Isolation of facultative anaerobic entophytic bacterial companioned Ca Las infected citrus tissues and evaluation of dominant bacterial populations

Abstract:

To analysis the entophytic bacterial diversity of citrus and find the companion bacteria populations associated with Huanglongbing pathogen-infected and healthy citrus plant tissues for decipher the co-cultivation of HLB pathogen, we selected varied parts of citrus tissues collected from different loca-tions of citrus planted area. The facultative anaerobic entophytic bacteria were isolated and purified based on bacterial morphology, physiology, biochemistry characteristics and the molecular method of PCR-DGGE (Denaturing gradient gel electrophoresis) analysis based on the sequence of 16S rRNA V6-V8 fragment gene. By the directional isolation of the facultative anaerobic entophytic bacteria and 16S rDNA amplification, total 12 genera of bacteria were identified from 19 cultivable bacterial popula-tions. The dominant bacterial population in infected citrus plants were Curtobacterium sp. (IF: 29.07%), Bacillus sp. (IF: 23.12%), Microbacterium sp. (IF: 21.09%) while in healthy citrus tissues belonged to Bacillus sp. (IF: 21.03%), Planococcus sp. (IF: 20.69%), Pseudomonas sp. (IF: 17.44%). From 50 target bands obtainded by the DGGE approach, 9 genera of cultivable bacteria were recognized. The dominant bacterium population belonged to Serratia sp. (IF: 28%) and Pantoea sp. (IF: 14%) followed by it. Can-didatus Liberibacter asiaticus was only found in tangerine pith of deformed orange fruit, which sug-gested that the content (>1%) of Huanglongbing was more in diseased fruits and other tissues of citrus had low abundance percentage. The density and species of entophytic bacteria were also observed in re-markable difference between infected and healthy citrus plant from the PCR-DGGE profiles.

Key words:Huanglongbing, Entophytic bacteria, Directional isolation, 16S rRNA diversity analysis