柑橘黄龙病赣南脐橙内生菌种群结构分析

Endophytic bacterial community in Gannan navel orange affected by huanglongbing

【目的】分析赣南脐橙黄龙病植株和健康植株叶片内生菌,对比不同培养条件下培养出的内生菌,为筛选出对柑橘黄龙病原菌有影响的伴生菌奠定理论基础。【方法】通过PCR方法对脐橙中黄龙病菌进行验证,并基于16S r RNA基因高通量测序技术对患病与健康赣南脐橙叶片内生菌以及不同培养基富集培养后的内生菌进行多样性分析。【结果】所采集样品中有5株患病株,5株健康株。5株病株中共同含有的细菌属有13个,其中7个在5株健株中也共同存在。Defluviicoccus属和Granulicella属在病健株植物中都是优势菌属,且在健株中的平均含量高于病株。病株与健株的样品相似度存在明显界线。富集培养后不同样本和不同培养基中菌属分布不同。肠杆菌属(Enterobacter)、短小杆菌属(Curtobacterium)、假单胞菌属(Pseudomonas)和泛菌属(Pantoea)得到了大量富集,不动杆菌属和沙雷氏菌属等9个菌属富集量较少。另外,培养和未培养各样本间未分类菌(Unclassified)含量差异也较大。【结论】赣南脐橙患病植株和健康植株叶片内生菌有着明显差异,黄龙病菌的存在改变了脐橙叶片原有内生细菌的菌群结构。从活体植物组织内直接检测才能得到真正的植物内生菌群落分布情况。通过分析菌群的差异,有望找到与柑橘黄龙病菌生长相关的伴生菌。

[Objective] To analyze the endophytic bacteria in leaves of Huanglong disease affected and healthy Gannan navel oranges, and to compare the bacterial community under different culture conditions. [Methods] Candidatus Liberibacter was verified by PCR. Based on 16 S r RNA gene high throughput sequencing, the diversity of endophytic bacteria in the leaves of diseased and healthy plants（each five plants） cultured in different media was analyzed. [Results] Thirteen strains existed in diseased plants and seven of them were also found in all healthy plants. The dominant bacterial population of both diseased and healthy plants was mainly from Defluviicoccus sp. and Granulicella sp., whose average contents in healthy plants were higher than that in diseased ones. The similarity of diseased and healthy samples had obvious boundary. The strains obtained after cultivation in different samples and media were different. Enterobacter sp., Curtobacterium sp., Pseudomonas sp. and Pantoea sp. got a lot of enrichment, whereas the enrichment of 9 genera including Acinetobacter sp. and Serratia sp. was less. Besides, the amount of unclassified bacteria in the samples had obvious differences. [Conclusion] There were obvious differences between endophytic bacteria in disease and healthy Gannan navel oranges. The existence of Ca. Liberibacter changed the bacterial community structures. Only direct detection from the living plant tissue could obtain the real endophytic bacterial distribution. It is expected to find the bacteria associated with Ca. Liberibacter growth by analyzing the community difference of bacteria.