Anguita-Maeso Manuel, Olivares-García Concepción, Haro Carmen, Imperial Juan, Navas-Cortés Juan A, Landa Blanca B
Institute for Sustainable Agriculture, Spanish National Research Council (CSIC), Córdoba, Spain.
Institute of Agricultural Sciences, Spanish National Research Council (CSIC), Madrid, Spain.
Front Plant Sci. 2020 Jan 21;10:1708. doi: 10.3389/fpls.2019.01708. eCollection 2019.
Microbial endophytes are well known to protect host plants against pathogens, thus representing a promising strategy for the control of xylem-colonizing pathogens. To date, the vast majority of microbial communities inhabiting the olive xylem are unknown; therefore, this work pursues the characterization of the xylem-limited microbiome and determines whether the culture isolation medium, olive genotype, and the plant material used to analyze it can have an effect on the bacterial populations retrieved. Macerated xylem tissue and xylem sap extracted with the Scholander chamber from olive branches obtained from two cultivated and a wild olive genotypes were analyzed using culture-dependent and -independent approaches. In the culture-dependent approach using four solid culture media, a total of 261 bacterial isolates were identified after performing Sanger sequencing of 16S rRNA. Culturable bacteria clustered into 34 genera, with some effect of culture media for bacterial isolation. The cultivated bacteria belonged to four phyla and the most abundant genera included (18.8%), (16.4%), and (14.6%). On the other hand, in the culture-independent approach conducted using Illumina MiSeq 16S rRNA amplicon sequencing [next-generation sequencing (NGS)] of the xylem extracts, we identified a total of 48 operational taxonomic units (OTUs) belonging to five phyla, being (30.1%) (24.1%) and (22.4%) the most representative genera (>76% of reads). In addition, the results indicated significant differences in the bacterial communities detected in the xylem sap depending on the genotype of the olive tree studied and, to a minor extent, on the type of sap extraction method used. Among the total genera identified using NGS, 14 (41.2%) were recovered in the culture collection, whereas 20 (58.8%) in the culture collection were not captured by the NGS approach. Some of the xylem-inhabiting bacteria isolated are known biocontrol agents of plant pathogens, whereas for others little information is known and are first reported for olive. Consequently, the potential role of these bacteria in conferring olive tree protection against xylem pathogens should be explored in future research.
众所周知，微生物内生菌可保护宿主植物免受病原体侵害，因此是控制定殖于木质部的病原体的一种很有前景的策略。迄今为止，绝大多数栖息在橄榄木质部的微生物群落仍不为人所知；因此，本研究旨在对局限于木质部的微生物组进行表征，并确定培养分离培养基、橄榄基因型以及用于分析的植物材料是否会对所获得的细菌种群产生影响。使用依赖培养法和非依赖培养法，对从两种栽培橄榄基因型和一种野生橄榄基因型的橄榄枝条中提取的木质部组织浸出物和用 Scholander 室提取的木质部汁液进行了分析。在使用四种固体培养基的依赖培养法中，对 16S rRNA进行桑格测序后，共鉴定出 261 株细菌分离株。可培养细菌聚为 34 个属，培养基对细菌分离有一定影响。培养出的细菌属于四个门，最丰富属包括（18.8%）、（16.4%）和（14.6%）。另一方面，在用 Illumina MiSeq 1对木质部提取物进行 16S rRNA 扩增子测序 [下一代测序（NGS）] 的非依赖培养法中，我们共鉴定出属于五个门的 48 个可操作分类单元（OTU），最具代表性的属是（30.1%）、（24.1%）和（22.4%）（>76% 的读数）。此外，结果表明，根据所研究的橄榄树基因型以及在较小程度上根据所用汁液提取方法的类型，在木质部汁液中检测到的细菌群落存在显著差异。在使用 NGS 鉴定出的所有属中，14 个（41.2%）在培养物收集中被回收，而培养物收集中的 20 个（58.8%）未被 NGS 方法捕获。一些分离出的栖息在木质部的细菌是已知的植物病原体生物防治剂，而其他一些细菌的信息很少，并且是首次在橄榄中报道。因此，这些细菌在赋予橄榄树抵御木质部病原体保护方面的潜在作用应在未来研究中加以探索。
