Summary Plant‐beneficial Pseudomonas spp. competitively colonize the rhizosphere and display plant‐growth promotion and/or disease‐suppression activities. Some strains within the P. fluorescens species complex produce phenazine derivatives, such as phenazine‐1‐carboxylic acid. These antimicrobial compounds are broadly inhibitory to numerous soil‐dwelling plant pathogens and play a role in the ecological competence of phenazine‐producing Pseudomonas spp. We assembled a collection encompassing 63 strains representative of the worldwide diversity of plant‐beneficial phenazine‐producing Pseudomonas spp. In this study, we report the sequencing of 58 complete genomes using PacBio RS II sequencing technology. Distributed among four subgroups within the P. fluorescens species complex, the diversity of our collection is reflected by the large pangenome which accounts for 25 413 protein‐coding genes. We identified genes and clusters encoding for numerous phytobeneficial traits, including antibiotics, siderophores and cyclic lipopeptides biosynthesis, some of which were previously unknown in these microorganisms. Finally, we gained insight into the evolutionary history of the phenazine biosynthetic operon. Given its diverse genomic context, it is likely that this operon was relocated several times during Pseudomonas evolution. Our findings acknowledge the tremendous diversity of plant‐beneficial phenazine‐producing Pseudomonas spp., paving the way for comparative analyses to identify new genetic determinants involved in biocontrol, plant‐growth promotion and rhizosphere competence.

The phenazine-1-carboxylic acid (PCA)-producing Pseudomonas fluorescens strain LBUM223 shows biocontrol potential against Streptomyces scabies, which causes common scab of potato. To better characterize the impact of inoculating this specific biocontrol agent under field conditions, the microbiomes of the rhizosphere and the geocaulosphere of potato plants were characterized using next-generation sequencing. A single initial application or biweekly applications of LBUM223 were performed up to 11 weeks after planting. Rhizosphere and geocaulosphere soils (when potato tubers were produced) were sampled every 2 weeks. Following soil DNA extractions, 16S rRNA gene amplification and sequencing were performed using the Illumina MiSeq technology. The QIIME pipeline was used for data analyses. Results were generated from 45 rhizosphere and 27 geocaulosphere samples, for which 63,502 and 44,469 different operational taxonomical units were observed. Diversity comparisons between both datasets were performed. To our knowledge, this is the first time that the geocaulosphere microbiome is characterized and compared with the rhizosphere microbiome following inoculation with a specific microorganism. Eleven phyla accounted for 95% of the diversity, with Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria being the most abundant ones. Overall, the results obtained suggest that P. fluorescens strain LBUM223 does not significantly alter the autochthonous rhizosphere nor geocaulosphere microbiomes.

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Plant-beneficial phenazine-producing Pseudomonas spp. are effective biocontrol agents, thanks to the broad-spectrum antibiotic activity of the phenazine antibiotics they produce. These bacteria have received considerable attention over the last 20 years, but most studies have focused only on the ability of a few genotypes to inhibit the growth of a limited number of plant pathogens.

Pseudomonas protegens Pf-5 is an effective biocontrol agent that protects many crops against pathogens, including the fungal pathogen Botrytis cinerea causing gray mold disease in Cannabis sativa crops. Previous studies have demonstrated the important role of antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DAPG) in Pf-5-mediated biocontrol. To assess the potential involvement of PLT and DAPG in the biocontrol exerted by Pf-5 against B. cinerea in the phyllosphere of C. sativa, two knockout Pf-5 mutants were generated by in-frame deletion of genes pltD or phlA, required for the synthesis of PLT or DAPG respectively, using a two-step allelic exchange method. Additionally, two complemented mutants were constructed by introducing a multicopy plasmid carrying the deleted gene into each deletion mutant. In vitro confrontation assays revealed that deletion mutant ∆pltD inhibited B. cinerea growth significantly less than wild-type Pf-5, supporting antifungal activity of PLT. However, deletion mutant ∆phlA inhibited mycelial growth significantly more than the wild-type, hypothetically due to a co-regulation of PLT and DAPG biosynthesis pathways. Both complemented mutants recovered in vitro inhibition levels similar to that of the wild-type. In subsequent growth chamber inoculation trials, characterization of gray mold disease symptoms on infected cannabis plants revealed that both ∆pltD and ∆phlA significantly lost a part of their biocontrol capabilities, achieving only 10 and 19% disease reduction respectively, compared to 40% achieved by inoculation with the wild-type. Finally, both complemented mutants recovered biocontrol capabilities in planta similar to that of the wild-type. These results indicate that intact biosynthesis pathways for production of PLT and DAPG are required for the optimal antagonistic activity of P. protegens Pf-5 against B. cinerea in the cannabis phyllosphere.