Generation of spontaneous rifampicin-resistant Pseudomonas mutants

Abstract

Plants are colonised by numerous microorganisms collectively termed the microbiome. Globally efforts are being made to engineer the microbiomes of plants to enhance the traits delivered by microorganisms. Synthetic communities or ‘SynComs’ are formulations of selected microbial strains that are prepared in the laboratory and designed to mimic natural microbial ecosystems. The research presented here is part of a study aimed to monitor the colonisation dynamics of a synthetic bacterial community (Syncom) composed of Pseudomonas strains with antagonistic potential against grapevine trunk disease (GTD). We describe the production of rifampicin resistance strains (rif+) to enable the quantitative tracking bacteria when introduced into plants and to allow for clear differentiation from the native microflora. Spontaneous rif⁺ mutants were generated from previously identified Pseudomonas isolates with known biocontrol activity against the GTD causal agent Eutypa lata. Genetic stability was assessed and only mutants that retained resistance were considered stable and heritably resistant. The inhibitory activity of rifampicin-resistant Pseudomonas mutants towards E. lata was evaluated using a dual plate assay. Assays were conducted in parallel with non-mutant (wild-type) Pseudomonas isolates to verify that their biocontrol efficacy had been maintained. PCR sequencing targeting the rpoB gene (RNA polymerase βsubunit) was used to identify rif+ mutations. This method is a cost-effective and reliable strategy for studying the behaviour and stability of applied microbial consortia in planta. These rif⁺ mutants will be used to explore the persistence and distribution of the bacteria in planta with the goal of engineering Syncoms that enhance plant defence against pathogens.