size was 4,106,799 bp.In this study,the genome-specific virulence factors of the Pseudomonas syringae pv.tabaci FX strain were compared with that of Pseudomonas syringae pv.tabaci yuexi-1,which has landed on the NCBI in Sichuan’s Yuexi area.Pseudomonas syringae pv.tabaci yuexi-1 strain has the sequence number JWJF00000000.A total of 423 different factors,such as hptA,ptxR,rtx D,rfa E,htp B,Icm F1,clp V,farB,mtr D and so on.3.Inhibitory rate of 7 kinds of biological fungicides such as Bacillus subtilis and Trichoderma biocontrol agent,6 % kasugamycin,3 % Zhongshengmycin,and 1.8 % Cytosinpeptidemycin polyoxin through indoor plate diffusion method.The assay was performed: 1.8 % Cytosinpeptidemycin polyoxin > 3 % Zhongshengmycin > 6 % kasugamycin> HBL> Bacillus subtilis > 10 % jinggangmycin> Trichoderma biocontrol agent.Through tobacco field trials,the results showed that the best control effect was 6 % of kasugamycin and 1.8 % of pyridine-pigmented polyclonal antibody,and the prevention and control rate was 61.47 % and 59.10 %,and the effect was good. |
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