Diversity of Endophytic Bacterial Populations and Their Interaction with Xylella fastidiosa in Citrus Plants

Citrus variegated chlorosis (CVC) is caused by Xylella fastidiosa, a phytopathogenic bacterium that can infect all Citrus sinensis cultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis as Bacillus pumilus, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium spp. (including Methylobacterium extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, and M. zatmanii), Nocardia sp., Pantoea agglomerans, and Xanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species of Methylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolated C. flaccumfaciens significantly more frequently from asymptomatic plants than from those with symptoms of CVC while P. agglomerans was frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G+C ratio was observed only in asymptomatic plants. The higher frequency of C. flaccumfaciens in asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.

柑橘杂色褪绿病（Citrus variegated chlorosis, CVC）由苛养木杆菌（Xylella fastidiosa）引发，该植物病原细菌（phytopathogenic bacterium）可侵染所有甜橙（Citrus sinensis）品种。本研究采用培养法（cultivation）与非培养依赖技术（cultivation-independent techniques），对健康、抗病及感染CVC的柑橘植株的内生细菌群落（endophytic bacterial communities）展开分析。通过涂布平板法（plating）与变性梯度凝胶电泳（denaturing gradient gel electrophoresis, DGGE），对表面消毒后的柑橘枝条中的内生细菌群落进行评估。优势分离菌株经脂肪酸甲酯分析（fatty-acid methyl ester analysis）鉴定为：短小芽孢杆菌（Bacillus pumilus）、枯萎短小杆菌（Curtobacterium flaccumfaciens）、阴沟肠杆菌（Enterobacter cloacae）、甲基杆菌属（Methylobacterium spp.，涵盖扭脱甲基杆菌Methylobacterium extorquens、藤泽甲基杆菌M. fujisawaense、嗜中温甲基杆菌M. mesophilicum、耐辐射甲基杆菌M. radiotolerans以及扎氏甲基杆菌M. zatmanii）、诺卡氏菌属（Nocardia sp.）、成团泛菌（Pantoea agglomerans）与野油菜黄单胞菌（Xanthomonas campestris）。研究观察到CVC症状与甲基杆菌属（Methylobacterium）菌株的分离频率存在相关性，该属是我们从发病植株中分离得到的最优势类群。与之相对，枯萎短小杆菌（C. flaccumfaciens）在无症状植株中的分离频率显著高于发病植株；而成团泛菌（P. agglomerans）则可高频分离自橘（Citrus reticulata）与甜橙（C. sinensis）植株，不受植株是否表现CVC症状的影响。对从植物总DNA（total plant DNA）中扩增获得的16S rRNA基因片段（16S rRNA gene fragments）进行DGGE分析，所得多条条带与分离得到的细菌菌株条带相匹配，表明DGGE图谱可用于检测柑橘植株中的部分内生细菌。然而，部分条带未与任何已知分离菌株匹配，提示存在其他未培养或尚未被培养的内生细菌类群。一条具有高鸟嘌呤-胞嘧啶（G+C）含量的特异性条带仅在无症状植株中被观测到。无症状植株中枯萎短小杆菌（C. flaccumfaciens）的更高分离频率，提示该菌体在植株抵御CVC的过程中发挥了一定作用。