Isolation and characterization of endophytic bacteria from huanglongbing’s hidden host plant- Murraya paniculata

Abstract:

[Objective] Our goal is to analyze the bacterial endophytic diversity in Murraya paniculata, which was huanglongbing’s hidden host plant, and find the endophytic bacteria that may have potential to suppress the HLB pathogen. [Methods] The endophytic bacteria were isolated from surface-sterilized M. paniculata midribs of leaves and phloem of stems by plating and restriction fragment length polymorphism (RFLP). The functions of 14 culturable bacteria had been tested using different substrates and cultural medium. [Results] By the artificial anaerobic culture, we obtained 26 strains that were grouped into 9 genus according to GenBank. Enterobacter sp. (IF=19.23%) and Bacillus sp. (IF=38.46%) were the dominant bacterial population in M. paniculata. However, the bacterial colony number of endophytic bacteria in the stems was higher than in the leaves. The endophytic bacteria 16S rDNA library of M. paniculata was established. Coverage C of the clone library was 94.97% and the rarefaction curve of the clone library tended to plateau, which indicate that the library are large enough to re?ect the bacterial endophytic diversity of the respective samples. 179 positive clones in 16S rRNA gene library were chosen and digested by restriction endonuclease Hae Ⅲ, MspⅠ, RsaⅠrespectively. Twenty OTUs (Operational taxonomic units) were observed based on the similarity of the RFLP banding profiles. In the observed clones, 63.69% of the clones were Serratia sp., which means that the Serratia sp. was absolute preponderant endophytic bacteria in M. paniculata. The functional analysis of the 14 endophytic bacteria showed that 9 isolates can produce IAA and 3 of them had amylolysis activity. The frequency of endophytic bacteria which can produce ACC deaminase, siderophore and protein activity was 1, 8 and 2 respectively. Three of bacteria isolates with N-fixation ability were screened by using N-free medium and nifH gene and 4 of endophytic bacteria cansynthesis antibiotic (phlD). A total of 4 bacterial isolates was provided with four of mentioned traits. [Conclusion] The endophytic bacteria isolated from M. paniculata have abundant diversity and genetic characterization, and they may have important functions for the plant development, anti abio-stresses and bio-stresses.