Isolation And Identification Of Pathogenic Bacteria In Batocera Lineolata Chevrolat And The Preliminary Research On Their Pathogenic Mechanism

Batocera lineolata Chevrolat is a major trunk borer with multiple hosts,and there are few reports on its pathogen mining.In order to screen out biological control microbial resources for the prevention and control of B.lineolata,this study collected natural diseased and dead B.lineolata larvae from the Walnut plantation in Hechi City,Guangxi,and used a method combining traditional microbial identification with molecular biology identification to isolate and identify the bacteria and fungi in vivo.Pathogenic bacteria of B.lineolata larvae was screened out by reinfection experiments.The most virulent strain was selected for indoor insecticidal virulence testing,and the fermentation culture conditions were optimized.The physiological mechanism of pathogenic bacteria pathogenicity was revealed by measuring the effects of pathogenic bacteria treatment on proteins and major enzymes in the midgut and hemolymph of B.lineolata larvae.The results were as follows:（1）Twenty-one bacterial strains were isolated from B.lineolata larvae that died of natural disease and belonged to 12 genera,including Serratia,Klebsiella and Alcaligenes;The obtained 8 fungi belonged to 6 genera,including Cordyceps,Clonostachys and Fusariums.（2）In the reinfection experiments,strains B5,F1,F2 and F3 were found to be the pathogenic bacteria of B.lineolata larvae,with the most significant insecticidal activity of strain B5,which has the species status of S.marcescens of the genus Serratia in the Enterobacteriaceae.Through the indoor insecticidal test,it was found that the virulence of the insecticide was related to the concentration of bacteria,the higher the concentration,the stronger the insecticidal activity,the greater the cumulative corrected mortality rate and the shorter the median lethal time.At a concentration of 4.8×10¹¹cfu/m L,the cumulative corrected mortality rate reached 100%at 15 d,and the LT₅₀was 4.77 d.（3）The optimal medium compositions and fermentation conditions of strain B5 were as follows:sucrose was the carbon source,and the addition amount was1%.Yeast powder was the nitrogen source,and the addition amount was 6%.The Na Cl addition amount was 1%.The temperature was 32°C,and the initial p H was 7,shaker speed was 240 rmp.（4）The protein content,protective enzymes and detoxification enzymes activity in the midgut and hemolymph were significantly changed after B.lineolata larvae were infected by strain B5.The protein content in both midgut and hemolymph showed a trend of increasing and then decreasing.In the protective enzymes of the midgut,POD and SOD tended to increase,then decrease,then increase,while the activity of CAT tended to increase,then decrease.In the protective enzymes of the hemolymph,the activity of POD and SOD tended to increase,then decrease,while the activity of CAT tended to change in"M"shape.In the detoxification enzyme system of midgut,the activity of Car E and GSTs tended to change in"M"shaped,while the activity of Ach E showed an increasing,then decreasing,then increasing trend.In the detoxification enzyme system of hemolymph,the activity of GSTs tended to change in"M"shaped trend,while the activity of Ach E showed an increasing,then decreasing trend,with the activity of Car E always lower than that of the control group,showing a decrease and then an increase.The activity of SOD,GSTs and Ach E in the hemolymph was much higher than that in the midgut,while the activity of Car E in the midgut was higher than that in the hemolymph.The above results show that strain B5 has strong insecticidal activity,which leads to metabolic disorders in B.lineolata larvae,and the protein content and the ability of detoxification resistance are significantly decreased.Therefore,strain B5 has the potential to be applied in biological control of B.lineolata.