| Kiwifruit industry is one of the pillar industries that promote farmers in Western Hunan to get rid of poverty,but in recent years the area kiwifruit canker disease frequent occurrence,resulting in many kiwifruit orchards destroyed,huge economic losses,seriously restrict the healthy development of kiwifruit industry,however,healthy plants were still present in diseased orchards,and we hypothesized that the rhizosphere soil of healthy kiwifruit may contain strains of Kiwifruit canker disease preventing bacteria.In order to solve this problem,we collected typical symptom branches,leaves and rhizosphere soil samples from three growing areas with frequent occurrence of Kiwifruit canker disease in western Hunan(Qianling Town Baojing County,Songbai Town Yongshun County,and Alaying Town Fenghuang County)points three times(symptom appearance,fastigium,lag phase).The main obtained results were as follows:(1)Five candidate strains were isolated from infected kiwifruit branches of "Miliang No.1" and "Hongyang" from three sampling sites by traditional culture method,and were numbered L211,L212,L321,L322 and L323,respectively.Their 16 S r DNA sequence and 16S-23 S r DNA inter transcribed spacer region were amplified by PCR,and five strains were identified as Pseudomonas syringae pv.actinidae(Psa)by sequencing and phylogenetic analysis.Five bacterial strains were found to cause typical kiwifruit canker symptoms after 30 d of incubation using isolated branch inoculation experiments,confirming the pathogenicity of these isolates for kiwifruit canker disease.Single-factor experimental analysis revealed that the optimum growth temperature was20°C,the optimum p H was 7.0,and the optimum medium was SPA and LB medium.(2)Whole genome sequencing of strain L211 was performed using the Illumina platform.The splicing assembly yielded 284 Contigs with a total length of 6,263,093 bp;gene annotation yielded 5741 genes with 58.53% GC content.Through VFDB database comparison,121 virulence factors were found,including secretion system,adhesins,toxins,biofilm formation,Iron transport absorption,and immune regulation.71 genes of pathogenic factors interacting with host plants were identified by PHI-base database comparison,including genes encoding enzymes for plant cell wall degradation,biofilm formation,and motility-related genes.Through CARD database comparison,482 drug resistance genes were found,involving 29 kinds of antibiotic resistance functions,and antibiotic effusion system was the main drug resistance mechanism of pathogens.The phylogenetic tree constructed by Single Nucleotide Polymorphism(SNP)further identified the genotype of the pathogen as biovar 3.(3)The bacterial community structure and abundance in rhizosphere soil of healthy and diseased kiwifruit plants were analyzed by metagenomic sequencing.It was found that although there were some differences between diseased and healthy kiwifruit rhizosphere soil bacterial community,the dominant groups were all Acidobacteria group,Gemmatimonas,Gaiella,etc.The species abundance of Acidobacteria Gp2 in rhizosphere soil showed an obvious decreasing trend over time,while that of Sphingomonas showed an obvious increasing trend over time.The species abundance of Pseudomonas and Arthrobacter increased first and then decreased,and the abundance was the highest in June.The species abundance of sphinomonas and flavobacterium in diseased rhizosphere soil was higher than that in healthy samples,suggesting that the two bacteria had auxiliary functions in the occurrence of kiwifruit canker disease.The proportion of Bacillus and Streptomyces in healthy rhizosphere soil was higher than that in diseased samples,suggesting that these bacteria played an antagonistic role in the occurrence of kiwifruit canker disease.Pseudomonas was significantly higher in the diseased samples than in the healthy samples in the first two periods,and then recovered to be consistent with the healthy samples in September.The abundance of pseudomonas was consistent with the pathogenesis of Kiwifruit canker disease,suggesting that soil bacteria were one of the sources of infection.(4)142 bacterial strains were isolated from healthy kiwifruit rhizosphere soil by pure culture method,and were initially identified as belonging to 4 phyla,6 orders,11 families and 28 genera based on 16 S r RNA gene sequence alignment analysis;the16S r RNA gene homology with typical strains ranged from 92.03% to 100%,suggesting the existence of potential new species or genera.4 bacterial strains(A11,D1 h,A4 and B15)with antagonistic effect against Psa were screened from the isolated strains using Oxford cup method,and it was tentatively determined that strains A11 and D1 h belonged to Rhodococcus,strain A4 belonged to Bacillus and B15 belonged to Streptomyces.This work elucidates the causes of kiwifruit bacterial canker disease in western Hunan,broadened the understanding of the mechanism of kiwifruit bacterial canker disease,and revealed the difference of bacterial community in rhizosphere soil of healthy kiwifruit and diseased kiwifruit and the correlation with the occurrence of kiwifruit bacterial canker disease.This work not only accumulated germplasm resources for the research and development of kiwifruit canker biocontrol agents,but also provided important theoretical basis for the formulation of kiwifruit canker biocontrol strategy. |
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