Search for: All records

Although our understanding of the microbial diversity found within a given system expands as amplicon sequencing improves, technical aspects still drastically affect which members can be detected. Compared with prokaryotic members, the eukaryotic microorganisms associated with a host are understudied due to their underrepresentation in ribosomal databases, lower abundance compared with bacterial sequences, and higher ribosomal gene identity to their eukaryotic host. Peptide nucleic acid (PNA) blockers are often designed to reduce amplification of host DNA. Here we present a tool for PNA design called the Microbiome Amplification Preference Tool (MAPT). We examine the effectiveness of a PNA designed to block genomic Medicago sativa DNA (gPNA) compared with unrelated surrounding plants from the same location. We applied mitochondrial PNA and plastid PNA to block the majority of DNA from plant mitochondria and plastid 16S ribosomal RNA genes, as well as the novel gPNA. Until now, amplifying both eukaryotic and prokaryotic reads using 515F-Y and 926R has not been applied to a host. We investigate the efficacy of this gPNA using three approaches: (i) in silico prediction of blocking potential in MAPT, (ii) amplicon sequencing with and without the addition of PNAs, and (iii) comparison with cultured fungal representatives. When gPNA is added during amplicon library preparation, the diversity of unique eukaryotic amplicon sequence variants present in M. sativa increases. We provide a layered examination of the costs and benefits of using PNAs during sequencing. The application of MAPT enables scientists to design PNAs specifically to enable capturing greater diversity in their system. 

To understand factors that influence the assembly of microbial communities, we inoculatedMedicago sativawith a series of nested bacterial synthetic communities and grew plants in distinct nitrogen concentrations. Two isolates in our eight‐member synthetic community,Williamsiasp. R60 andPantoeasp. R4, consistently dominate community structure across nitrogen regimes. WhilePantoeasp. R4 consistently colonizes plants to a higher degree compared to the other six organisms across each community and each nutrient level,Williamsiasp. R60 exhibits nutrient specific colonization differences.Williamsiasp. R60 is more abundant in plants grown at higher nitrogen concentrations, but exhibits the opposite trend when no plant is present, indicating plant‐driven influence over colonization. Our research discovered unique, repeatable colonization phenotypes for individual microbes during plant microbiome assembly, and identified alterations caused by the host plant, microbes, and available nutrients.