Study On The Change Of Endophytic Bacterial Community Structure And Screening Of Antagonistic Bacteria Against Kiwifruit Canker Pathogen

Kiwifruit industry is one of the pillar industries for farmers in Western Hunan to get rid of poverty,but in recent years kiwifruit canker disease outbreak frequently,a large number of orchards were destroyed,causing huge economic losses to local farmers,seriously affecting the development of the industry,rapid,effective and environmentally friendly prevention and control technology is in urgent need.Endophytic bacteria play an important role in protecting host plants from pests and diseases,promoting growth and improving stress resistance,and are important germplasm resources for biocontrol agent development.However,the mechanism of endophytic microorganisms of Kiwifruit in the occurrence of Kiwifruit canker disease have not been studied.Based on this,this study took the orchards with kiwifruit canker disease in the main kiwifruit growing areas in western Hunan as sampling sites,and collected infected and healthy kiwifruit branches and leaves samples in three stages:germination stage,growth stage and fruit maturity stage.The following studies were carried out:The structure and changes of the endophytic bacterial community of infected and healthy kiwifruit branches were investigated by macrogenome sequencing,as well as the differences in the relative content and specific taxa of endophytic bacteria between infected and healthy kiwifruit branches during the three stages.The endophytic bacteria were found to be distributed in six phylum,nine family and ten genus in infected samples,and in six phylum,eight family and eight genus in healthy samples.At the genus level,the dominant taxa in infected branches were Streptophyta,unclassified bacteria,and unclassified＿Enterobacteriaceae;the dominant taxa in healthy branches were Streptophyta,unclassified bacteria,and Acinetobacter.The infected branch samples had their special taxa Oxalobacteraceae,Janthinobacterium and Buttiauxella.These differences and special taxa may be positively associated with the occurrence of kiwifruit canker disease.Regarding the succession pattern of microbial taxa with the growth of kiwifruit plants,Proteobacteria showed a decreasing trend in healthy branch samples and a increasing trend in diseased branch samples over the three stages.Pseudomonadaceae and Pseudomonas differed in abundance between healthy and infected kiwifruit branches,mainly during the germination stage,growth stage,with higher abundance of Pseudomonas in infected branch samples than in healthy branches,but not detected during fruit maturity stage,consistent with the corresponding period in healthy samples.These trends coincided with the occurrence of kiwifruit canker disease process.By analyzing the endophytic bacterial community structure of infected and healthy kiwifruit leaf samples,it was found that endophytic bacteria in infected samples were distributed in 13 phylum,36 family and 35 genus;in healthy samples,endophytic bacteria were distributed in 13 phylum,36 family and 36 genus.At the genus level,the dominant taxa in infected leaves were Streptophyta,Acinetobacter,and Pseudomonas;the dominant taxa in healthy branches were Streptophyta,Acinetobacter,and Bacillus.Analysis of the differential and special taxa between the two samples over the three stages showed that the differential taxa of endophytic bacteria in infected and healthy kiwifruit leaves were mainly concentrated in Firmicutes,Proteobacteria,Bacteroidetes,and Chloroflexi.The infected leaf samples contained the speical taxa Xanthomonas and the healthy leaf samples contained the special taxa Fusobacterium and Porphyromonas.It is hypothesized that endophytic bacteria of the genus Flavobacterium may assist in the development of kiwifruit canker disease,and that bacteria of the genus Clostridium and Porphyromonas play an active role in the growth and development of the leaves and in the resistance to the injection of kiwifruit canker disease.Methylobacteriaceae,Rhizobiaceae,Xanthomonadaceae,unclassified＿Sphingobacteriales,Methylobacterium showed downward trend in their relative contents during the growing germination stage-growth stage in injected leaf samples,but showed a significant upward trend in healthy leaf samples,presumably these taxa evolved symbiotically with kiwifruit and competed synergistically with kiwifruit canker bacteria.81 strains of endophytic bacteria were isolated from healthy kiwifruit branches and leaves by traditional culture methods,and were initially identified as belonging to 4phylum,19 family and 25 genus based on 16 S r RNA gene sequence analysis.The endophytic bacteria isolated were screened by Oxford Cup antagonism assay,and 9strains were obtained with antagonistic effect on Psa;after phylogenetic analysis,3strains were identified as Paenibacillus,4 as Bacillus,3 as Staphylococcus,and 1 as Bacillus.1 strain was Mycolicibacterium.The hypothesis of the presence of endophytic bacteria with antagonistic function against kiwi canker disease in healthy kiwifruit,as proposed previously,was confirmed.This work revealed structural differences in the endophytic bacterial community of injected and healthy kiwifruit,elucidated the pattern of endophytic bacterial community changes during the development of kiwifruit canker disease,broadened the understanding of the mechanism of kiwifruit canker disease,and provided an important theoretical basis for the development of kiwifruit canker disease prevention strategies.The screening of antagonistic strains of kiwifruit canker disease will accumulate germplasm resources for the development of kiwifruit canker disease prevention agents.