Anguita-Maeso Manuel, Haro Carmen, Navas-Cortés Juan A, Landa Blanca B
Department of Crop Protection, Institute for Sustainable Agriculture, Spanish National Research Council (CSIC), 14004 Córdoba, Spain.
Plants (Basel). 2022 May 16;11(10):1320. doi: 10.3390/plants11101320.
Understanding the unique and unexplored microbial environment of xylem sap is starting to be of relevant importance for plant health, as it could include microbes that may protect plants against xylem-limited pathogens, such as Verticillium dahliae and Xylella fastidiosa. In this study, we evaluated the effects that the method for extracting the xylem bacterial communities, the plant age and the PCR primers may have on characterizing the xylem-bacterial-community composition by using an NGS approach. Xylem sap was extracted from xylem vessels by using a Scholander pressure chamber, or by macerating wood shavings that were obtained from xylem tissues by using branches from 10-year-old olive trees, or the entire canopy of 1-year-old olive plantlets. Additionally, we compared four different PCR-primer pairs that target 16S rRNA for their efficacy to avoid the coamplification of mitochondria and chloroplast 16S rRNA, as this represents an important drawback in metabarcoding studies. The highest amplifications in the mitochondria and chloroplast reads were obtained when using xylem woody chips with the PCR1-799F/1062R (76.05%) and PCR3-967F/1391R (99.96%) primer pairs. To the contrary, the PCR2-799F/1115R and PCR4-799F/1193R primer pairs showed the lowest mitochondria 16S rRNA amplification (<27.48%), no chloroplast sequences and the highest numbers of bacterial OTUs identified (i.e., 254 and 266, respectively). Interestingly, only 73 out of 172 and 46 out of 181 genera were shared between the xylem sap and woody chips after amplification with PCR2 or PCR4 primers, respectively, which indicates a strong bias of the bacterial-community description, depending on the primers used. Globally, the most abundant bacterial genera (>60% of reads) included Anoxybacillus, Cutibacterium, Pseudomonas, Spirosoma, Methylobacterium-Methylorubrum and Sphingomonas; however, their relative importance varied, depending on the matrix that was used for the DNA extraction and the primer pairs that were used, with the lowest effect due to plant age. These results will help to optimize the analysis of xylem-inhabiting bacteria, depending on whether whole xylematic tissue or xylem sap is used for the DNA extraction. More importantly, it will help to better understand the driving and modifying factors that shape the olive-xylem-bacterial-community composition.
了解木质部汁液独特且未被探索的微生物环境，对植物健康正开始具有重要意义，因为其中可能包含能保护植物抵御木质部限制性病原体（如大丽轮枝菌和桑氏假单胞菌）的微生物。在本研究中，我们评估了提取木质部细菌群落的方法、植物年龄和PCR引物对通过使用NGS方法表征木质部细菌群落组成可能产生的影响。通过使用Scholander压力室从木质部导管中提取木质部汁液，或者通过将从10年生橄榄树的树枝或1年生橄榄幼苗的整个树冠获得的木质部组织制成的刨花进行浸软来提取。此外，我们比较了四种针对16S rRNA的不同PCR引物对，以评估它们避免线粒体和叶绿体16S rRNA共扩增的效果，因为这在宏条形码研究中是一个重要缺点。使用PCR1 - 799F/1062R（76.05%）和PCR3 - 967F/1391R（99.96%）引物对与木质部木屑时，线粒体和叶绿体读数的扩增最高。相反，PCR2 - 799F/1115R和PCR4 - 799F/1193R引物对显示出线粒体16S rRNA扩增最低（<27.48%），没有叶绿体序列，并且鉴定出的细菌OTU数量最多（分别为254和266）。有趣的是，分别用PCR2或PCR4引物扩增后，木质部汁液和木屑之间，172个属中只有73个属、181个属中只有46个属相同，这表明根据所用引物的不同，细菌群落描述存在很大偏差。总体而言，最丰富的细菌属（>60%的读数）包括嗜热栖热菌属、棒状杆菌属、假单胞菌属、螺菌属、甲基杆菌 - 甲基红菌属和鞘氨醇单胞菌属；然而，它们的相对重要性因用于DNA提取的基质和所用引物对而异，植物年龄的影响最小。这些结果将有助于根据用于DNA提取的是整个木质部组织还是木质部汁液，优化对木质部内生细菌的分析。更重要的是，这将有助于更好地理解塑造橄榄木质部细菌群落组成的驱动和修饰因素。
