Phytobiomes Journal

BackgroundMicrobial communities in the rhizosphere are key drivers of plant immunity, mediating plant responses to stress. Under specific stresses plants are capable of recruiting beneficial microorganisms into their rhizosphere with the potential to alleviate these stresses. Among these stresses, herbivorous pests remain a major agricultural challenge. Despite this, the impact of leaf herbivory on root-associated microbiomes, and how this impact can shape plant defense phenotypes are still understudied. In this study, our main objective was to determine the extent to which leaf herbivory affects the rhizosphere microbiome, and whether and how these herbivory-induced changes modulate plant defense phenotypes through plant-soil feedback. To that end, we designed a two-phase assay in which we challenged sunflower (Helianthus annuus L.) with Spodoptera exigua and later tested the effect of the microbial legacy after infestation on sunflower defense phenotype, considering resistance and tolerance as major drivers. ResultsWe found that herbivory triggered significant changes in the bacteriome structure and dynamics, and microbiome functional profile, while effects on mycobiome were comparatively less pronounced. Under herbivory, several bacterial taxa and functional groups were enriched, the bacterial co-occurrence network was more complex and assembly processes were slightly more stochastic. Furthermore, after evaluating the plant-soil feedbacks of herbivory-induced microbiomes we observed no effect on plant resistance proxies such as herbivore growth and survival, and leaf phenolic and flavonoid content. We did observe differences on tolerance proxies, while plants grown on herbivore-challenged microbiome were overall smaller, the biomass loss to herbivory was significantly lower while the elemental nutrient content and photosynthetic pigments content was enhanced. ConclusionsOur study demonstrates that insect herbivory by S.exigua reshapes sunflower rhizosphere microbiome and generates a soil legacy that promotes herbivory tolerance on subsequent plant generations. This highlights the broader potential of microbiome-mediated plant-soil feedbacks in shaping plant adaptation to herbivory.

BackgroundPotato is an important crop worldwide, yet its production is severely threatened by Phytophthora infestans, the causal agent of late blight. Alternatives to the current control strategies are needed, as these rely heavily on environmentally harmful treatments. The recruitment of beneficial microbes by plants upon stress ("cry-for-help" mechanism) may represent an opportunity to find new biocontrol agents but this has not yet been reported for potato. The aim of this study was to analyse whether foliar late blight infection induces shifts in the phyllosphere, rhizosphere and soil bacterial communities associated with two potato cultivars of differing sensitivity to late blight. Moreover, we aimed at isolating members of the plant microbiota to test whether bacteria putatively recruited upon infection would be particularly active in protecting the plant against late blight. ResultsControlled foliar infection triggered substantial, cultivar-specific shifts in the rhizosphere communities across two successive generations. Despite the number of differentially abundant ASVs detected being ten times higher in the second generation than in the first one, the same taxonomic groups were concerned by the shifts: Burkholderiales, Flavobacteriales, and Bacillales. Furthermore, the communities linked to the susceptible cultivar consistently shifted more strongly than the communities linked to the resistant cultivar. The obtained ASV sequences were used to identify 163 corresponding isolates. The inhibition potential of these strains against P. infestans spores was assessed through biological assays, which revealed the biocontrol potential of strains otherwise not yet known to inhibit phytopathogenic organisms, such as Advenella, Nocardioides and Phyllobacterium strains. Although we found no correlation between the relative abundance shift of the ASVs upon infection and the activity of the corresponding strains, we observed that the overall activity of strains isolated from the resistant cultivar was higher than that of the strains isolated from the susceptible one. ConclusionTaken together, the higher activity of the strains isolated from the resistant cultivar, along with its comparatively modest microbiome shifts upon infection suggest that the investigated resistant cultivar might harbour specific microbiota enriched in strains with efficient protective abilities against their host plants pathogens, which possibly contribute to its higher resistance against P. infestans.

Endophytic bacteria are increasingly recognised for their roles in plant health through symbiosis. However, methodological challenges, such as inconsistent root sterilisation, inefficient microbial DNA extraction, and co-amplification of plant organellar DNA, limit accurate characterisation of these communities, especially in wild grassland plants and non model plant in general. To address this, we developed and tested a streamlined protocol for bacterial endophyte detection from wild grassland plant roots, encompassing surface sterilisation of roots, DNA extraction, clamping of plant internal mitochondrial and chloroplast DNA, and 16S rRNA amplicon sequencing. Our approach minimises plant DNA contamination and yields high-quality microbial profiles. The protocol is adaptable and specific to grassland plant species, offering a standardised foundation for endophyte studies in wild and non-model plants. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=141 HEIGHT=200 SRC="FIGDIR/small/706108v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@157df36org.highwire.dtl.DTLVardef@1ff645aorg.highwire.dtl.DTLVardef@1580ecorg.highwire.dtl.DTLVardef@1c31b89_HPS_FORMAT_FIGEXP M_FIG C_FIG (Haskins and Ajaz, 2026) https://BioRender.com/47gd2xr

Controlled environment agriculture (CEA), including soilless farming systems, is rapidly expanding as a strategy to improve food security and resource efficiency. However, limited information is available on how different soilless farming system designs influence microbial populations relevant to plant health and food safety. This study investigated the effects of soilless growing systems and growing season on aerobic plate counts (APC) and bacterial community composition in nutrient solution and on bok choy (Brassica rapa subsp. chinensis) leaves. Five soilless systems, deep water culture (DWC), Kratky (KR), nutrient film technique (NFT), ebb and flow (EF), and drip irrigation (DI), were evaluated across fall and spring growing seasons. Soilless system type significantly influenced APC in nutrient solution, with the DI system consistently exhibiting the highest counts across both seasons. Increased nutrient solution pH was negatively associated with APC, whereas temperature did not significantly affect bacterial concentrations. In contrast, APC on bok choy leaves were not significantly influenced by system type, season, pH, or temperature. Bacterial community composition in nutrient solution was strongly shaped by season, soilless system type, sampling day, and temperature, as determined by 16S rRNA V4 amplicon sequencing. Microbial diversity varied primarily by system type, with limited influence of pH or temperature. Core microbiota analysis identified a small subset of taxa that persisted across systems and seasons, with Acidovorax detected in all samples. We found that soilless system design and seasonal conditions strongly influence microbial load and community structure in nutrient solution, providing a foundation for developing system-specific microbial management strategies. ImportanceUnderstanding factors that shape microbial community composition in soilless farming systems is critical for optimizing plant health, system productivity, and food safety. Microbial communities influence nutrient cycling, biofilm formation, and pathogen survival, all of which affect the ecological stability and performance of these systems. By identifying how system design, seasonal variation, and environmental conditions influence shifts in microbial populations, targeted strategies can be developed to promote beneficial microorganisms and mitigate risks associated with pathogens. This knowledge contributes to advancing safe and sustainable soilless farming practices that can meet the growing demand for fresh produce grown in controlled environments.

BackgroundThe root microbiome plays a critical role in nutrient acquisition, stress tolerance and overall plant health. Rice, a staple food for more than half of the worlds population, is commonly cultivated under flooded conditions. Despite its agronomical importance, our current understanding of root-associated microbiomes in rice grown under flooded conditions is limited. On the other hand, nitrogen (N) and phosphorus (P) fertilizers are routinely applied to maximize rice yield. It is also well known that root colonization by arbuscular mycorrhizal (AM) fungi enhances mineral nutrition in plants, but whether mycorrhizal associations influence the composition of the rice root microbiome remains poorly understood. In this study, shotgun metagenomic sequencing was used to characterize the root endosphere and rhizosphere microbiomes in two temperate japonica rice varieties (cv. Bomba and JSendra) grown under flooded conditions. The impact of colonization by the AM fungus Rhizophagus irregularis on the root microbiome was investigated. ResultsRoot-associated compartments harbour distinct microbial communities in rice with bacterial taxa comprising approximately 95% of the total microbia in rice roots. At the Phylum level, the root bacteriome was primarily composed of Pseudomonadota (Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria) followed by Actinomycetota. The fungal microbiome was dominated by Ascomycota (Sordariomycetes, Eurotiomycetes and Dothideomycetes) and Basidiomycota. Not only the root compartment, but also the host genotype can shape the root microbiome. Recruitment of specific microorganism mainly occurs at the species level. Genotype-specific and compartment-specific associations of microbial species in mycorrhizal rice roots were also observed supporting that root colonization by an AM fungus contributes to variations in the root microbiome. Further, key microbial species primarily associated to methane production and nutrient cycling (e.g. Phosphate Solubilizing Bacteria and Nitrogen cycling bacteria) colonizing root compartments in each rice genotype and mycorrhizal condition are described. ConclusionsThe rice genotype, root compartment and mycorrhizal condition markedly influence the microbiome in roots of rice plants growing in flooded rice fields. These findings illustrate the potential of the plant to shape its associated root microbiome, thus, offering valuable insights for the development of microbiome-based strategies to improve growth and performance in rice plants under flooded conditions.

1Synthetic microbial communities (SynComs) could help plants withstand biotic stress and reduce the need for pesticides. With this in mind, we created two SynComs, comprising bacterial strains isolated from the rhizospheres of barley and wheat. We then studied their potential to trigger induced systemic resistance against the barley pathogen Blumeria graminis f. sp. hordei (Bgh). To investigate the plant-microbial interactions from the perspective of both plants and microbes, we performed DAF staining to quantify Bgh propagation in plant leaves, analysed leaf transcriptomes and conducted rhizosphere 16S rRNA gene metabarcoding and rhizosphere metatranscriptome analysis. Our results demonstrate that the SynComs elicit defence responses in barley against Bgh in a manner similar to that of the positive control strain Pseudomonas simiae WCS417r. The SynComs act without triggering a strong gene response prior to inoculation with the plant pathogen or affecting plant-associated prokaryote communities; they only mildly influence bacterial gene expression in the rhizosphere. Instead, they act as priming agents, preparing the plant for further pathogen attack. These findings suggest that protective SynComs can be applied in the field without causing signficant disruption to native microbial communities.

Plant roots host defined microbial communities that differ from those found in the surrounding soil and these communities shift dynamically in response to plant development and environmental changes. Whilst it is widely accepted that root exudates play a key role in the assembly and dynamics of root-associated microbial communities, the underlying mechanisms are not well understood. This is partly due to a lack of controlled experimental systems that monitor both exudate- and microbiome-dynamics simultaneously. Here, we compared two microcosm systems commonly used in either root microbiome (clay particle-based) or root exudate studies (glass bead-based) for their suitability to simultaneously monitor both aspects. We evaluated these systems based on plant performance, bacterial growth, and time-resolved community and exudate profiling. In both systems, we reveal an exudate effect, characterised by higher bacterial diversity and Pseudomonas abundances in proximity to plant roots. While clay particles impeded exudate recovery, even when plants were removed from microcosms for exudate collection, the glass bead set-up allowed us to uncover dynamic exudate shifts during bacterial community establishment. This highlighted a transient increase of glucosinolates upon root colonisation by initially dominant Pseudomonas species. Overall, the comparison proved only the glass bead-based semi-hydroponic system to be suitable for the paralleled study of exudate and root microbiome dynamics.

Leaf diseases pose a serious threat to faba bean production. Leaf blotch of faba bean, caused by Alternaria spp., has become increasingly widespread and destructive in several countries. Leaf diseases pose a serious threat to faba bean production. The infection of plant by pathogens can be influenced by various factors associated with the host plant, environmental conditions and presence of other microorganisms. The phyllosphere and endosphere play a critical role in plant health and disease development. This study aimed to evaluate the factors shaping the structure and diversity of fungal communities associated with faba beans. Plant samples were collected in 2004 from two intensively managed faba bean production fields in the central region of Latvia. Fungal assemblages were characterized using an ITS region metabarcoding approach based on Illumina MiSeq sequencing. Among the assigned amplicon sequence variant (AVS), 65% belonged to the phylum Ascomycota, while approximately 4% were classified as Basidiomycota. Alternaria and Cladosporium were the dominant genera across samples. The alfa and beta diversities of fungal communities was higher during flowering of faba beans to compare with ripening. The higher abundance of Basidiomycota yeasts were observed during flowering, in contrast, Cladosporium genus was significantly more abundant during ripening. Alternaria DNA was found on leaves that showed no symptoms of the disease. The diversity and composition of fungal communities were significantly influenced by sampling time and presence of leaf blotch, caused by Alternaria spp.

Microbial communities can shift under drought in ways that enhance plant performance during drought ("microbe-mediated acclimation"). However, it is also possible for microbial communities to shift in ways that worsen the effects of drought ("mal-acclimation"). It is unclear how and where microbe-mediated acclimation vs. mal-acclimation occurs, or if there are types of soils or microbial communities that are more likely to harbor microbes that enhance plant acclimation and limit mal-acclimation. We tested for microbe-mediated plant acclimation/mal-acclimation to drought in soils from 21 maize farms in the midwestern United States, spanning a range of climate, soil types, and management practices. We first conditioned soil microbial communities to drought vs. well-watered conditions in a greenhouse and then tested for microbe-mediated acclimation by growing maize in soils inoculated with the conditioned microbial communities under drought and well-watered conditions. Drought-conditioned soils did not enhance plant performance under drought. In fact, one third of the farms exhibited mal-acclimation, especially under well-watered conditions where wet-conditioned soils reduced plant performance in well-watered contemporary conditions. Farm management practices, climate, soil texture, and microbial diversity generally did not predict when this microbe-mediated mal-acclimation occurred. Overall, these results suggest that in agricultural soils, microbes may frequently impede-rather than facilitate-plant acclimation to soil moisture levels. Open research statementThe plant and soil data used in this study are available via the Environmental Data Initiative repository at https://doi.org/10.6073/pasta/f4a0db3a076cf6d8cef908947f82736e. The bacterial and fungal amplicon sequence data are available via the European Nucleotide Archive under accessions PRJEB110071 and PRJEB109827, respectively.

Endophytic microbiomes of crop wild relatives (CWRs) adapted to extreme environments, such as halophytes, are promising sources of plant-beneficial bacteria and secondary metabolites for sustainable food production. Here, we analyzed 25 Bacilli isolates obtained from CWRs, halophytes, and other plant species in Crete, Greece. Using a hybrid Illumina-PacBio sequencing approach, we generated high-quality genomes and performed comparative genomics, phylogenetic, and pangenome analyses, complemented by in vitro assays. We identified 312 biosynthetic gene clusters (BGCs), nearly 60% of which showed no similarity to known clusters, revealing extensive unexplored biosynthetic potential. These unique BGCs may constitute an adaptive feature enabling endophytic Bacilli to colonize and interact with host plants. The isolates spanned diverse genera (Bacillus, Paenibacillus, Peribacillus, Neobacillus, Cytobacillus, Rossellomorea), including three novel species. Phenotypic assays of our isolates demonstrated high salinity tolerance (up to 17.5% w/v NaCl) and strong antagonism against major bacterial and fungal phytopathogens. Genome mining further revealed a broad array of putatively plant-beneficial traits related to growth promotion, stress adaptation, host interaction and inhibition of pathogens. Together, these findings show that Bacilli endophytes from wild and halophytic plants possess exceptional phylogenetic novelty, functional diversity, and biosynthetic capacity, providing new genomic and ecological insights into Bacilli associated with plants inhabiting extreme environments.

1.Structured Abstract1.1 AbstractSoybean red crown rot, caused by the soil-borne fungus Calonectria ilicicola, causes substantial yield losses, but the response of the root-associated bacterial microbiome remains poorly understood. Here, we combined 16S rRNA gene sequencing, shotgun metagenomics, and single-cell genomics to characterize bacterial communities in soybean root-associated soils. 16S rRNA gene sequencing showed that diseased plants had rhizosphere and, more strikingly, rhizoplane microbiomes distinct from those of healthy plants, often with increased Enterobacterales. Shotgun metagenomics further revealed enrichment of genes associated with antibiotic resistance, particularly cationic antimicrobial peptide resistance, in diseased rhizoplane samples. Single-cell genomics recovered seven nonredundant Enterobacterales genomes and showed that plant pathogenicity-related genes were broadly distributed across these lineages. In contrast, dlt genes, which are associated with cationic antimicrobial peptide resistance, were detected only in the Enterobacterales lineages enriched in diseased rhizoplane soils. These results support a model in which soybean red crown rot is accompanied by microbiome restructuring and opportunistic enrichment of specific Enterobacterales lineages carrying putative cationic antimicrobial peptide resistance genes. More broadly, this study highlights the value of strain-resolved single-cell genomics for linking disease-associated community shifts to specific bacterial traits. 1.2 ImportanceUnderstanding crop disease requires resolving not only the primary pathogen but also the root-associated bacteria that respond to infection. Here, we used 16S rRNA gene sequencing, shotgun metagenomics, and single-cell genomics to examine the soybean rhizoplane microbiome under red crown rot. Diseased plants showed reproducible shifts in bacterial composition, including frequent enrichment of Enterobacterales and antimicrobial resistance-related functions. Strain-resolved genomes further revealed that the Enterobacterales lineages enriched in diseased rhizoplane soils specifically carried putative dlt-mediated resistance to cationic antimicrobial peptides, whereas general pathogenicity-related genes were broadly shared. These findings suggest that host defense-associated selection, rather than pathogenicity genes alone, may help shape disease-associated root microbiomes. This study demonstrates how single-cell genomics can uncover strain-level traits hidden within bulk community data and thereby clarify plant-pathogen-microbiome interactions.

Plants exhibit clinal trait variation along aridity gradients driven by strong selective pressures, yet whether plant-microbiome associations follow similar patterns remains unclear. Brachypodium spp., a model for temperate cereals spanning environments from hyper-arid to humid, provides an ideal system to test this hypothesis. Here, we compare rhizosphere bacterial communities of Brachypodium growing across arid, semi-arid, and dry sub-humid zones in Israel with those of ecotypes collected along the same precipitation gradient and grown under common-garden conditions. Rhizosphere bacterial diversity was highest in plants from mid-precipitation sites within the Mediterranean semi-arid transition zone, where annual precipitation decreases from 600 to 400 mm. Together with higher stochasticity and fewer significantly associated taxa in rhizosphere microbiomes from mid-precipitation plants suggest weaker plant-driven microbiome selection in this transition zone, a pattern that persisted under common-garden conditions. The results may represent a promising avenue to develop microbiome-based strategies for drought resilience by advancing our understanding of host filtering across aridity gradients.

Australia is the largest producer of Pyrethrum (Tanacetum cinerariifolium) globally. Amongst the constraints on production are the fungal pathogens Didymella tanaceti and Stagonosporopsis tanaceti, which pose a significant threat to the industry, causing substantial yield losses. While the infection biology of S. tanaceti is well characterised, knowledge of D. tanaceti and its potential interaction with S. tanaceti on plants remains limited, hindering disease management. We developed fluorescently labelled strains of both pathogens via Agrobacterium tumefaciens-mediated transformation (ATMT). Binary vectors carrying the mNeonGreen or tdTomato fluorescent protein genes were introduced into D. tanaceti and S. tanaceti, respectively, and expression of the fluorescent proteins was confirmed by microscopy. Genome sequencing revealed single-copy T-DNA insertions in all transformants, with minor genomic rearrangements at insertion sites. Detached leaf assays demonstrated that transformed strains retained pathogenicity, producing disease symptoms indistinguishable from those of the wild type. These fluorescently labelled variants enabled detailed visualisation of D. tanaceti infection biology and its interactions with S. tanaceti, including co-infection dynamics. Co-infection assays using fluorescent strains further facilitated simultaneous visualisation and differentiation of both pathogens within host tissues. Importantly, these tools also allowed the first description of the early stages of infection by D. tanaceti in pyrethrum leaves. This study represents the first successful transformation of D. tanaceti and S. tanaceti, providing valuable resources to investigate their infection processes.

Plant-associated microbiota are composed of hundreds of microbial species. For many of them, little is known about their individual functions and even less is known about their emergent community-level traits. While culture-independent methods provide valuable insights into the composition, diversity, and functional potential of plant-associated microbiota, culture-dependent methods are essential for reductionist lines of inquiry into the roles of individual species and their interactions within a community. Here, we present ZeaMiC, a publicly available culture collection of root-associated bacteria from Zea mays (maize). This resource comprises 88 isolates obtained from diverse soils and several maize genotypes, with live cultures available through DSMZ (German Collection of Microorganisms and Cell Cultures) both as single stocks and as cost-effective bundles (https://www.dsmz.de/collection/catalogue/microorganisms/microbiota/zeamic). To maximize relevance, isolates were selected to be representative of maize root-associated microbiomes in the Corn Belt of the United States, based on abundance-occupancy patterns from previously published root microbiome data, phylogenetic diversity, and literature-based evidence of functional importance. Whole-genome sequencing and annotation revealed genes associated with root colonization, plant growth promotion, and nutrient cycling, including functions such as chemotaxis, biofilm formation, secretion systems, hormone modulation, and phosphate solubilization. This collection serves as a community resource for future mechanistic studies of plant-microbe and microbe-microbe interactions, filling the gap in our understanding of the ecological interactions in plant microbiomes.

Sustaining high agricultural productivity with minimal environmental impact requires innovative and sustainable strategies that reduce reliance on mineral fertilizers. Promoting root association with plant growth-promoting bacteria (PGPB), either within the native microbiome or through bioinoculant application, represents a promising strategy to improve crop performance while reducing mineral fertilizer inputs. The success of this strategy, however, is strongly influenced by plant genetic traits that regulate microbial recruitment and colonization. Here, we tested whether silencing the ABAP1 Interacting Protein (AIP10), a negative regulator that links cell division with primary metabolism, modulates the association of Arabidopsis thaliana to PGPB. Non-inoculated aip10-1 roots exhibited gene expression patterns similar to genotypes with enhanced microbial associations. AIP10 silencing reshaped root and rhizosphere bacterial communities, favoring beneficial PGPB associations and limiting potential pathogens. Consistently, aip10-1 plants showed greater colonization by inoculated diazotrophic PGPB, particularly in low fertilization conditions, leading to increased plant performance. These effects were accompanied by modulation of plant cell cycle and nitrogen assimilation pathways, together with increased bacterial colonization and nifH expression. Our findings suggest that AIP10 functions as a regulatory hub coordinating growth and metabolism with beneficial PGPB recruitment. Modulating AIP10 could enhance plant productivity and support more sustainable and regenerative agriculture practices. HighlightAIP10 silencing participates in a regulatory hub coordinating plant cell cycle and metabolism with recruitment of beneficial bacteria in the root microbiota, contributing to improved plant growth and productivity under nutrient-limited conditions.

BackgroundMicrobiota members collectively contribute to individual performance in humans, animals, and plants. This led to the quest for probiotics to improve host health and reproductive performance. Although the efficacy of probiotics is known to be strongly affected by environmental factors, microbe-microbe interactions, and microbial strain identity, the effects of host genotype and the underlying genetic architecture have been overlooked. In addition, the evolutionary causes of such genetic variation are typically not addressed. In this study, we aimed to describe the genetic architecture of the adaptation of the host plant Arabidopsis thaliana to commensal bacterial members of its native microbiota by identifying candidate genes associated with fitness proxies and presenting signatures of natural selection. ResultsA Genome-Wide Association study conducted under field conditions revealed extensive variation within a new mapping population of 162 genotypes of A. thaliana scored for total seed production and its two underlying components, namely fruit number and mean seed number per fruit, in response to 13 commensal strains. In agreement with the strong host genotype x commensal strain identity interactions observed for each reproductive trait, the polygenic genetic architecture was highly flexible among the 13 commensal strains. Candidate genes exhibited a significant enrichment in signatures of both local adaptation and balancing selection. In line with the phenotyped reproductive traits, we identified seven candidate genes with functions specifically and strongly linked to seed germination and fertility. ConclusionsOur findings reveal the importance of genotype-by-genotype interactions when measuring fitness proxies on a wild plant species inoculated with key members of its native microbiota. In addition, this study improves our understanding of the genetic signatures of natural selection acting on native host-microbiota adaptive interactions.

Water scarcity is an increasing constraint on agricultural productivity and demands scalable strategies that improve crop performance under reduced irrigation. As soil microorganisms regulate key processes at the soil-plant interface, microbial inoculants may help sustain plant growth and physiological function during water limitation. Here, we assembled five functionally diverse microbial consortia containing taxa selected to support rhizosphere colonization, soil structural stabilization, and fungal-mediated nutrient and water foraging. These consortia were evaluated in greenhouse trials with lettuce and spinach grown under full irrigation or a 30% deficit irrigation regime (70% of crop water requirement). Crop responses were assessed using yield, harvest delay, root length, wilting incidence, chlorophyll content, and Water Band Index (WBI). Across both crops, microbial consortium treatments improved performance under deficit irrigation relative to untreated water-stressed controls. In lettuce, yield increased by 3-9%, while in spinach yield increased by 4-13%, with several treatments restoring performance to levels not significantly different from the fully irrigated control. Microbial treatments also reduced harvest delay by an average of three to four days, improved root length, lowered wilting incidence, and reduced WBI, indicating reduced plant water stress. In several cases, these physiological responses approached those observed under full irrigation despite 30% lower water input. Higher application rates (500 vs 250 g h-1) generally produced stronger responses, although this trend was not always statistically significant. Together, these results show that complex microbial consortia can buffer the negative effects of deficit irrigation and improve crop performance in leafy greens. These findings support the development of microbial inoculants as biologically based tools to enhance agricultural resilience under increasing water scarcity. TeaserMicrobial soil inoculants help crops maintain yield and harvest synchrony under reduced irrigation.

1Microbial symbionts have the potential to contribute to host-plants ecological and evolutionary success, especially in plants adaptions to harsh environments, however their role has often been overlooked. Conversely, how host local adaptation (e.g., trait divergence across elevation) shapes the composition of associated microbial symbiont communities remains poorly understood. We explored how foliar endophytic fungi (FEF) abundance, richness, and community composition in three sympatric Monkeyflowers, an ecologically diverse group of flowering plants, change across elevation in the Sierra Nevada, CA, USA. We asked: Q1) Are there differences in leaf functional traits and FEF communities among sympatric Mimulus species populations at similar elevations? Q2) How do traits and FEF communities change across elevation within species? Q3) Are FEF richness, diversity and community composition correlated with leaf functional traits and/or elevation? Q4) How does FEF community composition differ with geographic distance within each species? We collected M. guttatus, M. nasutus, and M.laciniatus individuals from natural populations across the Sierra Nevada, measured leaf functional traits and used ITS sequencing to describe the leaf endophyte community. We found significant associations of FEF community composition with host species, and elevation, suggesting that these factors influence fungal community composition. Furthermore, FEF community dissimilarity was correlated with geographic distance indicating isolation by distance and limited dispersal of fungal endophytes. We detected the prevalence of Vishniacozyma victoriae, an endophyte found most commonly in Antarctica, across all Sierran Mimulus populations. The presence of V. victoriae could play a role in the adaptation of Mimulus to cold, high elevation environments. Our findings offer novel insights into the intricate interactions between fungal endophyte communities, plant traits, and elevation.

Colletotrichum sublineola (Cs) is a hemibiotrophic fungal pathogen that causes anthracnose in Sorghum bicolor, leading to significant yield losses. To enable infection, Cs secretes effectors - proteins, small RNAs, and metabolites - that damage the plant cell wall or enter the plant cell to suppress immune responses and manipulate host metabolism. Effectors can detoxify host antimicrobials, alter nutrient processing, and evade host immunity. Paradoxically, some effectors can also trigger pattern-triggered immunity (PTI), especially in biotrophic and necrotrophic fungi. More than half of fungal protein effectors lack conserved domains and functional network annotations. In this study, we identified prospective Cs effectors, separating those with non-conserved domains and classifying those with conserved domains by protein families. Comparative genomics is employed to predict effector functions and analyze their roles. Using their predicted locations and domains, we mapped the effectors into functional subsystems related to PTI. These include interactions in the apoplast, oxidative stress response, protein modification and degradation systems, and Cysteine-rich Fungus-specific Epidermal Growth Factor-like Module (CFEM) domain proteins involved in immune regulation. Our functional network analysis advances the understanding of Cs pathogenicity and offers insights into effector infection mechanisms.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense race TR4 (Foc), is one of the most destructive diseases threatening global banana production, particularly the Cavendish cultivar. Conventional control strategies, including chemical treatments and quarantine, remain largely ineffective and unsustainable, underscoring the urgent need for alternative approaches. Biological control using rhizosphere-associated microorganisms offers a promising and environmentally friendly strategy. In this study, we isolated 436 bacterial strains from the rhizosphere of healthy banana plants and screened them for antifungal activity against Foc. Out of the screened isolates, 93 exhibited significant in-vitro inhibitions, and 64 of these were subsequently evaluated in greenhouse assays. We found that 22 strains reduced Fusarium wilt severity by 45-85% compared to untreated controls. Among them, two isolates, DDC20 and DDC_NEW2, consistently demonstrated strong biocontrol activity. In addition, cell-free culture media (CFCM) and crude extracts inhibited spore germination in fluorescence-based assays, indicating the involvement of secreted antifungal metabolites. Microscopy and confocal observations of GFP-tagged Foc revealed hyphal abnormalities in the presence of bacterial treatments, including swelling, irregular branching, and distortion, accompanied by excessive sporulation characterized by abundant microconidia, macroconidia, and chlamydospores. Whole-genome sequencing and comparative analyses placed both isolates within the genus Bacillus. Genome mining using antiSMASH identified multiple biosynthetic gene clusters encoding known antifungal compounds such as surfactin, fengycin, bacillibactin, and difficidin, as well as putative novel clusters. LC-MS confirmed the presence of surfactin and fengycin in bacterial extracts, supporting the genomic predictions. Collectively, these findings highlight the potential of DDC20 and DDC_NEW2 (related to Bacillus spp.) from the banana rhizosphere as effective biocontrol agents against Foc TR4. This integrated approach, combining phenotypic assays, microscopy, and genome mining, provides a strong foundation for the development of sustainable strategies to manage Fusarium wilt in banana cultivation.