Pan-genome analysis identifies intersecting roles for Pseudomonas specialized metabolites in potato pathogen inhibition

Abstract

23Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, 24 including the inhibition of plant pathogens. Pseudomonas are one of the most abundant 25 bacterial genera in the soil and rhizosphere and play important roles in promoting plant growth 26 and preventing disease. However, the genetic determinants of this beneficial activity are only 27 partially understood, especially in relation to specialized metabolite production. Here, we 28 genetically and phenotypically characterize the Pseudomonas fluorescens population in 29 commercial potato field soils and identify strong correlations between specialized metabolite 30 biosynthetic pathways and antagonism of the potato pathogens Streptomyces scabies and 31 Phytophthora infestans. Genetic and chemical analyses identified hydrogen cyanide and cyclic 32 lipopeptides as key specialized metabolites associated with S. scabies inhibition. We show that 33 a single potato field contains a hugely diverse and dynamic population of Pseudomonas 34 bacteria, whose capacity to produce specialized metabolites is shaped both by plant 35 colonization and defined environmental inputs. 36 2 37 CFC agar and incubated overnight at 28 °C before streaking to single colonies on King's B (KB) 595 agar plates (96). Six isolates were selected at random per soil sample and subjected to 596 phenotypic/genomic analysis. 597 598 Amplicon sequencing 599 Genomic DNA was isolated from 3 g of pooled soil samples using the FastDNA™ SPIN Kit for 600 soil (MP Biomedicals, UK) following the manufacturer's instructions. Genomic DNA 601 concentration and purity was determined by NanoDrop spectrophotometry as above. Microbial 602 16S rRNA genes were amplified from soil DNA samples with barcoded universal prokaryotic 603 23 primers (515F/R806) targeting the V4 region, and then subjected to Illumina® MiSeq 604 sequencing (600-cycle, 2x300 bp) at the DNA Sequencing Facility, Department of Biochemistry, 605

A biofoundry workflow for the identification of genetic determinants of microbial growth inhibition

“…Prior analysis of the Pseudomonas sp. Ps652 genome determined that it features no BGCs for known antimicrobial compounds ( 10 ), with the exception of a hydrogen cyanide (HCN) BGC ( 17 ). However, in preliminary experiments (unpublished data), we determined that HCN production in Pseudomonas sp.…”

“…To develop biofoundry workflows for studying microbial interactions, we focused on a Pseudomonas strain for which previous investigations had not identified BGCs of known antimicrobials ( 10 , 24 ). The utility of our automated workflow was initially validated by growth curves and cross-streak assays ( Figure 3 ), and subsequently by the identification of a BGC highly likely to produce the tropolonoid antimicrobial 7-hydroxytropolone.…”

“…Ps652, recovered from a commercial potato field in Norfolk (UK). This isolate had previously been shown to display strong in vitro activity against Streptomyces scabies 87-22, a commercially relevant potato pathogen ( 9 , 10 ). The determinants of Pseudomonas sp.…”

Identification and Differentiation of Pseudomonas Species in Field Samples Using an rpoD Amplicon Sequencing Methodology

“…Also, some strains promote growth of plants by solubilizing inorganic nutrients, such as phosphate and iron, or by producing plant hormones ( 11 – 14 ). Members of the Pseudomonas fluorescens group, in particular, are a major source of bioactivity since they have markedly larger genomes than other pseudomonads ( 15 ) and a high number of biosynthetic gene clusters, as determined by genomic analysis ( 16 ). In addition to strains expressing these and other beneficial traits, it is also becoming clear that the structure and diversity of the Pseudomonas community in bulk and rhizosphere soils per se can be associated with suppression of crop fungal pathogens ( 17 , 18 ).…”

Identification and differentiation of Pseudomonas species in field samples using an rpoD amplicon sequencing methodology

“…Also, some strains promote growth of plants by solubilising inorganic nutrients such as phosphate and iron or by producing plant hormones (11)(12)(13)(14). Members of the Pseudomonas fluorescence group, in particular, are a major source of bioactivity since they have markedly larger genomes than other pseudomonads (15) and a high number of biosynthetic gene clusters as determined by genomic analysis (16). In addition to strains expressing these and other beneficial traits, it is also becoming clear that the structure and diversity of the Pseudomonas community in bulk and rhizosphere soils per se can be associated with suppression of crop fungal pathogens (17,18).…”