地黄连作对叶际细菌群落结构及多样性的影响

地黄为玄参科多年生草本药用植物,以块根入药,是我国著名的大宗药材.但是地黄在农业生产过程中存在严重的连作障碍问题,造成产量和品质急剧下降.细菌作为叶际微生物中最为丰富的一类,对宿主植物的生长发育与健康至关重要.叶际细菌区系研究为探索连作障碍形成机制及其消减措施提供了一个全新视角,同时差异菌群也可作为连作障碍发生的指示菌.本研究采用16S rDNA基因高通量测序结合传统可培养法分析地黄连作下叶际细菌群落结构及多样性变化.结果表明: 地黄连作导致叶际细菌群落结构发生明显变化,重茬地黄和病株地黄的叶际细菌群落结构较为相似,聚为一类,并明显区别于头茬地黄.同时,重茬地黄和病株地黄叶际细菌群落的均匀度指数、Shannon多样性指数、Simpson多样性指数均显著低于头茬地黄.物种注释分析显示,地黄叶际细菌主要由变形菌门(91.2%)、厚壁菌门(5.1%)和放线菌门(3.7%)组成.韦恩图分析发现,连作下地黄叶际细菌种类变化不大,但是结合相对含量来看,地黄连作导致叶际变形菌门含量增加,而厚壁菌门和放线菌门下降.在属水平上,头茬地黄叶际的微小杆菌属、芽孢杆菌属、节细菌属等潜在有益菌相对含量显著高于重茬地黄和病株地黄,而假单胞菌属呈现相反的变化趋势.传统可培养法结合致病性验证发现,在病株地黄叶片上广泛分离到的变形假单胞菌D9对地黄叶片表现出较强的侵染致病性.综上可见,地黄连作下叶际细菌群落结构发生偏移,导致有益菌含量下降而病原菌含量上升,造成连作地黄叶片病症频发,加剧了地黄再植病害的发生.

Effects of Rehmannia glutinosa consecutive monoculture on the community structure and diversity of phyllosphere bacteria

Rehmannia glutinosa, a perennial herbaceous species, belongs to the family Scrophularia-ceae. As a staple medicinal material, its tuberous roots are highly valued in traditional Chinese medicine. However, R. glutinosa suffers from serious consecutive monoculture problems in production, which leads to a decline in both productivity and quality. Phyllosphere bacteria, the most abundant component of phyllosphere microorganisms, play crucial roles in plant growth and health. Characterization of phyllosphere bacteria could provide new insights into the mechanisms of consecutive monoculture problems and their control measures. Meanwhile, the varied taxa could be served as an important indicator of consecutive monoculture problems. The barcoded pyrosequencing of 16S rDNA genes combined with a culture-dependent approach was applied to characterize the shifts of bacterial community structure and diversity in the phyllosphere under consecutive monoculture of R. glutinosa. The results showed that consecutive monoculture clearly affected bacterial community structure in the phyllosphere. The phyllosphere bacterial communities of the two-year monocultured (TY) and the diseased plants (DP) were more similar, and different from the one-year monocultured (OY). The evenness, Shannon and Simpson diversity indices were significantly lower in TY and DP than in OY. Species annotation showed that bacterial community in R. glutinosa phyllosphere mainly consisted of Proteobacteria (91.2%), Firmicutes (5.1%) and Actinobacteria (3.7%). There was no significant difference in the number of detected bacterial taxa. However, Proteobacteria was significantly increased while Firmicutes and Actinobacteria were significantly decreased under consecutive monoculture. At the genus level, the relative abundances of genera Exiguobacterium, Bacillus and Arthrobacter, potentially beneficial microorganisms, were significantly higher in OY than that in TY and DP, but it was opposite for the genus Pseudomonas. The results from the culture-dependent approach and pathogenicity test showed that Pseudomonas plecoglossicida D9, widely isolated from the diseased leaves, was highly pathogenic to leaves. In conclusion, R. glutinosa monoculture resulted in distinct phyllosphere bacterial community variation with the accumulation of pathogen loads at the expense of beneficial microorganisms, which could contribute to the occurrence of leaf disease symptoms,and aggravate R. glutinosa replant disease in a monoculture regime.