| Chinese dwarf cherry is a small deciduous shrub that is native to China.With the increasing scale of cultivation,the crown gall disease has gradually spread in recent years.However,there has been a lack of systematic research on it,including the identification and characterization of the pathogen,as well as the prevention and control of the disease.In this study,the pathogenic bacteria causing crown gall disease of Chinese dwarf cherry were isolated and identified,and their characteristics were clarified.Then,under artificial inoculation and natural disease conditions,the resistance of Chinese dwarf cherry germplasm resources to crown gall disease was evaluated.Between severe and light disease fields,Chinese dwarf cherry’10-06’ showed significant difference in resistance.So,we examined the diversity of rhizosphere soil and root endophytic bacterial communities of ’10-06’ in these two fields,using high-throughput sequencing technology.The analysis results provided directions for screening biocontrol bacteria.Finally,biocontrol bacterium was screened and its mechanisms of biocontrol were explored,providing a theoretical basis and material foundation for the biological control of Chinese dwarf cherry crown gall disease.The main research results are as follows:(1)This study isolated and identified 45 crown gall disease pathogenic strains from the rhizosphere soil and gall tissues of diseased Chinese dwarf cherry from five regions in China.For the first time,the crown gall disease pathogens of Chinese dwarf cherry were identified as three species: Agrobacterium radiobacter,Agrobacterium fabacearum,and Rhizobium rhizogenes through physiological and biochemical characteristics,16 S r RNA phylogenetic analysis,and multilocus sequence analysis based on the rpo B,atp D,and rec A housekeeping genes.Based on PCR amplification of opine synthesis and metabolism genes,five strains contained typical nopaline-type plasmid,while the other 40 strains contained atypical nopaline-type plasmids.Resistance to the agrocins produced by R.rhizogenes K1026 was observed in all 45 strains.Tracing by red fluorescent protein markers revealed that the pathogenic bacterium could migrate through the vascular system within the stem of Chinese dwarf cherry.Using the q-PCR detection system established in this study,19.44% of samples from newly emerged shoots inoculated with the pathogenic bacteria were found to contain the pathogens,demonstrating that the pathogens can migrate from old branches to new shoots.(2)A strong oncogenic bacterium was screened from 45 strains of pathogenic bacteria causing Chinese dwarf cherry crown gall disease.It was artificially inoculated onto 77 Chinese dwarf cherry germplasm resources(including a few related species),and the resistance of different resources was evaluated using a systematic clustering method.Based on the investigation results of three indicators including disease incidence,maximum and average diameter of crown gall tumors,differences in resistance to crown gall disease were found among different germplasm resources.70% of the resources had a disease incidence rate of 100%,while only 3.9% had an incidence rate lower than 50%.None of the 77 materials had immunity or high resistance,but four materials had moderate resistance: ‘Y03-09’,‘Nongda 5’,‘Nongda 7’,and ‘S-D’.There were 53 germplasms had moderate susceptibility,and 20 germplasm had high susceptibility.In Chinese dwarf cherry orchards under natural field conditions,there were six resources with disease incidence rates below 5%,namely ‘10-06’,‘3-39-17-1’,‘Dong12-3-49’,‘Dong11-3-47’,‘Dong8-1-9’,and ‘DG-5’.Among them,the incidence rate of ‘10-06’ was 26.61% in the severe disease field,while it was 0 in the light disease field.This suggests that there are some other factors besides genetic resistance that can resist invasion by crown gall pathogenic bacteria.(3)High-throughput sequencing technology was used to study the diversity of bacterial communities in the rhizosphere and root endophytic of Chinese dwarf cherry ‘10-06’ under severe and light crown gall disease field.At the phylum level,Actinobacteria and Proteobacteria were significantly enriched in both the rhizosphere soil and root endophytic of ‘10-06’ in both the severe and light disease fields.At the genus level,Bacillus,Marinobacter,and Actinoplanes were enriched in the rhizosphere soil of the light disease field,while Bacillus,Ectothiorhodospira,Rhizobium,and Sorangium were enriched in the root endophytic habitat of the light disease field.The principal coordinate analysis results showed that there were significant differences in bacterial community β-diversity between samples from the severe and light disease fields,regardless of whether they were in the rhizosphere soil or root endophytic habitat.LEf Se analysis showed that Bacillus,nitrogen-fixing bacteria,and Myxobacteria were significantly enriched in the rhizosphere soil and root of the light disease field,which might be related to the resistance of Chinese dwarf cherry ‘10-06’in the light disease field.(4)Ten Bacillus spp.strains with antagonistic activity against crown gall pathogenic strains were screened using selective media and in vitro antagonism assays,and strain 7-35 exhibited the most prominent comprehensive antagonistic ability.Through morphological,physiological and biochemical characteristics,as well as 16 S r RNA and gyr A gene phylogenetic analysis,strain 7-35 was identified as B.velezensis.In greenhouse,when the biocontrol strain 7-35 was mixed with three Chinese dwarf cherry crown gall disease pathogens(X-A282,X-AB1,and X-A271)at the quantity ratio of 1:1 and inoculated on the stem of Chinese dwarf cherry cuttings,the incidence rates were 61.0%,22.0%,and 0,respectively.In comparison,the incidence rates for the K1026 control group were 100%,75%,and 0,respectively.This suggests that the biocontrol effect of 7-35 is better than that of K1026.Additionally,taking simultaneous inoculation as the control,when biocontrol strain 7-35 was inoculated four hours before pathogenic strain X-A282,the incidence rate decreased from 61.0% to 44.3%.However,when it was inoculated 30 minutes after the pathogen,the disease incidence rate increased from 61.0% to 100%.This indicates that the order of inoculation between the biocontrol strain and the pathogen has a significant impact on the biocontrol effect.(5)B.velezensis 7-35 was marked with green fluorescent protein,and it was observed that the labeled strain 7-35-gfp could stably survive in both soil and roots for at least 60 days after root irrigation.7-35-gfp colonized the epidermis,lateral root emergence sites,exodermis,and the intercellular spaces of cortex parenchyma cell of Chinese dwarf cherry roots.After artificial inoculation,both 7-35-gfp and pathogens were able to colonize the epidermis and vessels of Chinese dwarf cherry stems,indicating that the strain 7-35 can compete with the pathogens for colonization spaces in roots and wounds.When strain 7-35 and pathogens were co-inoculated in liquid medium,the pathogen’s population was 5.04%,2.20%,0.83%,0.01%,0.01%,and 0.03% of those inoculated only with itself at 8 h,12 h,16 h,20 h,24 h and 48 h after inoculation.When co-inoculated on Chinese dwarf cherry stems,the pathogen’s population reached a maximum 7 days after inoculation and was four times of the initial inoculation amount,whereas its population was 188 times of the initial inoculation amount when inoculated alone.This indicates that B.velezensis 7-35 can significantly inhibit the growth of the pathogen.When 7-35 and pathogens were co-inoculated on Chinese dwarf cherry stems,the expression of four plasmid virulence genes(Vir A,Vir B4,Vir C1,and Vir D2)and three chromosome virulence genes(Chv A,Chv B,and Chv E)in the pathogen was inhibited,with expression levels decreasing by 3% to 95%.This demonstrates that B.velezensis 7-35 directly affects the induction of a wide range of virulence genes in the Chinese dwarf cherry crown gall disease pathogen,elucidating its molecular biocontrol mechanism. |
