Associated Diversity Of Phyllosphere Bacterium With Canker Disease

Citrus bacterial canker disease is one of worldwide phytosanitary diseases, the leaves, branches and fruit will have crater-like lesion after affected, and it causing severe defoliation and fruit dropping, affecting fruit growing influence, citrus yield and quality and causing serious economic losses. Citrus canker is closely related to varietal resistance, field management and prevention measures, but also has a certain relationship with the phyllosphere bacterial microbial community structure and diversity. The difference between canker-uninfected and canker-infected phyllosphere bacterial community and the correlation analysis of the quantitative change of citrus bacterial canker and phylloshere bacterial community have been rarely reported. Therefore, taking canker-infected bingtangcheng plant for the study, comparative analysis of the relationship between the phyllosphere bacterial community of canker-uninfected and canker-infected leaves, and the number of inter-bacterial leaf with citrus canker and the incidence of population based on the 16S rDNA V3-V4 variable region Miseq sequencing techniques and quantitative real-time PCR. Discuss of possible synergistic or antagonistic bacteria and the influence on phylloshere bacterial community when Citrus bacterial canker infected. Discuss of possible synergistic or antagonistic bacteria and the influence on phylloshere bacterial community caused by citrus bacterial canker disease.The main results and conclusions are as follows:(1)Through Miseq high-throughput sequencing, the amount of data table of canker-infected leaves are more than canker-uninfected leaves. Received total of 390,929 Tags from 20 samples, every sample has 19,546.45 tags averagely by Miseq platform and bioinformatics analysis. Canker-uninfected leaves have 187,012 reads and for each sample has 18,701.2 averagely tags. Canker-infected leaves have 233,917reads and for each sample has 23,391.7 tags. There are 262 OUTs form 20 samples.(2)Citrus bacterial canker disease can affect the phyllosphere bacterial community, OTU classified respectively comparative analysis on phylum, class, order, family, genus and species. The result shows:On the species level, there are 61.93% of the species are unclassed.Therefore, we chose to analyze on genus:Acinetobacter(52.24%) is the highest content in canker-uninfected leaves, belonging to Proteobacteria, only have 6.62% in canker-infected leaves. Pantoea(25.20%) is highest content belonging to Proteobacteria in canker-infected leaves, and is similar to canker-uninfected leaves(24.26%). In addition, the bacterial have relative content of more than 1.0% and belonging Proteobacteria are as follows:The content of Xanthomonas in canker-infected leaves(7.24%) is about twice than that in canker-uninfected (3.54%). Enterobacter in former(11.83%) is far than latter(2.09%). The canker-infected leaves have Pseudomonas(1.02%), Stenotrophomonas(1.43%), Phingomonas(1.63%), Agrobacterium(4.06%)and Acinetobacter(6.62%), which is undetected in canker-uninfected leaves. Alpha diversity analysis showed that canker-uninfected leaves have lower richness and evenness than canker-infected samples. Probably due to citrus bacterial canker disease causes a lot of open wounds, not only weakens the bingtangcheng’s disease resistance, as well as beneficial to saprophytic bacteria and other environmental microbial colonization.(3)There are many environment favorable microflora, for example, Pseudomonas can promote plant growth through nitrogen fixation; Pantoea can produce growth hormone promoting plant growth and play a role in solubilizing. But some pathogens are also detected in the canker-infected leaves. Under normal conditions, these pathogens do not cause danger to animls or plant, but encountered suitable conditions, they may be converted into pathogens, such as Agrobacterium、Enterobacter、Pseudomonas、Chryseobacteriu.(4)At the generic level, select microflora which has Fourfold differences in canker-uninfected leaves and canker-infected, make linear regression equation with genus Xanthomonas. The result shows:Stenotrophomonas, Pseudomonas, Sphingomonas, Agrobacterium, Pedobacter, Chryseobacterium and Sphingobacteriaceae;Other are positively correlated to the genus Xanthomonas and the correlation coefficients are greater than 0.9, which shows they may be related or synergistic to the occurrence of Citrus bacterial canker disease; Acinetobacter is negatively correlated with Xanthomonas and the correlation coefficient is greater than 0.9, which shows it is may have antagonistic effect to the disease.(5)Compare Miseq high-throughput sequencing and qPCR quantitative analysis, the consistency of both the higher. And the area of Lesion reflects the content of the leaf surface pathogens beastly. When the pathogen content is more than 8%, bingtangcheng shows obvious symptoms.