Phytobiomes Journal

Antibiotic use in agricultural systems can unintentionally disrupt beneficial rhizosphere microorganisms, yet the consequences of this dysbiosis for plant fitness remain insufficiently understood. Building on previous findings that application of streptomycin to the roots decreases cyanobacteria and increases tomato plant susceptibility to foliar Xanthomonas infection, this study aimed to determine whether this relationship reflects causation or correlation. We evaluated whether targeted inoculation with the filamentous nitrogen-fixing cyanobacterium Cylindrospermum sp. (CI) or a complex rhizosphere microbiome transplant (RMT) could mitigate antibiotic-induced dysbiosis. As expected, streptomycin treatment significantly increased bacterial spot disease severity and reduced microbial richness in the rhizosphere, marked by a pronounced decline in cyanobacterial and Cylindrospermum operational taxonomic units. Co-occurrence network analysis revealed that this dysbiotic state was defined by reduced community connectivity and increased negative associations, indicating a breakdown in cooperative microbial relationships. Notably, both CI and RMT reduced plant disease severity, though they caused distinct rhizosphere community reassembly outcomes. While RMT relied on microbial functional redundancy, the targeted CI approach achieved more robust colonization and effectively "patched" the functional gap left by dysbiosis. Microbiome restoration directly influenced host physiology, significantly reducing the overactivation of ethylene-mediated defense genes, such as ERF1, and partially reinstating auxin-responsive signaling pathways (IAA21) that were disrupted under dysbiosis. These findings suggest that targeted microbial inoculation could reverse dysbiosis and enhance plant resilience under pathogen pressure as effectively as complex microbial transplants. This work highlights a shift in microbiome management: from the complex rebuilding of communities to the strategic repair of specific functional gaps.

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.

Agricultural management practices act as ecological disturbances that can restructure soil and plant-associated microbial communities, but the functional consequences of these microbial shifts on crop performance remain poorly understood. Here, we examined how common orchard inputs, including wood mulch, glyphosate, and humic acid, affect citrus root and rhizosphere microbiomes and tree performance over a three-year field experiment. Mulch emerged as the dominant driver of microbiome structure, significantly altering bacterial and fungal community composition and increasing rhizosphere alpha diversity. Root microbiomes remained comparatively stable, suggesting stronger host selective forces within root tissues. Mulched rhizospheres were enriched with saprotrophic fungi and metabolically diverse bacteria, while non-mulched soils contained taxa typically associated with nutrient cycling, like Rhizobium, Sphingomonas, and Nitrososphaera. Interactions between mulch and glyphosate further reshaped bacterial communities and corresponded with reduced tree physiological performance, including photosynthesis rates. To verify whether these microbial shifts were contributing to these plant phenotype changes, we conducted a greenhouse experiment using field-derived soil microbiota. Active microbiota from mulch-treated soils reduced citrus seedling establishment and root growth relative to microbiota from non-mulched soils, whereas heat-killed controls eliminated these negative effects, demonstrating a causal relationship between management-induced microbiota changes and decreases in plant performance. In contrast, humic acid influenced plant growth primarily through direct abiotic effects rather than microbial community-level traits. Together, our results show that orchard management practices can restructure citrus microbiomes and generate community-level traits that influence plant performance, highlighting the importance of incorporating microbial ecology and microbiome information when designing and testing crop management strategies.

Plants recruit microorganisms to form mutually beneficial associations that enhance their health, productivity, and resilience. The composition of the plant microbiome is shaped by factors such as host species, developmental stage, genotype, and tissue type, with microbial recruitment mediated by plant exudates and secondary metabolites. Eugenia uniflora, a Myrtaceae species native to Brazils Atlantic Forest, produces pharmacologically relevant secondary metabolites and holds ecological and economic value. However, little is known about its associated microbiome, particularly from a metagenomic perspective. In this study, we investigated the phyllosphere bacterial communities, both epiphytic and endophytic, of E. uniflora across two developmental stages (young and mature trees). We also examined the core microbiome shared between E. uniflora and other Myrtaceae genera to better understand microbial diversity and structure within this family. Amplicon sequencing of the V3-V4 region of the 16S rRNA gene was conducted on 19 E. uniflora samples and 13 additional samples from three other Myrtaceae genera. In E. uniflora, we identified 1,456 bacterial ASVs representing 17 phyla, 115 families, and 171 genera. Alpha and beta diversity analyses revealed significant differences in bacterial community composition between developmental stages. Genera such as Massilia and Hymenobacter were more abundant in mature trees, while Aureimonas and Terriglobus were more common in young plants. Leaf microbiome functional potential shifted with plant age, with older leaves favoring secondary metabolite production and younger leaves emphasizing microbial interactions and defense. A total of 16 genera formed the Myrtaceae core microbiome, with five, Methylobacterium-Methylorubrum, Hymenobacter, Sphingomonas, Bdellovibrio, and Terriglobus, present in 100% of samples. Notably, [~]0.7% of the bacterial diversity remained poorly classified, highlighting the underexplored nature of Myrtaceae-associated microbiomes and their potential for bioprospecting.

Common bean (Phaseolus vulgaris) is an important crop in Canada and globally. Like other legumes, common bean (Phaseolus vulgaris) establishes symbiotic interactions with nitrogen fixing bacteria called rhizobia. However, nitrogen fixation by rhizobia in association with common bean is often suboptimal, constraining its productivity and necessitating the application of nitrogen fertilizer. To support the development of high-performing, locally adapted rhizobial inoculants for Ontario common bean growers, we isolated 216 common bean-nodulating rhizobia from southern Ontario soils using a nodule trapping approach with four common bean cultivars. Whole genome sequencing followed by phylogenomic analyses of the 216 rhizobial isolates revealed substantial diversity, assigning them to 11 Rhizobium species, including two novel species. Nearly all isolates belong to the symbiovar phaseoli, spanning the nodC {gamma}-a, {gamma}-b, and alleles, with four isolates belonging to the symbiovar gallica. Soil origin had a significant impact on the species-level community composition recovered during the nodule trapping experiments, indicative of biogeographical structuring of common bean-nodulating rhizobia across southern Ontario. In contrast, host trapping cultivar had only a minor influence of the recovered Rhizobium population diversity. Greenhouse assays demonstrated that one of the novel Rhizobium species exhibited the highest average symbiotic effectiveness, although high-quality isolates were found across multiple species. Together, these results revealed a diverse and genomically variable Rhizobium community capable of forming effective symbioses with common bean in southern Ontario soils. Importantly, our genome-sequenced Rhizobium collection will serve as a valuable resource for identifying competitive and high-quality strains for the development of inoculants tailored to Ontario common bean production. IMPORTANCECommon bean is a globally important food crop, yet its productivity is often limited by suboptimal nitrogen fixation, forcing growers to rely on synthetic fertilizers. Consequently, identifying high-performing, locally adapted inoculant strains is essential for reducing dependence on synthetic nitrogen fertilizers and improving the sustainability of temperate agroecosystems. Our study provides a genome-sequenced collection of common bean-nodulating Rhizobium from southern Ontario, revealing substantial species and genomic diversity across sampling locations. Greenhouse studies allowed us to identify multiple isolates, including isolates from a novel Rhizobium species, that consistently fix nitrogen with, and enhance the growth of, common bean plants. Our findings highlight strong biogeographical structuring of rhizobial communities and demonstrate that Ontario soils already harbour strains with high symbiotic potential. In addition, our Rhizobium collection represents a foundational resource to support future inoculant development and enables future work on the ecology, evolution, and applied optimization of legume-rhizobium symbioses.

Seed microbiota play a crucial role in plant health and development, yet remain understudied compared to other plant-associated microbial communities. This study aimed to characterize seed microbiota diversity across four major crops (common bean, rapeseed, tomato, and wheat) and establish a comprehensive strain collection of seed-borne microorganisms (bacteria and fungi). We employed a combination of culture-dependent and culture-independent approaches to analyze 68 seed samples representing diverse genotypes and production modes. Our results revealed highly variable seed microbiota, with bacterial colonization ranging from 10 to 100 million bacterial CFUs per gram of seeds, and microbial richness varying from 4 to 351 bacterial and 16 to 138 fungal amplicon sequence variants (ASVs) per sample. Both plant genotype and production mode significantly influenced microbiota composition, with each seed sample produced harboring a distinct microbial assemblage. Interestingly, seeds produced in confined environments exhibited lower bacterial colonization but higher microbial richness compared to field-produced seeds. We observed divergent ecological drivers shaping bacterial and fungal communities. Bacterial assemblages were more host-specific and variable, while fungal communities showed greater stability and a substantial core microbiome shared across plant species. Our culturomics approach yielded a collection of 2,510 bacterial and 837 fungal isolates, representing 10-21% of the seed microbiota diversity detected by metabarcoding and the majority of the prevalent and abundant taxa. Notably, 44-60% of cultured bacterial isolates were not detected by metabarcoding, highlighting the complementary nature of these approaches to detect rare or under amplified taxa in PCR. This study provides insights into the complexity and variability of seed microbiota across different crops and production conditions. Our findings emphasize the importance of combining culturomics and sequencing methods for comprehensive characterization of seed microbiota to uncover the potential of seed-borne microorganisms as bioinoculants for sustainable agriculture.

Phyllosphere microbiomes are increasingly recognized as key regulators of plant health and stress responses, although they are also known to change considerably over both space and time. In the phyllosphere, members of the genus Methylobacterium are often abundant and ecologically important as plant growth promoting bacteria. However, knowledge about the temporal abundances and community dynamics of Methylobacterium in agricultural systems remains limited. To address this gap, we characterized seasonal shifts in Methylobacterium-specific and total phyllosphere bacterial loads and community structure on two common summer crops and one overwintering cover crop. Leaf samples of Zea mays (corn), Glycine max (soybean), and Thlaspi arvense L. (pennycress) plants were collected over one year in Minnesota, USA and analyzed with host-associated microbial PCR (hamPCR). Microbial loads and community composition varied strongly among hosts and across growing seasons. Corn supported the highest Methylobacterium and total bacterial loads, increasing towards senescence, while pennycress exhibited the lowest loads and the most distinct communities. While there were strong host-specific patterns, a group of most abundant genera were shared across all crops (Methylobacterium, Sphingomonas, Pseudomonas, and Massilia) and the most abundant Methylobacterium amplicon sequence variants were present on all three hosts. Our findings highlight how microbial loads and community composition change during phyllosphere assembly across diverse summer and overwintering crops, with a small core of versatile taxa dominating multiple agricultural hosts. Understanding these host and season-linked patterns provides a foundation of harnessing Methylobacterium strains to enhance crop productivity and resilience.

Bacterial canker, caused the Pseudomonas syringae species complex, is a major constraint on sweet cherry production worldwide. However, the influence of agronomic practices on pathogen ecology, dispersal and evolution under field conditions remains poorly understood. Here, we combined a factorial-design field experiment with whole-genome sequencing to investigate the effects of polytunnel covering and nitrogen fertigation on phyllosphere populations and the dynamics of a key pathogen, P. syringae pathovar syringae 9644 (Pss9644) in young cherry trees. Epiphytic P. syringae populations initially resembled those in surrounding woodland environments. Over time, pathogenic phylogroup 2d lineages became dominant, particularly on uncovered trees. Diversity of P. syringae populations was higher in uncovered treatments. Polytunnel covering markedly altered community composition and limited rain-splash dispersal of Pss9644 from stem cankers to leaves, thereby interrupting a key stage of the disease cycle. By contrast, nitrogen fertigation had no detectable effect on phyllosphere community structure, but enhanced plant growth and reduced lesion expansion following inoculation. Whole-genome sequencing of re-isolated Pss9644 strains revealed limited short-term genomic diversification, with single-nucleotide polymorphisms detected in 22 re-isolates. In total, 36 mutations were identified across the chromosome although no mutation affected virulence or motility. Taken together, our results show that agronomic practices influence both pathogen ecology and disease outcomes through distinct mechanisms: polytunnel covering primarily limits pathogen dispersal and reshapes phyllosphere communities, while nitrogen fertigation enhances plant growth and reduces disease severity. These findings highlight the potential to integrate canopy management and nutrient strategies to mitigate bacterial canker risk in commercial cherry production.

Endophytic bacteria from arid medicinal plants represent a promising source of stress-adapted, plant growth-promoting (PGP) microorganisms. Here, we investigated the cultivable endophytic microbiota of Peganum harmala using both nutrient-rich and diluted media to maximize taxonomic recovery. Isolates were dominated by Bacillota and Gammaproteobacteria, with higher diversity in roots than in shoots. Venn analysis revealed a shared core fraction between compartments, forming the basis for consortium assembly. Nine representative strains belonging to Phyllobacterium, Bacillus, Brevibacillus, Burkholderia, Ralstonia, and Amycolatopsis were selected for functional screening. Pairwise antagonism assays showed high compatibility among Bacillus-related strains, whereas certain taxa exhibited inhibitory interactions, guiding rational consortium design. Functional characterization demonstrated complementary PGP traits, including nitrogen-related activity, phosphate, potassium, and silicate solubilization, siderophore and indole-3-acetic acid production, and ammonia production. No single isolate performed optimally across all traits, supporting a consortium-based strategy. A synthetic bacterial consortium (C2), reconstructed from the core endophytic microbiota using compatibility-guided selection, was evaluated in two crop systems. In vitro flax germination assays showed accelerated radicle emergence and improved vigor index, particularly with C2. Under greenhouse conditions, C2 significantly enhanced flax shoot and root biomass, root architecture, leaf area expansion, and photosystem II performance in sterile soil. In faba bean under natural soil, C2 increased leaf number (p = 0.02) relative to the control. These results indicate that consortia derived from core endophytes of arid medicinal plants can promote plant growth across diverse crops and soil contexts, although effects remain context-dependent and require rigorous field validation. IMPORTANCEEndophytic bacteria can serve as sustainable bioinoculants to enhance crop performance under stress conditions. This study demonstrates that the core microbiota of the arid medicinal plant Peganum harmala can be rationally assembled into a functionally complementary consortium that improves germination, nutrient acquisition, and whole-plant physiological performance in flax and faba bean. By combining compatibility-guided assembly with functional screening, we show that consortium-based strategies may outperform single-strain inoculants. These findings provide insight into the development of scalable, plant growth-promoting microbial consortia and highlight the importance of testing microbial inoculants under multiple environmental contexts to ensure consistent benefits.

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.

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.

Understanding how host-microbiome interactions influence tree disease is critical for understanding forest resilience. Here, we present foliar microbiome ITS2 metabarcoding transcriptomic datasets from Pinus sylvestris to investigate susceptibility to Dothistroma needle blight (DNB), a globally important foliar disease caused by Dothistroma septosporum. We hypothesised that host genotype shapes foliar microbial communities and their interactions, thereby influencing disease outcomes. Samples were collected from a progeny-provenance field trial in the south of Scotland representing a broad spectrum of disease susceptibilities. The dataset comprises ITS2 metabarcoding samples from 200 genotypes across three timepoints and RNAseq samples from 48 genotypes across two timepoints. Sampling captured key stages of pathogen exposure and disease progression. Both standardised and bespoke protocols were used for nucleotide extraction, sequencing, and quality control, including multiple negative and positive controls. These datasets, available in the European Nucleotide Archive (project accession PRJEB88228), enable analysis of temporal dynamics in foliar fungal communities, host-microbiome transcriptional responses, and genotype-dependent variation in disease susceptibility.

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.

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.

Ralstonia pseudosolanacearum (Rps) belongs to the Ralstonia solanacearum species complex (RSSC). It is a vascular pathogen that causes lethal bacterial wilt disease in many plants, including tomato and eggplant. In this study, we infiltrated tomato leaves with the phytopathogenic bacterium at 109 CFU/mL and observed the development of necrotic scars in the infiltrated area at 48 hours post-infiltration. Interestingly, this response was followed by petiole bending toward the ground of the compound leaf. This was followed by the gradual senescence of the infiltrated leaflet only. In addition, the terminal leaflet infiltrated with the pathogen exhibited epinasty. None of the above symptoms were observed in leaves infiltrated with the known virulent deficient hrpB::{Omega} mutant. Surprisingly, all of the above symptoms were observed in leaves infiltrated with another well-known virulence-deficient mutant phcA::{Omega}. It indicated that the necrotic lesion caused in tomato leaves was hrp-dependent. Infiltration in eggplant leaves caused necrotic scarring and leaf senescence, which were relatively delayed. Necrotic scarring without petiole bending or senescence in tomato leaves was also observed due to infiltration of Pseudomonas aeruginosa SPT08, a tomato endophyte having plant growth promotion activity. The patho-phenotypes such as petiole bending, epinasty, and senescence observed in the case of tomato in this study were not reported earlier. We believe these phenotypes produced in tomato after leaf infiltration may be useful to study the virulence of this pathogen.

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.

CoverCress is a new winter annual oilseed crop developed from field pennycress within the past 20 years. Field pennycress is commonly considered to be self-pollinated but little basic research has been published and there is some misalignment of conclusions. Our experience working with pennycress plant growth in greenhouse and field conditions over the past 13 years suggests that outcrossing is uncommon. We conducted lab, greenhouse, and field experiments to strengthen the body of work. Pollen viability kinetics analysis showed that longevity of pollen viability is negatively impacted by increasing temperatures and by direct exposure to light. Samples treated at 4C declined to 50% viability in 12 hours while it took just 2.5 hrs at 37C, and 1.6 hrs in full sunlight on a cool early April day. Cross-pollination was absent among greenhouse-grown plants flowering inside an agitated plastic pollen-containment covering. Across greenhouse tests, high rates of cross-pollination occurred only in an emasculation treatment that rendered flowers male sterile and opened the pistil to cross-fertilization. Field trials designed to measure pollen flow distance using a trackable fae1 knockout reporter gene failed to show detectable movement of pollen under field conditions in two locations. This data strongly suggests that domesticated field pennycress may be considered a self-pollinated crop and managed as such.

O_LISymbiosis between legumes and rhizobia is beneficial on nutrient-poor soils, as it enables the fixation of atmospheric N2. To establish this symbiosis, gene expression in both the host plant and the symbiont has to be regulated. To understand the underlying RNA-mediated regulation of host gene expression, we designed experiments to identify competing endogenous networks involving circular RNA, microRNA, and linear transcripts during symbiosis, using wt and symbiosis-deficient Lotus japonicus mutants with the rhizobium Mesorhizobium loti (M. loti). C_LIO_LICircRNA, miRNA, and linear transcripts were identified from Lotus japonicus wildtype and CCamK mutant (ccamk-13; snf-1) seedlings without inoculation or with M. loti inoculation using deep short-read sequencing with rRNA-depletion and random primers. C_LIO_LIDifferentially expressed miRNAs showed negative correlations to predicted target genes and may regulate symbiotic processes. The symbiosis essential iron-sensor LjnsRING/BRUTUS expresses a circRNA which was upregulated in symbiotic treatments. This circRNA may act as a target mimic and contribute to nodule longevity. CircRNAs are predicted to act predominantly as trans-regulatory molecules with similar frequencies in Arabidopsis thaliania, Oryza sativa, and Lotus japonicus. C_LIO_LIWe identified novel miRNAs, long noncoding RNAs, and circRNAs, and nominated several as potential new regulatory non-coding RNAs that may act as target mimics to stabilize genes and support symbiosis. C_LI SummarySymbiosis between Lotus japonicus and Mesorhizobium loti involves treatment-specific regulation of competing endogenous RNA networks involving circular RNA, miRNA, and linear transcripts.