Biology subjects

NAVAS-CORTES, J. A.

Publications and source records attributed to NAVAS-CORTES, J. A..

2 recordsLinked to original sources

bioRxiv2020

Evaluation of established methods for DNA extraction and primer pairs targeting 16S rRNA gene for bacterial microbiome profiling of olive xylem sap

Next Generation Sequencing has revolutionized our ability to investigate the microbiota composition of diverse and complex environments. However, a number of factors can affect the accuracy of microbial community assessment, such as the DNA extraction method, the hypervariable region of 16S rRNA gene targeted or the PCR primers used for amplification. The aim of this study was to assess the influence of commercially available DNA extraction kits and different primer pairs to provide a nonbiased vision of the composition of bacterial communities present in olive xylem sap. For that purpose, branches from Picual and Arbequina olive cultivars were used for xylem sap extraction using a Scholander chamber device. The DNA extraction protocol significantly affected xylem sap bacterial community assessment. That resulted in significant differences in alpha (Richness) and beta diversity (UNIFRAC distances) metrics among DNA extraction protocols, with the 12 DNA extraction kits evaluated being clustered in four groups behaving differently. Although the core number of taxa detected by all DNA extraction kits included four phyla, seven classes, 12 orders, and 16 or 21 families, and 12 or 14 genera when using the Greengenes or Silva database for taxonomic assignation, respectively, some taxa, particularly those identified at low frequency, were detected by some DNA extraction kits only. The most accurate depiction of a bacterial mock community artificially inoculated on sap samples was generated when using the PowerPlant DNA extraction Kit, the combination of 799F/1193R primers amplifying the hypervariable V5-V7 region and the Silva 132 database for taxonomic assignation. The DESeq2 analysis displayed significant differences among genera abundance between the different PCR primer pairs tested. Thus, Enterobacter, Granulicatella, Prevotella and Brevibacterium presented a significant higher abundance in all PCR protocols when compared with primer pair 799F/1193R, while the opposite was true for Pseudomonas and Pectobacterium. The methodological approach followed in this study can be useful to optimize plant-associated microbiome analysis, especially when exploring new plant niches. Some of the DNA extraction kits and PCR primers selected in this study will contribute to better characterize bacterial communities inhabiting within the xylem sap of olives or other woody crop species.

microbiology↗

bioRxiv2020

Verticillium dahliae inoculation and in vitro propagation modify the xylem microbiome and the resistance to Verticillium wilt of a wild olive genotype

Host resistance is the most practical, long-term and economically efficient disease control measure for Verticillium wilt in olive caused by the xylem-invading fungus Verticillium dahliae (Vd), and it is at the core of the integrated disease management. Plants microbiome at the site of infection may have an influence on the host reaction to pathogens; however, the role of xylem microbial communities in the olive resistance to Vd has been overlooked and remain unexplored to date. This research was focused on elucidating whether in vitro olive propagation may alter the diversity and composition of the xylem-inhabiting microbiome and if those changes may modify the resistance response that a wild olive clone shows to the highly virulent defoliating (D) pathotype of Vd. Results indicated that although there were differences in microbial communities among the different propagation methodologies, most substantial changes occurred when plants were inoculated with Vd, regardless whether the infection process took place, with a significant increase in the diversity of bacterial communities when the pathogen was present in the soil. Furthermore, it was noticeable that olive plants multiplied under in vitro conditions developed a susceptible reaction to D Vd, characterized by severe wilting symptoms and 100% vascular infection. Moreover, those in vitro propagated plants showed an altered xylem microbiome with a decrease in total OTU numbers as compared to that of plants multiplied under non-aseptic conditions. Overall, 10 keystone bacterial genera were detected in olive xylem regardless infection by Vd and the propagation procedure of plants (in vitro vs nursery), with Cutibacterium (36.85%), Pseudomonas (20.93%), Anoxybacillus (6.28%), Staphylococcus (4.95%), Methylobacterium-Methylorubrum (3.91%), and Bradyrhizobium (3.54%) being the most abundant. Pseudomonas spp. appeared as the most predominant bacterial group in micropropagated plants and Anoxybacillus appeared as a keystone bacterium in Vd-inoculated plants irrespective of their propagation process. Our results are first to show a breakdown of resistance to Vd in a wild olive that potentially maybe related to a modification of its xylem microbiome, and will help to expand our knowledge of the role of indigenous xylem microbiome on host resistance which can be of use to fight against main vascular diseases of olive.

microbiology↗