Tecia solanivora (The Guatemalan potato tuber moth) is a major potato pest, responsible for up to 20% of crop losses and a significant economic impact. Certain Pseudomonas exhibit insecticidal activity and produce virulence factors with cytotoxic and antimicrobial properties, positioning them as promising candidates for biological control. This study evaluated seven Pseudomonas strains with insecticidal activity and identified key virulence factors involved. The strains demonstrated varying degrees of insecticidal activity, with Pseudomonas protegens strains CHA0 and 59C being the most lethal, causing over 75% mortality and triggering a systemic melanization response in the insects. Genomic analysis revealed 175 virulence-related genes shared across all strains and 16 genes specific to the highly insecticidal ones, including genes for antimicrobial compounds and insect toxins. Mutational analysis confirmed the roles of hydrogen cyanide, 2,4-diacetylphloroglucinol, pyoluteorin, Fit toxin, and two-partner secretion systems in P. protegens CHA0 insecticidal activity. This strain also exhibited insecticidal effects on adult T. solanivora and delayed egg hatching and pupal emergence. In microcosm assays, P. protegens CHA0 reduced tuber damage caused by T. solanivora larvae by up to 38%. These results suggest that P. protegens CHA0 is a promising biocontrol agent, providing a sustainable alternative to chemical pesticides to control T. solanivora.

In disease-suppressive soils, the rhizosphere microbiota protects plants from root disease(s). However, the soil microbiome follows distinct spatial patterns, and the biogeographic factors shaping plant–microbe interactions and soil suppressiveness remain poorly understood. Here, we use Swiss and Savoie soils suppressive or conducive to Thielaviopsis basicola-mediated black root rot of tobacco, to test the hypothesis that plant–microbe interactions and suppressiveness are influenced by both the geological origin and geographic positioning of soils. Soils are compared based on tobacco health, soil physicochemistry and organic matter profiles, taxonomic and functional microbial diversity, and plant physiological responses. Soil physicochemistry and metabolomic profiling of soil organic matter show differences based on suppressiveness status, soil geology and geography. The taxonomic (metabarcoding of prokaryotes and fungi) and functional (metagenomics) diversity of the tobacco rhizosphere reveals that the microbiota is influenced by geography and geology which, in turn, affects suppressiveness. Additionally, shoot metabolomics shows that tobacco responses are impacted by soil geography and geology, particularly in Savoie soils regarding two nicotinic derivatives. Overall, suppressiveness is influenced by both the geological origin and geographic positioning of the soils, with distinct patterns in the two regions. In Swiss soils, suppressiveness is primarily associated with major differences in rhizosphere microbiota composition and functions between suppressive and conducive soils. In contrast, in Savoie soils, suppressiveness is linked to distinct plant physiological responses (pointing to induced systemic resistance) rather than strong microbial shifts. This study highlights the importance of considering the biogeographic features shaping disease-suppressive soils and their microbiota-plant interactions.

Since the 1980s, soils in a 22-km2 area near Lake Neuchâtel in Switzerland have been recognized for their innate ability to suppress the black root rot plant disease caused by the fungal pathogen Thielaviopsis basicola. However, the efficacy of natural disease suppressive soils against insect pests has not been studied. We demonstrate that natural soil suppressiveness also protects plants from the leaf-feeding pest insect Oulema melanopus. Plants grown in the most suppressive soil have a reduced stress response to Oulema feeding, reflected by dampened levels of herbivore defense-related phytohormones and benzoxazinoids. Enhanced salicylate levels in insect-free plants indicate defense-priming operating in this soil. The rhizosphere microbiome of suppressive soils contained a higher proportion of plant-beneficial bacteria, coinciding with their microbiome networks being highly tolerant to the destabilizing impact of insect exposure observed in the rhizosphere of plants grown in the conducive soils. We suggest that presence of plant-beneficial bacteria in the suppressive soils along with priming, conferred plant resistance to the insect pest, manifesting also in the onset of insect microbiome dysbiosis by the displacement of the insect endosymbionts. Our results show that an intricate soil–plant-insect feedback, relying on a stress tolerant microbiome network with the presence of plant-beneficial bacteria and plant priming, extends natural soil suppressiveness from soilborne diseases to insect pests.

Since the 1980s, soils in a 22-km2 area near Lake Neuchâtel in Switzerland have been recognized for their innate ability to suppress the black root rot plant disease. Their efficacy against insect pests has not been studied. We demonstrate that natural soil suppressiveness also protects plants from the leaf-feeding pest insect Oulema melanopus . Plants grown in the most suppressive soil have a reduced stress response to Oulema feeding, reflected by dampened levels of herbivore defense-related phytohormones and benzoxazinoids, and enhanced salicylate levels in plants without the insect indicate defense-priming. The rhizosphere microbiome network of the suppressive soils was highly tolerant to the destabilizing impact of insect exposure. The presence of plant-beneficial bacteria in the suppressive soils along with priming conferred plant resistance to the insect pest, manifesting also in the onset of insect microbiome dysbiosis. This intricate soil-plant-insect feedback extends natural soil suppressiveness from soilborne diseases to insect pests.### Competing Interest StatementThe authors have declared no competing interest.

Strains belonging to the Pseudomonas protegens phylogenomic subgroup have long been known for their beneficial association with plant roots, notably antagonising soilborne phytopathogens. Interestingly, they can also infect and kill pest insects, emphasising their interest as biocontrol agents. In the present study, we used all available Pseudomonas genomes to reassess the phylogeny of this subgroup. Clustering analysis revealed the presence of 12 distinct species, many of which were previously unknown. The differences between these species also extend to the phenotypic level. Most of the species were able to antagonise two soilborne phytopathogens, Fusarium graminearum and Pythium ultimum, and to kill the plant pest insect Pieris brassicae in feeding and systemic infection assays. However, four strains failed to do so, likely as a consequence of adaptation to particular niches. The absence of the insecticidal Fit toxin explained the non-pathogenic behaviour of the four strains towards Pieris brassicae. Further analyses of the Fit toxin genomic island evidence that the loss of this toxin is related to non-insecticidal niche specialisation. This work expands the knowledge on the growing Pseudomonas protegens subgroup and suggests that loss of phytopathogen inhibition and pest insect killing abilities in some of these bacteria may be linked to species diversification processes involving adaptation to particular niches. Our work sheds light on the important ecological consequences of gain and loss dynamics for functions involved in pathogenic host interactions of environmental bacteria.

Strains belonging to the Pseudomonas protegens and Pseudomonas chlororaphis species are able to control soilborne plant pathogens and to kill pest insects by producing virulence factors such as toxins, chitinases, antimicrobials or two-partner secretion systems. Most insecticidal Pseudomonas described so far were isolated from roots or soil. It is unknown whether these bacteria naturally occur in arthropods and how they interact with them. Therefore, we isolated P. protegens and P. chlororaphis from various healthy insects and myriapods, roots and soil collected in an agricultural field and a neighbouring grassland. The isolates were compared for insect killing, pathogen suppression and host colonization abilities. Our results indicate that neither the origin of isolation nor the phylogenetic position mirror the degree of insecticidal activity. Pseudomonas protegens strains appeared homogeneous regarding phylogeny, biocontrol and insecticidal capabilities, whereas P. chlororaphis strains were phylogenetically and phenotypically more heterogenous. A phenotypic and genomic analysis of five closely related P. chlororaphis isolates displaying varying levels of insecticidal activity revealed variations in genes encoding insecticidal factors that may account for the reduced insecticidal activity of certain isolates. Our findings point towards an adaption to insects within closely related pseudomonads and contribute to understand the ecology of insecticidal Pseudomonas.

Pseudomonas protegens shows a high degree of lifestyle plasticity since it can establish both plant-beneficial and insect-pathogenic interactions. While P. protegens protects plants against soilborne pathogens, it can also invade insects when orally ingested leading to the death of susceptible pest insects. The mechanism whereby pseudomonads effectively switch between lifestyles, plant-beneficial or insecticidal, and the specific factors enabling plant or insect colonization are poorly understood. We generated a large-scale transcriptomics dataset of the model P. protegens strain CHA0 which includes data from the colonization of wheat roots, the gut of Plutella xylostella after oral uptake and the Galleria mellonella hemolymph after injection. We identified extensive plasticity in transcriptomic profiles depending on the environment and specific factors associated to different hosts or different stages of insect infection. Specifically, motor-activity and Reb toxin-related genes were highly expressed on wheat roots but showed low expression within insects, while certain antimicrobial compounds (pyoluteorin), exoenzymes (a chitinase and a polyphosphate kinase), and a transposase exhibited insect-specific expression. We further identified two-partner secretion systems as novel factors contributing to pest insect invasion. Finally, we use genus-wide comparative genomics to retrace the evolutionary origins of cross-kingdom colonization.

Minor differences in the previously obtained genome of Pseudomonas protegens CHA0 were detected after resequencing the strain. Based on this, the genome size slightly increased. Additionally, we performed a manual annotation of genes involved in biocontrol and insect pathogenicity. This annotation version will be the basis for upcoming genome studies.

The discovery of insecticidal activity in root-colonizing pseudomonads, best-known for their plant-beneficial effects, raised fundamental questions about the ecological relevance of insects as alternative hosts for these bacteria. Since soil bacteria are limited in their inherent abilities of dispersal, insects as vectors might be welcome vehicles to overcome large distances. Here, we report on the transmission of the root-colonizing, plant-beneficial and insecticidal bacterium Pseudomonas protegens CHA0 from root to root by the cabbage root fly, Delia radicum. Following ingestion by root-feeding D. radicum larvae, CHA0 persisted inside the insect until the pupal and adult stages. The emerging flies were then able to transmit CHA0 to a new plant host initiating bacterial colonization of the roots. CHA0 did not reduce root damages caused by D. radicum and had only small effects on Delia development suggesting a rather commensal than pathogenic relationship. Interestingly, when the bacterium was fed to two highly susceptible lepidopteran species, most of the insects died, but CHA0 could persist throughout different life stages in surviving individuals. In summary, this study investigated for the first time the interaction of P. protegens CHA0 and related strains with an insect present in their rhizosphere habitat. Our results suggest that plant-colonizing pseudomonads have different strategies for interaction with insects. They either cause lethal infections and use insects as food source or they live inside insect hosts without causing obvious damages and might use insects as vectors for dispersal, which implies a greater ecological versatility of these bacteria than previously thought.

Particular groups of plant-beneficial fluorescent pseudomonads are not only root colonizers that provide plant disease suppression, but in addition are able to infect and kill insect larvae. The mechanisms by which the bacteria manage to infest this alternative host, to overcome its immune system, and to ultimately kill the insect are still largely unknown. However, the investigation of the few virulence factors discovered so far, points to a highly multifactorial nature of insecticidal activity. Antimicrobial compounds produced by fluorescent pseudomonads are effective weapons against a vast diversity of organisms such as fungi, oomycetes, nematodes and protozoa. Here, we investigated whether these compounds also contribute to insecticidal activity. We tested mutants of the highly insecticidal strains Pseudomonas protegens CHA0, Pseudomonas chlororaphis PCL1391, and Pseudomonas sp. CMR12a, defective for individual or multiple antimicrobial compounds, for injectable and oral activity against lepidopteran insect larvae. Moreover, we studied expression of biosynthesis genes for these antimicrobial compounds for the first time in insects. Our survey revealed that hydrogen cyanide and different types of cyclic lipopeptides contribute to insecticidal activity. Hydrogen cyanide was essential to full virulence of CHA0 and PCL1391 directly injected into the hemolymph. The cyclic lipopeptide orfamide produced by CHA0 and CMR12a was mainly important in oral infections. Mutants of CMR12a and PCL1391 impaired in the production of the cyclic lipopeptides sessilin and clp1391, respectively, showed reduced virulence in injection and feeding experiments. Although virulence of mutants lacking one or several of the other antimicrobial compounds, i.e. 2,4-diacetylphloroglucinol, phenazines, pyrrolnitrin, or pyoluteorin, was not reduced, these metabolites might still play a role in an insect background since all investigated biosynthetic genes for antimicrobial compounds of strain CHA0 were expressed at some point during insect infection. In summary, our study identified new factors contributing to insecticidal activity and extends the diverse functions of antimicrobial compounds produced by fluorescent pseudomonads from the plant environment to the insect host.

BACKGROUND:AmrZ, a RHH transcriptional regulator, regulates motility and alginate production in pseudomonads. Expression of amrZ depends on the environmental stress sigma factor AlgU. amrZ and algU mutants have been shown to be impaired in environmental fitness in different pseudomonads with different lifestyles. Considering the importance of AmrZ for the ecological fitness of pseudomonads and taking advantage of the full sequencing and annotation of the Pseudomonas fluorescens F113 genome, we have carried out a ChIP-seq analysis from a pool of eight independent ChIP assays in order to determine the AmrZ binding sites and its implication in the regulation of genes involved in environmental adaption.RESULTS:154 enriched regions (AmrZ binding sites) were detected in this analysis, being 76% of them located in putative promoter regions. 18 of these peaks were validated in an independent ChIP assay by qPCR. The 154 peaks were assigned to genes involved in several functional classes such as motility and chemotaxis, iron homeostasis, and signal transduction and transcriptional regulators, including genes encoding proteins implicated in the turn-over of c-diGMP. A putative AmrZ binding site was also observed by aligning the 154 regions with the MEME software. This motif was present in 75% of the peaks and was similar to that described in the amrZ and algD promoters in P. aeruginosa. We have analyzed the role of AmrZ in the regulation of iron uptake genes, to find that AmrZ represses their expression under iron limiting conditions.CONCLUSIONS:The results presented here show that AmrZ is an important global transcriptional regulator involved in environmental sensing and adaption. It is also a new partner in the complex iron homeostasis regulation.