Phytobiomes Journal

Pyrroloquinoline quinone (PQQ) is known to promote plant growth, yet how the rhizosphere microbiome varies with PQQ concentration remains poorly understood. We applied metagenomic sequencing to characterize the taxonomic composition and functional potential of the tobacco rhizosphere microbiome across PQQ treatments at 0.5, 1 and 2 mol/L plus an untreated control. The rhizosphere microbiome was dominated by Pseudomonadota and Actinomycetota and differed among treatments in a concentration-associated, non-monotonic manner. The most pronounced differences were observed at 1 mol/L, where alpha diversity showed dimension-specific variation and beta-diversity analysis resolved the most distinct community profile. Taxonomic and functional profiles varied concurrently, with Streptomyces, Sphingomonas, Rhodanobacter and Lysobacter, together with functions related to biosynthesis, energy metabolism, colonization and environmental sensing, showing higher relative abundance at this concentration. No broad-spectrum antimicrobial effect was detected at the community level, and antibiotic resistance gene abundance showed no significant differences among treatments after correction for multiple testing. These findings provide a basis for further evaluation of PQQ in modulating the rhizosphere microbiome in continuous cropping systems.

Microbial community assembly on leaves (the phyllosphere) is influenced by deterministic, stochastic, and historical processes. However, the importance of historical processes such as priority effects, where the timing and order of colonization influence community assembly, has not been extensively investigated in field-based phyllosphere studies. We designed a field experiment in a temperate forest in Quebec where we inoculated five strains of Methylobacterium onto the leaves of red maple and American beech trees, either early or late during the growing season. We quantified microbial community structure throughout the season using metabarcoding of a Methylobacterium-targeting rpoB primer as well as a 16S rRNA primer targeting the entire bacterial community. We show that the effects of inoculation on bacterial communities depend on inoculation timing: early-season inoculation led to strain-specific shifts in community composition, while late-season inoculation had no significant effect. Although the effect of early-season inoculation weakened over time, early- and late-season inoculation still led to different community compositions at the end of the growing season. Our study demonstrates that priority effects contribute to the assembly of phyllosphere bacterial communities under field conditions, with arrival timing and strain identity shaping community trajectories. Considering historical processes will thus be crucial for successful microbiome management.

Abstract Background: Soil microbiomes play critical roles in plant health and resilience, yet it is still poorly understood how disruptions in local microbial communities, such as during soil management, impact these ecological functions. In this study, we investigated how compost addition, a widely used soil management practice, and microbiome origin influence microbiome-mediated plant resistance to insects. We combined surveys of working organic farms across New York with controlled laboratory experiments, amplicon sequencing, plant-insect bioassays, and mass spectrometry. Results: Compost use was associated with shifts in the relative abundance of 20 fungal and 2 bacterial genera across ten working farms. However, no consistent changes in bacterial or fungal diversity or community structure in fields with compost were observed across the farms. Under controlled conditions using a common compost source, compost increased rhizosphere microbial diversity and significantly altered rhizosphere community structure, including the relative abundance of 6 fungal and 41 bacterial genera. Despite these substantial community-level changes, compost did not affect populations of green peach aphids (Myzus persicae) on kale (Brassica oleracea). Instead, aphid populations and plant salicylic acid concentrations were more strongly associated with microbiome origin, demonstrating that compost can substantially restructure soil and rhizosphere microbial communities without corresponding changes in microbiome-mediated plant resistance to insects. Conclusions: Our findings demonstrate that microbial diversity and compositional responses to management are not necessarily reliable indicators of all microbiome functions. More broadly, this work highlights the importance of local soil microbiome variation in shaping plant-insect interactions and emphasizes the need to link community-level responses to direct functional measurements when evaluating microbiome responses to agricultural management.

Carbon farming practices are increasingly adopted as part of regenerative agriculture to enhance soil carbon sequestration and improve agricultural sustainability. However, their effects on the phyllosphere microbiome, and how these interact with field biodiversity, remain poorly understood. We investigated how field biodiversity and carbon farming practices (cover crops, all-in mixes, adaptive grazing and ley mixture) influence leaf microbial communities across 19 Finnish farms using ITS and 16S amplicon sequencing. Foliar fungal - but not bacterial - diversity increased with plant species richness. Carbon farming altered both fungal and bacterial community composition, but only fungal community was positively associated with disease load. Network analysis identified three candidate keystone taxa: Alternaria sp. (FASV 34) and Sporobolomyces roseus (FASV 2070), which were mutually exclusive, and Methylobacterium sp. (BASV 75), which co-occurred with FASV 2070. Together, these findings demonstrate that different carbon farming practices shape distinct leaf microbiomes cover crop systems tending to favor potential fungal pathogens, whereas ley mixture and adaptive grazing strategies promote microbial taxa associated with healthier phyllosphere communities. Our study underscores the importance of understanding the complex microbial interactions within leaf ecosystems to inform sustainable agricultural practices.

Background Symbiosis with microbes is a key strategy that has enabled plants to colonize extreme environments. Since the benefits conferred by root-associated microbes depend on both environmental conditions and host-microbe combinations, plant adaptation to harsh environments is closely linked to the assembly of root microbial communities. Understanding how environmental and host filtering jointly shape these communities is therefore fundamental to elucidating the mechanisms underlying plant adaptation to extreme environments. Results In this study, we investigated the differentiation of root-associated prokaryotic and fungal communities and individual operational taxonomic units (OTUs) across two contrasting habitats surrounding fumaroles, solfatara-field and forest-edge habitats, and six dominant Ericaceae and ectomycorrhizal plant taxa. Prokaryotic and fungal OTUs rarely exhibited strong preferences for both habitat and host identity. Instead, many of prokaryotic and fungal OTUs specialized to one of these niches, collectively generating root microbial communities differentiated by both factors. Nonetheless, striking specializations in habitat and host niches were observed in the fungal family Hyaloscyphaceae (Helotiales). To gain insight into the evolutionary basis of microbial specialization, we examined phylogenetic signals in preference phenotypes. The resulting weak phylogenetic signals in these preference phenotypes further suggest that this fungal clade has undergone substantial ecological divergence. Conclusion Overall, our findings indicate that root-associated microbial communities in extreme environments are assembled through the accumulation of microbial taxa specialized to either habitat or host, and that strong ecological specialization in fungi can arise with little phylogenetic constraint.

Stemflow transports water and biological material from forest canopies to the stem base, yet fungal studies of this flux have largely relied on microscopy of conidia. We used ITS2 metabarcoding to characterize fungal environmental DNA (eDNA) in stemflow from 15 trees - five each of American beech (Fagus grandifolia), sugar maple (Acer saccharum), and tulip poplar (Liriodendron tulipifera) in a closed-canopy forest in northeastern Ohio, USA. Stemflow was sampled during eight rain events in the leafless season (November 2024-May 2025), along with simulated bark and gutter washes to assess near-collar and collector signals. Natural stemflow communities were compositionally distinct from both controls and occupied greater ordination space. Ascomycota dominated across all samples, with Parafenestella, Cytospora, Microcera, Nectria, and Aureobasidium among the most abundant resolved genera and 13 of the 25 most abundant taxa differed significantly among host species. Woody-plant-associated endophytes, canker fungi, and saprobes dominated ecological guild composition. Storm event explained 28.6% of taxonomic variation, host tree species 24.1%, and their interaction 10.0%. Guild composition was more strongly structured by host species (35.4%) than storm event (20.4%). Lichenized fungi and lichen-associated fungi were detected. Thus, leafless-season stemflow integrates broad bark-associated fungal eDNA, including recurrent lichen-derived material, beyond the morphologically distinctive conidia captured by traditional methods. Stemflow eDNA therefore offers a minimally invasive means of monitoring canopy fungal diversity and the strong host and temporal filtering of DNA exported from woody surfaces.

Spontaneous vegetable fermentation is driven by the indigenous microbiota associated with the raw ingredients. The process often involves an initial heterofermentative phase followed by a predominantly homofermentative phase, both ensured by lactic acid bacteria (LAB). While microbial dynamics during fermentation are well characterized, phage communities remain poorly understood, despite evidence linking phage activity to LAB succession. We monitored viral and bacterial succession during sauerkraut and carrot fermentations using a metagenomics approach to characterize community dynamics. To further assess the impact of phage PLE001 on bacterial community dynamics, we developed a simplified carrot fermentation model comprising representative strains of Rahnella aquatilis, Leuconostoc mesenteroides and Lactiplantibacillus plantarum. Two consortia were assembled: one containing the PLE001-susceptible strain L. mesenteroides CIM3004, and the other containing also its phage-resistant derivative, CIM3004 BIM. We observed major shifts in the viral community during the first week of fermentation, coinciding with changes in bacterial structure during hetero- and homofermentative phases transition. In the simplified model, phage PLE001 reduced the population of the host during the heterofermentative phase but did not affect overall fermentation dynamics nor metabolite production. These results suggest that bacteriophages contribute to bacterial succession during fermentation, while communities remain resilient to phage infection, preserving fermentation stability.

Sinorhizobium meliloti is a nitrogen-fixing soil bacterium that forms a facultative mutualism with its host alfalfa. S. meliloti strains exhibit diverse colony morphologies on solid culture media that span a continuum from dry to mucoid. Studies on how this trait affects symbiosis rely on mutations in exopolysaccharide II production genes; however, whether natural variation in morphology confers a selective advantage on rhizobia in their natural habitats remains unknown. Here, we used 90 mucoid and 27 dry natural isolates to test whether colony morphology is associated with rhizobial fitness in the soil and in the host, or with the nitrogen-fixation benefits they provide to the plant. Colony morphologies were widely distributed across a phylogenetic tree, suggesting that morphological shifts occurred repeatedly and no single genetic variant was strongly associated with this trait. We inoculated soil mesocosms and plants with a mixture of dry and mucoid bacteria. We observed that dry rhizobia have higher fitness, particularly at 22{degrees}C and 32{degrees}C, but not under cold or drought conditions, independent of the initial rhizobial load. Conversely, mucoid bacteria have a modestly higher relative fitness in host nodules, and single inoculations showed that they tend to confer greater benefits to all three alfalfa varieties. Over time, we also observed rare, repeated conversion from mucoid to dry morphologies in soil and host plants. Taken together, our study reveals that morphological variation within a species in a single agricultural field is important for bacterial fitness in the soil and can have cascading effects on the rhizobia's plant benefits.

Pectobacterium, a member of the soft rot Pectobacteriaceae (SRP), can cause highly destructive soft rot and blackleg diseases on many important crop and other host plants, particularly potato (Solanum tuberosum). Pectobacterium and its sister genus Dickeya have common virulence strategies, yet a highly diverse "open" pangenome containing many unique and hypothetical proteins. Here, we used randomly-barcoded transposon insertion-site sequencing (RB-TnSeq) on six Pectobacterium species to identify genes involved in bacterial survival and growth in potato tubers, as well as in minimal media conditions containing different single carbon sources. The strains evaluated varied broadly in genomic content as well as host plant and year of isolation. We focused on identifying conditionally essential genes across all strains tested, as well as genes that were important for growth in one or few of the tested strains. These results highlight the importance of many common genes involved in metabolism and maintaining cellular homeostasis, as well as shared and rare virulence traits including polygalacturonate catabolism, genes involved in polysaccharide biosynthesis, immunity proteins, and (for P. brasiliense) self-tolerance of the antibiotic carbapenem. This large dataset of conditionally essential genes, generated across Pectobacterium species during soft rot infection of the agriculturally important plant host potato, can serve as a rich dataset for further hypothesis generation and identification of important and/or novel in planta fitness factors.

Heritable variation in microbiome composition could theoretically evolve in response to selection acting on hosts, but empirical evidence of this is scarce. We tested for effects of allelic variation in Boechera stricta on microbiome composition and downstream consequences for host fitness. In the greenhouse, near-isogenic lines with contrasting phytochemistry alleles differentially conditioned soil microbiota in ways that changed the relative fecundity of host genotypes. In the field, root microbiome differences mediated allelic effects on host fitness. Selection gradients on individual taxa and multivariate axes of root microbiome composition suggest that independent components of the bacterial community favor each allele, especially under drought. These results show that microbiota contribute to natural selection on a major-effect phytochemistry gene, empirically closing the loop between microbiome heritability and host trait evolution.

Microbial biological control agents (mBCAs) offer a promising alternative to conventional disease management, but identifying effective strains requires screening large microbial collections against many pathogens. Here, we developed a scalable screening approach to characterize the antagonistic activity of potato-associated bacteria against multiple pathogens and investigated how bacterial origin and phylogeny relate to antagonistic phenotypes. A collection of 600 bacterial strains isolated from two potato cultivars, three plant compartments using four cultivation media was screened against six major potato pathogens: the bacteria Dickeya solani and Pectobacterium carotovorum, the fungi Alternaria solani and Rhizoctonia solani, and the oomycetes Phytophthora infestans and Pythium ultimum. A scalable confrontation method was developed for non-filamentous bacteria, while complementary lower-throughput assays were used for filamentous strains. This approach enabled successful assessment of 92% of non-filamentous and 97% of filamentous strain-pathogen combinations. Isolation cultivar, plant compartment and cultivation medium had only limited effects on the proportion of antagonistic strains. In contrast, pathogen sensitivity varied markedly: P. infestans was the most susceptible pathogen, whereas D. solani and P. carotovorum were generally resistant to bacterial antagonism. Antagonistic activity clustered phylogenetically, with Bacillus and Streptomyces containing many broad-spectrum inhibitors. In addition, several taxa showed preferential activity against the oomycetes, including P. infestans-specific antagonists from the Pseudomonas genus and P. ultimum-specific antagonists from the Frigoribacterium, Curtobacterium, Pedobacter and Phyllobacterium genera. Overall, multi-pathogen screening revealed distinct generalist and specialist antagonistic profiles and identified candidate strains and taxa for further evaluation as mBCAs.

Soil nematodes play key roles in regulating organic matter decomposition, nutrient cycling and trophic interactions in the soil ecosystem, and are very useful bioindicators of soil condition and soil health. But to characterise them, we need reliable measurements of nematode biodiversity in soils. DNA-based techniques such as metabarcoding have recently explored their biodiversity, with successful results. However, there are many methodological steps that can modify the outcome considerably and require informed decisions. In this study, we tested the effect on nematode alpha and beta diversity metrics of three methodological steps, namely i) type of extraction of genetic material from the matrix, either from soil directly with different starting amount of material or extracting nematodes first via the Baermann or Oostenbrink methods, ii) the choice of taxonomically-relevant DNA segment for amplification, with a nematode specific primer (NemF/18Sr2b) and a universal eukaryotic primer (3NDf/1132rmod), both within 18S rDNA, and iii) the selection of appropriate reference library for taxonomic assignment (comparing three nematode specialised databases and three broad taxonomic databases). Observed differences in alpha diversity were more pronounced when comparing the nematode extraction vs soil extractions. In particular, direct soil extractions co-extracted many non-nematode taxa, especially if amplified with a universal primer, which can significantly reduce the number of nematode reads obtained and therefore the accuracy of nematode community indexes and other nematode-based metrics. The choice of the reference database strongly affected the taxonomic identification, which calls for extreme caution when making this selection. Notably, specialised databases tended to over assign reads to nematodes that were detected as non-nematodes by broad taxonomic databases. However, beta diversity metrics were consistent across methods, highlighting these metrics as robust against methodological choices. Our study clearly demonstrates that the choices of methods for nematode biodiversity studies using molecular tools can have a big impact on the results, and decisions should be made consciously and carefully.

The Fusarium oxysporum species complex (FOSC) comprises numerous plant pathogens infecting crops such as tomato, lettuce, melon, and banana. Despite its economic and ecological importance and decades of research, the species names linked to different phylogenetic lineages within this species complex remain largely controversial. Earlier taxonomic frameworks relied on a limited number of marker genes, variable taxon sampling and inconsistent species definitions, leading to a fragmented and unstable taxonomy. To address these challenges, we here analysed nearly 2,500 conserved marker genes from 660 genomes, including genome assemblies from 22 ex-type strains that were sequenced using long-read sequencing technology, representing nearly half of the 51 currently described species within FOSC. Maximum likelihood- and coalescence-based analyses of the full phylogenomic data matrix as well as subsets yielded a robust phylogeny of FOSC, which was furthermore corroborated by whole genome comparisons. Many previously described species are redundant, while two novel lineages remain undescribed. Direct comparisons to traditional marker gene-based approaches demonstrate their inadequacy for species delimitation in FOSC. We show that our phylogenomic framework can guide the detection of novel marker genes able to distinguish the major lineages within FOSC, which will be essential for taxon delineation and classification in future studies. Thus, our phylogenomic analyses provide a stable cornerstone for revising FOSC taxonomy and offers a critical resource for the broader research community.

The growth and performance of cattle is dependent on efficient feed conversion, facilitated by the gut microbiome, including both prokaryotic and eukaryotic organisms. Extrinsic factors such as management practices and intrinsic factors like age and genetics can shape these communities. To characterize the relative effects of such factors on the prokaryotic and eukaryotic microbiomes, fresh fecal samples from dairy heifers (n = 8), beef heifers (n = 8), bred beef heifers (n = 8), and adult beef cows (n = 8) were subjected to paired 16S rRNA (V4) and a novel 18S rRNA (V4-V5) metabarcoding assay, and 18S rRNA amplicons were annotated using two databases in parallel. Taxonomic resolution of 18S rRNA amplicons was excellent, with SILVA and EUKARYOME databases each providing better resolution of different groups of organisms. While no differences in eukaryotic richness or alpha diversity were detected, beta diversity differed in all pairwise comparisons except between beef heifers and bred beef heifers. Prokaryotic richness was lower in adult beef cows than the other groups, and beta diversity differed in all pairwise comparisons. Adult beef cows were the most distinct, characterized by replacement of fungal organisms with protozoa and loss of thermophilic bacteria in the eukaryotic and prokaryotic compartments, respectively. Among numerous differentially abundant features, dairy heifers harbored greater abundance of Cooperia oncophora while beef heifers contained greater abundance of Buxtonella sulcata and Mucor. Correlation network analysis revealed multiple significant positive and negative associations between bacterial, fungal, and protozoal features during maturation of the fecal microbiome. Collectively, these data highlight the richness and diversity of both the prokaryotic and eukaryotic compartments of the bovine fecal microbiome, and the influence of management practices and age on both communities. From a technical standpoint, these data demonstrate the ability to resolve fungal, protozoal, and metazoan organisms, including potential parasites and pathogens, with a single metabarcoding assay. They also highlight the nuanced differences between databases and the value of parallel analyses using more than one database.

Engineering bioenergy crops for enhanced biomass digestibility and high-value co-products is a promising approach to sustainable biofuel production. However, the impact of these engineered crops on the belowground microbiome remains poorly understood. In this study, we characterized the composition and functional responses of the belowground microbiome associated with transgenic sorghum lines: one with reduced cell wall crosslinking (AT10) and another with elevated protocatechuic acid (PCA) production (Qsub). The lines were grown under field conditions with four irrigation levels, and samples were collected twice during the growing season. We found that the two transgenic types drove a subtle but significant divergence in bacterial communities, both compositionally and functionally. Community-wide functional profiling showed that AT10 rhizospheres were consistently depleted in genes associated with mobile genetic elements, while Qsub rhizospheres were enriched in TonB-dependent transport and vanillate monooxygenase, an enzyme that funnels lignin-derived monomers toward PCA degradation, alongside reduced pectin- and cellulose-degradation genes. Genome-resolved analysis linked these community-level shifts to specific taxa: in Qsub, the enriched MAGs were taxonomically diverse, ranging from genome-reduced Patescibacteria with an episymbiotic lifestyle to taxa enriched in lignin-associated auxiliary activity enzymes. In AT10, the depleted Azospirillaceae MAGs carried unusually high mobile genetic element content, consistent with the community-wide pattern. Collectively, these findings offer new insights into how genetic modifications aimed at improving bioenergy traits shapes the composition and function of the rhizosphere microbiome, with potential downstream consequences for soil carbon cycling.

Diverse ecological communities can reduce pathogen transmission; a phenomenon known as the dilution effect of biodiversity. This effect, in turn, depends on variation in host species' ability to spread disease (i.e., host competence) and which host species persist under biodiversity loss. Mutualistic symbionts can influence host competence and are sensitive to variation in host biodiversity; yet whether mutualists can modify dilution effects is unknown, partially because quantifying host competence during epidemics remains challenging. We experimentally tested whether mutualists could modify dilution effects by establishing gradients of host species richness, community competence, and access to the below-ground arbuscular mycorrhizal fungus (AMF), Rhizoglomus irregulare. Plant hosts were then exposed to the fungal root pathogen, Rhizoctonia solani, and disease spread was monitored over the course of an epidemic. Overall, host richness did not affect epidemic outcomes. Instead, AMF inoculation increased disease risk via species-specific increases in host competence. Moreover, communities containing highly competent host species experienced more disease, but only in the presence of mutualistic AMF. These results underscore the importance of plant community composition in mycorrhiza-mediated disease dynamics and suggest that predicting disease in our changing world might require a deeper understanding of host-symbiont and pathogen-symbiont interactions under global change.

An increasing number of researchers, educators, and at-home growers are turning to hydroponics as an alternative method of food production, diversifying and strengthening the food supply chain. This has led to a substantial increase in the amount of academic work involving hydroponic production of plants for a variety of purposes, including but not limited to a more controlled and customizable environment than conventional production methods, utilizing hydroponics as a method of wastewater treatment, and investigating the vertical production of plants on a much smaller footprint than traditional commercial-scale production. Because so many unique stakeholders are interested in hydroponics for a variety of reasons, academic studies use a wide variety of hydroponic systems with varying levels of technology and automation, making comparisons across studies and systems challenging. A few institutions have sophisticated plant growth and research facilities, like those used by commercial companies, but these systems are often prohibitively expensive, costing upwards of $10,000 USD for a very limited amount of growing space for experimental trials. To enable more researchers, educators, and citizen scientists to conduct experiments and easily compare their results, this study details three pilot-scale hydroponic production systems (Kratky, NFT, and DWC), utilizing entirely off-the-shelf components, for under $5,000 USD each, that are capable of maintaining the desired consistent temperature and relative humidity levels, and have space for 216, 108 (6 irrigation loops), and 64 (16 irrigation bins) plants, respectively. The material lists and prices are provided along with assembly instructions and general guidelines for each hydroponic system. The testing and optimization process of environmental conditions within each growth system is also detailed, with suggested customizations for potential experimental setups. The authors developed these systems during their Masters and Doctoral degree programs, enabling improved hydroponic research at the University of Illinois Urbana-Champaign. They hope that other aspiring hydroponic researchers can benefit from this work in the development and operation of their hydroponic systems, increasing research efficiency and collaboration opportunities in this field of study.

Cucurbit yellow vine disease (CYVD) is a phloem disease caused by a group of Serratia ureilytica bacteria within the S. marcescens complex. The CYVD pathogen is distinct from other phloem pathogens in two fundamental ways: i) unlike other bacterial phloem pathogens, the CYVD pathogen readily grows on standard laboratory media; and ii) whereas most bacterial phloem diseases are transmitted solely by hemipteran insects, CYVD can be transmitted by both hemipteran (squash bug) and non-hemipteran (cucumber beetle) vectors. The phloem of cucurbits is also distinct, with cucurbits having a unique secondary phloem system, the extrafascicular phloem (EFP), for which a role in CYVD has not been investigated. The distinctive features of this pathosystem prompted a re-evaluation of pathogen localization in planta using a green fluorescent protein-tagged CYVD strain during infection of squash (Cucurbita pepo). In epifluorescence and confocal microscopy images of transverse and longitudinal sections of stem and petiole tissues, the fluorescence patterns of the CYVD pathogen were consistent with pathogen colonization of all four known EFP tissues, namely the peripheral, entocyclic, ectocyclic, and commissural sieve tubes. The CYVD pathogen may enter the EFP during herbivory by pathogen-infected vectors that cause mechanical damage, as the EFP exhibits much slower blockage following injury than the conventional fascicular (bundle) phloem. Moreover, although EFP has been proposed to have an antimicrobial function, EFP exudate collected from the stem and petioles of C. pepo supported rapid pathogen growth. These results provide evidence for a unique tissue tropism of the newly emerged plant pathogen S. ureilytica.

Soil bacteria play a crucial role in supporting ecosystem health, yet little is known about how wild populations adapt to abiotic stressors, like limited water availability. We used 186 Luteibacter isolates from maize roots grown in never-irrigated prairie soils collected from a steep precipitation gradient in Kansas, US to test whether climate history is associated with osmotic stress tolerance and genomic differentiation. In vitro, strains from semi-arid sites were more tolerant to osmotic-stress treatment than those from humid sites. Comparative genomics of a subset of 96 isolates identified four strongly biogeographically structured lineages and extensive accessory-genome diversity. Semi-arid climate-associated strains were enriched for genes involved in osmotic homeostasis, oxidative stress protection, motility, and type IV secretion. Humid climate-associated strains were enriched in environmental sensing, metabolic versatility, biofilm formation, iron acquisition, type VI secretion, and resource competition. GWAS identified 22 candidate genes associated with osmotic-stress tolerance, including those related to type IV pili, biofilm production, DNA repair, and phage. No candidates were shared between semi-arid and humid strains, suggesting distinct genetic mechanisms of osmotic tolerance. Predicted prophage composition was also geographically structured, although total prophage gene content did not predict osmotic-stress tolerance. Together, these results show that adaptation to long-term differences in water availability likely involves coordinated shifts in stress physiology, genome content, and ecological interactions in wild bacterial populations, providing insight into predicting microbial responses to changing climates.

Several pathogenic viruses benefit plants under specific circumstances, e.g., by inducing drought resistance. It is hypothesised that RNA viruses in the Endornaviridae and Partitiviridae families, sometimes called persistent since they are exclusively seed-transmitted at ~100% efficiency, have followed evolutionary trajectories towards mutualism. However, evidence comes from crossing non-identical host backgrounds making it impossible to distinguish definitively between the effects of viral versus host genotypes. We used virus-induced gene silencing (VIGS) to cure pepper plants of bell pepper endornavirus (BPEV) and the partitiviruses pepper cryptic virus 1 (PCV1) and PCV2 and compared these with genetically identical plants retaining these viruses. We found that PCV1, PCV2 and BPEV can significantly affect aerial and root growth and fruit/seed characteristics in ways that could benefit host fitness. VIGS-mediated curing allows unambiguous hypothesis-testing of potential benefits of persistent viruses and exploration of how viral and host genotypes shape persistent virus-host relationships.