Soil Microbial Bacteria Induced Pine Wood Nematode Disease Resistance In Pinus Massoniana

Pine wood nematode disease,also known as pine wilt disease(PWD),is a systematic invasive disease caused by pine wood nematode Bursaphelenchus xylophilus,which spreads quickly and can cause large-scale death and destruction of pine plants in a short period of time.In our field investigation,we found that there are untreated healthy Pinus massoniana around pine wood nematode disease,this phenomenon is not uncommon,each plant ecosystem microbial community is an important functional component,plays many roles conducive to plant growth or as a biocontrol agent for pathogenic organisms,stimulating the natural resistance of plants to pathogens is one of the main mechanisms of biological control.In this study,the differences between rhizosphere soil microorganisms of healthy resistant Pinus massoniana,healthy Pinus massoniana,diseased Pinus massoniana and Pinus massoniana were compared and analyzed without chemical treatment,and the differential microorganisms were isolated and purified and screened out,and the isolated differential microorganisms were screened for antagonist bacteria,and then the screened linecides were inoculated on 2 a raw Pinus massoniana seedlings to detect the changes in the defensive enzyme activity of Pinus massoniana seedlings.Finally,double antibacterial properties with both pine wood nematode effect and induction of Pinus massoniana resistance to wood nematode disease were screened.Finally,the media and culture conditions of the screened double antibacterial were optimized,and the best double antibacterial effect was finally obtained through pot experiments.The results of this trial are as follows:(1)Analysis of soil microbial differences in the rhizosphere of different diseased Pinus massoniana and preliminary screening of bactericidal bacteria:The results of highthroughput sequencing showed that the OTU richness of the four groups showed that the OTU of the diseased Pinus massoniana was the richest,and the OTU richness of Pinus massoniana was the lowest.The community structure of each group at different levels is different,and at the classification level of the phylum,the main ones are Proteobacteria,Actinobacteria,Acidobacteria,and Chloroflexi.Among them,the abundance of Acidobacterium phylum was significantly higher than that of the other three groups in healthy resistant Pinus massoniana soil,and the abundance of Acidobacillus phylum and Curvularia vulgaris was the highest in healthy Pinus massoniana soil,and the abundance of Curvularia aerugula was higher than that of the other three groups.In the rhizosphere soil of Pinus massoniana,the abundance of actinomycetes was higher than that of the other three groups of actinomycetes,and the abundance of microorganisms in the rhizosphere of Pinus massoniana was generally smaller than that of the other three groups of rhizosphere soil microorganisms,among which the abundance of proteobacteria,actinomycetes and acidobacteria was relatively large.In addition,856 cultureable microorganisms were isolated and purified from the collected samples,including 236 fungi and 620 bacteria.Through the nematicide activity assay,42 strains of bacteria with linear effect were preliminarily screened,7 strains were from the rhizosphere soil of resistant healthy wetland pine,20 strains were from healthy Pinus massoniana rhizosphere soil,10 strains were from dead Pinus massoniana rhizosphere soil,and 5 plants were from diseased Pinus massoniana rhizosphere soil.After identification,the results showed that the main strains were Bacillus,Burkholderia,Acidobacterium and Pseudomonas.(2)Screening and identification of linear bacteria and antibacterial induce:from the 42 strains of bacteria with linear killing effect in the initial screening,13 strains of bacteria with a linear killing effect of up to 80%at 24 h were rescreened,and then the four strains with the highest linear killing activity at 24 h were selected from the 4 strains 13-4,strain 45,strain 49 and strain 60,and the corrected mortality rates were 91%,90%,94%and 90%,respectively.Through morphological identification and molecular identification,it was finally determined that strain 13-4 was Lysinibacillus capsici,strain 45 was Bacillus paramycoides,strain 49 was Delftia tsuruhatensis,and strain 60 was Lelliottia jeotgali.Subsequently,the suspensions and fermentation filtrates of these four strains of the strains were verified,and the results showed that the fermentation filtrate and suspension of the four strains had strong linear killing effects.In order to screen out the antibacterial inducers with strong inducing Pinus massoniana resistance from the four strains of Pinus massonide,the bacterial suspension and fermentation filtrate of the four strains of Pinus massonide were respectively connected to the biennial Pinus massoniana seedlings,and the changes of the activities of three defense enzymes of Pinus massoniana POD,PAL and CAT within 96 h were observed,and it was found that the fermentation filtrate of strain 13-4,strain 45 and the fermentation filtrate and suspension of strain 60 effectively improved the activity of defense enzymes.The fermentation filtrate of strain 13-4 and the suspension of strain 60 were screened for the changes of the activities of the three defense enzymes of POD and CAT,which had good ability to induce pine wood nematode disease resistance of Pinus massoniana,and its ability to induce the resistance of Pinus massoniana could last until 28 days after infection with pine wood nematode disease.Finally,strains 13-4 and 60 were determined to be double antibacterial with both linear killing effect and inducing the ability of Pinus massoniana to resist pine wood nematode disease.(3)Optimization of double antibacterial culture and verification of pot experiments:Through single-factor experiments,the effects of carbon and nitrogen sources,inorganic salts,incubation time,inoculation amount,temperature and pH value on the fermentation culture of strains 13-4 and 60 were systematically studied,and finally the best medium for strain 13-4 was glucose,beef paste and sodium chloride,with an optimal pH of 6.5,an optimal inoculation amount of 2%,an optimal culture dimension of 35℃,and an optimal incubation time of 24 h.The optimal medium for strain 60 is glucose,beef paste and potassium chloride,the optimal pH is 7,the optimal inoculation amount is 4%,the optimal incubation temperature is 35℃,and the optimal incubation time is 24 h.The results showed that the linear killing effect of the optimized strain on pine wood nematodes and the resistance induction ability of Pinus massoniana were improved to a certain extent,and compared with the pre-optimization strain,the pathogenesis rate of pine wood nematode disease in Pinus massoniana seedlings was significantly inhibited.In summary,by analyzing the differences and antagonistic effects of rhizosphere soil microorganisms,this study screened out dual-resistant bacteria with nematicidal effect and induced resistance of Pinus massoniana to pine wilt disease,which laid a theoretical foundation for the screening of biocontrol strains against pine wilt disease.