Dissecting Disease-Suppressive Rhizosphere Microbiomes by Functional Amplicon Sequencing and 10× Metagenomics.

Disease-suppressive soils protect plants against soilborne fungal pathogens that would otherwise cause root infections. Soil suppressiveness is, in most cases, mediated by the antagonistic activity of the microbial community associated with the plant roots. Considering the enormous taxonomic and functional diversity of the root-associated microbiome, identification of the microbial genera and mechanisms underlying this phenotype is challenging. One approach to unravel the underlying mechanisms is to identify metabolic pathways enriched in the disease-suppressive microbial community, in particular, pathways that harbor natural products with antifungal properties. An important class of these natural products includes peptides produced by nonribosomal peptide synthetases (NRPSs). Here, we applied functional amplicon sequencing of NRPS-associated adenylation domains (A domains) to a collection of eight soils that are suppressive or nonsuppressive (i.e., conducive) to Fusarium culmorum, a fungal root pathogen of wheat. To identify functional elements in the root-associated bacterial community, we developed an open-source pipeline, referred to as dom2BGC, for amplicon annotation and putative gene cluster reconstruction through analyzing A domain co-occurrence across samples. We applied this pipeline to rhizosphere communities from four disease-suppressive and four conducive soils and found significant similarities in NRPS repertoires between suppressive soils. Specifically, several siderophore biosynthetic gene clusters were consistently associated with suppressive soils, hinting at competition for iron as a potential mechanism of suppression. Finally, to validate dom2BGC and to allow more unbiased functional metagenomics, we performed 10× metagenomic sequencing of one suppressive soil, leading to the identification of multiple gene clusters potentially associated with the disease-suppressive phenotype. Soil-borne plant-pathogenic fungi continue to be a major threat to agriculture and horticulture. The genus Fusarium in particular is one of the most devastating groups of soilborne fungal pathogens for a wide range of crops. Our approach to develop novel sustainable strategies to control this fungal root pathogen is to explore and exploit an effective, yet poorly understood naturally occurring protection, i.e., disease-suppressive soils. After screening 28 agricultural soils, we recently identified four soils that were suppressive to root disease of wheat caused by Fusarium culmorum. We also confirmed, via sterilization and transplantation, that the microbiomes of these soils play a significant role in the suppressive phenotype. By adopting nonribosomal peptide synthetase (NRPS) functional amplicon screening of suppressive and conducive soils, we here show how computationally driven comparative analysis of combined functional amplicon and metagenomic data can unravel putative mechanisms underlying microbiome-associated plant phenotypes.