柑橘黄龙病隐症寄主九里香内生细菌分离及功能鉴定

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

摘要:

【目的】分析黄龙病高发区的九里香植株叶及茎干中内生细菌, 为寻找具有抗柑橘黄龙病的内生细菌奠定基础。【方法】利用平板培养法及基于16S rDNA的限制性酶切长度多态性(Restriction fragment length polymorphism, RFLP)序列分子鉴定法, 对九里香植物内生细菌进行多样性分析。【结果】在兼性厌氧的生长环境下, 从九里香植株中分离获得可培养内生细菌26株, 分属于9个细菌属的14个种, 其中肠杆菌属(Enterobacter sp.) (IF=19.23%)、芽胞杆菌属(Bacillus sp.) (IF=38.46%)为九里香可培养内生细菌的优势菌属。茎干中内生细菌丰度高于叶中内生菌丰度。建立了九里香内生细菌16S rDNA文库, 对文库质量检测显示, 该克隆文库的覆盖度(Coverage C)为94.97%, 结合Rarefaction曲线分析, 表明所构建的克隆文库是相对充分的。对文库中179个阳性克隆进行Hae Ш、MspⅠ、RsaⅠ3种限制性内切酶分析, 得到20个不同的操作分类单元(Operational taxonomic units, OTUs) , 其中沙雷氏菌属(Serratia sp.)为九里香植株中内生细菌的绝对优势菌属。测定了14株内生细菌的功能, 其中9株菌能产生吲哚-3-乙酸(IAA); 具有抗生素(phlD)合成能力的内生细菌有4株; 结合nifH和NFb固氮培养基确定有3株内生细菌具有固氮能力; 1株内生细菌具有ACC脱氨酶合成能力; 8株内生细菌具有铁细胞合成能力; 3株内生细菌具有淀粉水解能力; 2株内生细菌显示强阳性的蛋白酶合成能力, 4株内生细菌具有以上4种功能。【结论】九里香植株中内生细菌具有丰富的多样性, 并且可能对九里香植株生长发育及抗生物和非生物胁迫有着重要的生理功能。

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.