柑橘黄龙病罹病植株显症差异组织内生细菌群落结构分析

Endophytic bacterial community in the symptoms and symptomless tissues of HLB-affected citrus plant

摘要:

摘要:【目的】分离鉴定同株罹病柑橘黄龙病植株不同显症状况组织的内生细菌，寻找与黄龙病菌［‘Candidatus Liberibacter asiaticus’(Ca．Las)］相互作用的优势菌株。【方法】利用基于16S rDNA的PCＲDGGE(Denaturing gradient gel electrophoresis)分析同一柑橘黄龙病罹病植株的显症和未显症组织内生细菌多样性，并用定量PCR 方法，对果、枝、叶3种组织黄龙病菌、优势菌株及细菌总数进行检测。【结果】结果显示显症和无症组织所带黄龙病菌差异很大，显症部位病菌量明显高于无症部位。分析显症和无症组织内生细菌DGGE图谱显示，同一组织内生菌群结构基本相同;对图谱中17条明显条带回收克隆测序，发现其中8个条带均属于沙雷氏菌属(Serratia)，占总条带数的47.06%。序列分析显示这8条序列为粘质沙雷氏菌(Serratia marcescens)不同的菌株(序列相似性为99.63%)。定量分析各差异显症部位单位组织内的粘质沙雷氏菌和细菌总数，发现相同部位的总细菌量差异不显著，但粘质沙雷氏菌与黄龙病菌的量呈负相关。【结论】柑橘黄龙病病株中，各部位所带病菌量不均匀，是否显症与组织内柑橘黄龙病菌的量呈正相关，内生菌群总量与显症无相关性，但粘质沙雷氏菌与黄龙病菌的量呈负相关。粘质沙雷氏菌与黄龙病菌在韧皮部细胞内增殖过程中的相互作用值得深入研究。

Abstract:

Abstract:［Objective］ To analyze the endophytic bacteria in symptoms and symptomless tissues，reveal dominant bacterial population which may interact with Ca. Las. ［Method］ The population structure and diversity of endophytic bacteria in symptoms and symptomless tissues of single HLB-affectedcitrus plant were studied based on 16S rDNA sequences analyzed by PCＲ-DGGE (PCR-denaturing gradient gel electrophoresis). Quantitative analysis of Ca．Las，dominant bacteria and total amount of bacterial population in symptoms and symptomless tissues of branches，leaves and fruits was done by quantitative PCR．［Result］ The amount of Ca．Las is significantly different in symptoms and symptomless tissues; bacterial population in symptoms citrus tissues was significantly higher than in symptomless tissues．PCR-DGGE analysis result shows that endophytic bacterial structures are basically the same in symptoms and symptomless tissues．Extraction，cloning and sequencing of 17 obvious bands in DGGE electrophoretogram reveal that 8 of 17 bands belonged to the genus Serratia，which accounted for 47.06%．Sequence alignment shows that they belonged to different strains of Serratia marcescens with the 99. 63% similarity．Quantitative analysis of the total bacteria and Serratia marcescens shows that there is no significant difference in total bacterial count between symptoms and symptomless tissues whereas the amount of Serratia marcescens was negatively correlated with the amount of Las.［Conclusion］The Ca. Las is heterogeneously distributed among various tissues of HLB-affected citrus plant．The amount of Ca．Las was negatively correlated with Serratia marcescens and total amount of endophytic bacteria have no relevance with symptom of HLB disease．How the interaction of Serratia marcescens and Ca．Las in tissue of citrus needs further study.