Study On Diversity Of Endophytic Microorganisms In The Leaf Midrib Of Sugar Orange Infected With Huanglongbing

Citrus Huanglongbing,is a destructive disease caused by the phloem refractory bacteria(‘Candidatus Liberibacter' asiaticus),which poses a serious threat to the citrus industry.Amplifier sequencing(including 16 S rDNA and ITS)was used to analyze the diversity of citrus endophytic microorganisms(including bacteria and fungi)which were isolated from healthy sugar orange plants and Huanglongbing infected plants in three different orchards.The species with significant differences between the groups were excavated and correlated statistical analysis was carried out in an effort to find microbial species that were antagonistic/synergy with the pathogen of Huanglongbing.Lays a foundation for exploring microbial genome resources,special genes and their metabolic pathways,gene functions of related strains,and revealing the synergistic/antagonistic mechanism with pathogenic bacteria of Huanglongbing.The research results were showed as follows.1.Compared the diversity of endophytic microflora in the middle veins of sugar orange leaves infected with Huanglongbing and healthy among different orchards.20 samples were selected from F orchard(Fengcun Town,the same as below)and C orchard(Chang'an Town,the same as below),10 samples were selected from B orchard(Bozhi town,the same as below),which used for sequencing analysis of 16 S rDNA and ITS.The percentage of Effective Tags obtained from each samples in the original data reached more than 92.15% and88.64% respectively.The diversity of endophytic bacteria and endophytic fungi in the orchards between healthy group and infected group was inconsistent.As for endophytic bacteria,the diversity in healthy group of F orchard was higher than infected group,while the B and C orchards were opposite.As for Endophytic fungi,the diversity in C orchard healthy group was higher than infected group,while the F orchard and B orchard ware opposite.2.Significant differences in the community structure among endophyte in the middle vein of sugar orange leaves.In different orchards,the endophytic bacteria in the middle vein of sugar orange leaves with healthy and infected Huanglongbing pathogens showed obvious differences in community structure,the difference of community structure between groups was greater than that within groups.As for the Endophytic fungi,there were also some differences in community structure between the healthy and infected Huanglongbing pathogens,but the differences were not consistent.Among them,the difference between healthy and infected Huanglongbing pathogens groups in F orchard was obvious and reached the extremely significant level(P<0.01),but the difference between B and C orchards wasrelatively smaller and didn't reach the significant level(P>0.05).3.Significant differences among endophyte communities in the middle vein of sugar orange leaves.the abundance of Actinomycetospora and Cyphellophora were higher in healthy group,the abundance of ‘Candidatus Liberibacter' Massilia,Setosphaeria,Erythrobasidium and Phaeosphaeriopsis were species significantly higher in infected group;Significant differences among different orchards were: Halomonas,Methylobacterium,Sphingomonas,Sporobolomyces,Symmetrospora;Significant differences species between the healthy and infected groups in each orchards were also large in endogenous microbial species,except for the pathogen ‘Candidatus Liberibacter',there were no significant differences species in common.The trends of abundance some species in different orchards were different between healthy and infected groups: for example,Sphingomonas in the infected group of F and B orchards were significant,but significantly higher in healthy group of C orchards;Nocardioides was significantly higher in healthy group of F and C orchards,but significantly higher in infected group of B orchard;Pestalotiopsis was significantly higher in the B orchard healthy group,while in F was significantly higher in infected group.Some genus were significantly higher in infected group or healthy group of a certain orchard: such as Actinomycetospora,Jatrophihabitans,etc.,only increased significantly in healthy group of F orchard;Erythrobasidium,Phaeosphaeriopsis,Penidiella,etc.,only increased significantly in infected group of F orchard,Aureobasidium only significantly elevated in the B orchard.4.Associated statistical analysis of the endophyte in the middle vein of sugar orange leaves between healthy and infected Huanglongbing pathogens.the difference of the microbial species that were antagonistic/synthesized with the pathogen of Huanglongbing in different orchards was very large,among them,there were 25 strong related species with huanglongbing pathogen in F orchard which absolute correlation coefficient greater than 0.5,1 in C orchard,and 13 in B orchard.At the same time,the consistency of the antagonistic/synergy were also poor,For example,Angustibacter were strongly negatively correlated with the pathogen of Huanglongbing in F orchard(-0.6773),but strongly positively correlated in B orchard(0.5194);Coniothyrium and Pestalotiopsis were strongly positively correlated with Huanglongbing pathogen in F orchard(0.5360,0.5684),but in B group was just the opposite(-0.7675,-0.6190).Pseudoncardia(-0.5361,-0.5392),Cyphellophora(-0.6175,-0.6816)were negatively correlated with Huanglongbing pathogen in F and B orchards,They may be a potential endophytic strain for controlling Huanglongbing.