Plant Disease

Background: Alternaria leaf spot (ALS), caused by fungi in the genus Alternaria (family Pleosporaceae), is a significant fungal disease that affects cotton production in the Lake Zone of Tanzania, the country's main cotton-producing region. The disease reduces photosynthetic capacity, boll retention, and fiber quality, leading to substantial yield losses in affected fields. Despite its increasing prevalence, systematic baseline epidemiological data remain scarce. Objective: This study aimed to determine the occurrence, incidence, and severity of Alternaria leaf spot across the major cotton-growing districts of the Lake Zone, Tanzania. Methods: Field surveys were conducted during the 2024-2025 growing season in the districts of Bariadi, Bunda, Chato, and Magu; 20 representative cotton fields were systematically assessed using a diagonal sampling approach. Disease incidence, prevalence, and severity were recorded for 1,000 cotton plants. Fungal isolates recovered from symptomatic leaves were sent to the laboratory for isolation and morphological identification. Results: Field prevalence was 100%, and the overall mean incidence was 64.4%, with significant inter-district variation: Bariadi (77.6%), Bunda (68.8%), Chato (60.0%), and Magu (52.4%). Mean DSI ranged from 44.0% (Magu) to 70.1% (Bariadi), indicating moderate-to-severe infection. Disease incidence and Disease severity (DSI) were strongly correlated when uninfected plants were included (r = 0.942, P < 0.001). However, this relationship weakened substantially and became non-significant when restricted to infected plants only (r = 0.396, P = 0.084). This suggests that higher incidence reflected more plants becoming infected rather than greater severity among already infected plants. Morphological characterization identified all 40 isolates as belonging to the genus Alternaria. Conclusion: These findings provide the first systematic epidemiological baseline for Alternaria leaf spot across the major cotton-growing districts of Tanzania's Lake Zone. Disease incidence and severity varied significantly across districts, underscoring the need for tailored disease management strategies. This study recommends ongoing surveillance and integrated disease management to safeguard cotton productivity among smallholder farmers in the region.

Gray leaf spot can cause severe turfgrass damage, particularly in intensively managed sports fields, while disease management remains challenging due to the rapid development of symptoms under favorable environmental conditions. Early detection of Pyricularia oryzae could therefore provide valuable information for anticipating disease development and supporting management decisions. This study aimed to develop a digital PCR (dPCR) molecular tool to support the monitoring and management of gray leaf spot disease in stadium turfgrass. The assay showed high sensitivity, consistently detecting P. oryzae when infected tissue represented only 0.05% of a mixture with healthy turfgrass, and enabled pathogen detection before symptom appearance under both controlled and field conditions. Different matrices were also investigated across 15 stadiums to identify the most relevant material for disease monitoring. Roots showed the highest prevalence of P. oryzae detection, including during asymptomatic periods, highlighting their potential as a relevant matrix for pathogen surveillance. In a stadium monitored several months after a gray leaf spot outbreak, P. oryzae DNA was still detected in roots from 27 of the 32 sampled zones despite the absence of visible symptoms. Overall, these results demonstrate the potential of dPCR for the early detection and monitoring of P. oryzae in stadium turfgrass, particularly through root sampling during periods when environmental conditions are not favorable for gray leaf spot development, and a spatio-temporal surveillance at stadium scale.

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.

To reduce reliance on pesticides in potato production, alternative partial control methods are needed, yet their efficacy and reliability are seldom quantified over multiple years. Canopy architecture is a potential lever against late blight, caused by Phytophthora infestans, a pathogen whose infection and development are highly driven by humidity and temperature. However, its practical value under field conditions in pure crop stands remains poorly documented. We compared two commercial potato cultivars with contrasting canopy architectures but similar leaf tissue susceptibility to P. infestans, in replicated field trials conducted over six years (2011 2016) under irrigation, and over three of these years also without irrigation. Disease and canopy development (height, closure, leaf area, stem number) were monitored throughout the growing seasons and analysed using non linear growth models. Monalisa, an erect and ramose cultivar, consistently slowed epidemic progress relative to Bintje, a semi-erect and leafy cultivar. This effect was significant in every non irrigated trial but in only half of the irrigated ones. No single canopy trait explained this suppression across all years and conditions. However, canopy closure at the time of inoculation was the most consistent correlate, supporting the hypothesis of a microclimate mediated effect. Disease reduction seldom translated into a yield benefit, except under irrigation, where Monalisa occasionally out yielded Bintje. Our results confirm that canopy architecture can complement other levers within integrated late blight management. Nevertheless, its significant year to year variability and dependence on environmental conditions must be accounted for if it is to be deployed as a reliable control method.

Cowpea, a crop of great importance in several regions in West Africa, faces many biotic and abiotic challenges. Several species of the genus Colletotrichum cause cowpea anthracnose disease (CAD), a major constraint to cowpea production severely affecting foliage and yield. Three Colletotrichum species: C. cliviicola, C. truncatum, and C. chrysophilum recently reported as causal agents of CAD in Nigeria were investigated in their infective processes and pathogenicity on cowpea leaves using histopathological and microscopic examination of stained leaf tissues. Inoculated leaves were examined at intervals to monitor fungal establishment, colonization, and reproductive development within host tissues. The three species completed the infection cycle, progressing from conidial germination and host penetration to tissue colonization, and sporulation. However, differences in both infective ability and pathogenicity were observed in representative isolates of each species. C. truncatum isolate was the most virulent, had extensive branching hyphae, produced severe tissue necrosis, abundant acervuli, and prominent seta formation. C. cliviicola also had substantial colonization and sporulation ability with dense conidial masses and mature acervuli. In contrast, C. chrysophilum had moderate infectivity with less extensive reproductive structures and tissue degradation. Here we provide the first histopathological report of infection and pathogenic development of these three species recently reported as causal agents of CAD

Fusarium oxysporum f. sp. lactucae (Fola), the cause of lettuce fusarium wilt, poses an increasing threat to global production due to the rapid emergence of resistance breaking isolates. However, the evolutionary origins and mechanisms underpinning this diversification remain poorly understood. We characterised 103 Fola isolates using molecular profiling, selecting ten key isolates for long-read whole-genome sequencing and performed in planta virulence assays for selected isolates. This included the first assemblies for Fola2 and Fola3, recent resistance-breaking variants and a highly divergent isolate from France designated as a putative novel race, FolaX. Phylogenomic analysis revealed that Fola is polyphyletic, with lettuce pathogenicity acquired independently across divergent F. oxysporum clades. Despite this, all lineages share a conserved "PanFola" effector set with near-identical sequences, indicating extensive horizontal gene transfer. Resistance-breaking Fola variants within established races were linked to copy number variations and targeted deletions, such as the loss of the SIX8/PSE1 locus in Fola4+. Conversely, FolaX possessed a divergent core genome and a chimeric accessory genome derived from multiple Fola lineages via segmental chromosome transfer and transposon-mediated reshuffling. Our findings highlight the remarkable evolutionary plasticity of Fola and the need for breeding strategies that address the dynamic nature of effector repertoires.

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.

Background: Plant defensins are small cysteine-rich antimicrobial peptides. In Arabidopsis thaliana, the inducible Class I gene AtPDF1.2a is widely used as a transcriptional marker of jasmonate/ethylene immunity, whereas the constitutively expressed Class II members have received less functional attention. AtPDF2.2 was compared with AtPDF1.2a during infection of Arabidopsis by the vascular fungal pathogen Verticillium longisporum, the necrotrophic fungus Sclerotinia sclerotiorum and the bacterium Pseudomonas syringae pv. tomato DC3000. Results: Profiling of the AtPDF family showed that AtPDF2.2, together with the Class II genes AtPDF2.3 and AtPDF2.5, was significantly reduced during V. longisporum infection, whereas AtPDF1.2a was induced. A promoter-reporter line showed a corresponding decrease in AtPDF2.2 promoter activity during V. longisporum infection, strongest in infected roots. KD-pdf2.2 plants contained more V. longisporum DNA, whereas OE-PDF2.2 plants contained less. The same reciprocal disease pattern was observed during S. sclerotiorum and Pst DC3000 infection. Histochemical Prom::AtPDF2.2::GUS reporter signal decreased during S. sclerotiorum infection but increased following Pst DC3000 infection. Basal AtPDF2.2 expression was lower in JA and ET-signaling mutants and higher in the SA-deficient sid2 background, while the analyzed promoter lacked the GCC-box that mediates inducible AtPDF1.2a regulation. AtPDF1.2a overexpression reduced symptoms across the three pathosystems, but loss of AtPDF1.2a increased disease only during V. longisporum infection under the conditions tested and did not alter Pst DC3000 DNA abundance. AtPDF2.2 overexpression was not associated with reduced vegetative growth under the conditions tested, whereas AtPDF1.2a overexpression reduced rosette size and leaf number. Conclusions: AtPDF2.2 is a constitutively expressed Class II defensin whose expression level is associated with disease outcome across two fungal pathogens and a bacterium. Its promoter shows tissue-dependent regulation during V. longisporum infection, while histochemical reporter signal decreases during S. sclerotiorum infection and increases during Pst DC3000 infection. This regulatory behavior differs from that of the inducible reference gene AtPDF1.2a. The reciprocal disease phenotypes of the AtPDF2.2 transgenic backgrounds support a protective association that may involve direct antimicrobial activity, modulation of host responses or both. The absence of reduced vegetative growth in OE-PDF2.2 under the conditions tested supports further evaluation of PDF2-type genes in Brassica.

Stink bugs (Hemiptera Pentatomidae) are major agricultural pests worldwide, including both native and invasive species. In France, for instance, Halyomorpha halys has rapidly expanded its range, affecting key crops such as hazelnuts, apples, and vegetables. Meanwhile, several native, polyphagous species - Nezara viridula, Palomena prasina and, to a lesser extent, Graphosoma italicum - are re-emerging and impacting diverse crops because of changes in agricultural practices and climatic conditions. Egg parasitoids represent promising biological control agents for these pests, be it through conservation, augmentation or introduction biological control. However, their actual potential is not precisely documented due to a lack of ambitious surveys and possible taxonomic issues. This study aims to address this gap by compiling results obtained from various of egg parasitism surveys of Pentatomidae in France and implementing a large-scale DNA Barcoding approach (2000 individuals) for the emerged parasitoids. Our results indicate a high diversity of egg parasitoids, and detected some taxonomic issues and molecular inconsistencies that warrant further study. Among the three families of egg parasitoids recovered (Encyrtidae, Eupelmide, and Scelionidae), Scelionidae was the most abundant and diverse with nine species of Trissolcus (including the two adventive species, Tr. japonicus and Tr. mitsukurii) and four Telenomus species (including two unidentified to species). Scelionidae dominated the parasitoid assemblages associated with most of the Pentatomidae host species, except for H. halys for which the eupelmid Anastatus bifasciatus was the most common species.

Red crown rot (RCR), caused by Calonectria ilicicola, is an emerging soybean disease in the U.S. Midwest for which scalable approaches to characterize within-field disease distribution are lacking. This study evaluated high-resolution PlanetScope satellite imagery for mapping RCR-affected soybean canopies across 15 commercial fields in Illinois surveyed during the 2024 and 2025 growing seasons. A total of 2,921 georeferenced canopy plots were classified as asymptomatic or RCR-affected and paired with six multispectral bands and seven vegetation indices. Spectral differences between classes were evaluated using linear mixed-effects models, and seven machine-learning classifiers representing linear, tree-based, neural-network, kernel, and probabilistic approaches were compared using spatially independent leave-one-field-out cross-validation. RCR-affected canopies exhibited increased reflectance in the visible and red-edge regions, reduced near-infrared reflectance, and lower vegetation-index values relative to asymptomatic canopies. All classifiers showed strong discrimination, with ROC-AUC values ranging from 0.963 to 0.982. Regularized logistic regression achieved the highest overall performance, with an accuracy of 0.945, balanced accuracy of 0.945, F1-score of 0.948, and ROC-AUC of 0.982 at the optimized decision threshold. Permutation analysis identified EVI, NDVI, and red reflectance as the most influential predictors across representative model architectures. Satellite-derived probability and classification maps generally corresponded with symptomatic canopy patterns observed in high-resolution UAV imagery, although mixed pixels reduced precision near disease-patch boundaries. These results demonstrate the potential of high-resolution satellite imagery for within-field mapping of RCR-associated canopy symptoms across independent commercial soybean fields.

Pectobacterium comprises Gram-negative, pectinolytic plant pathogens that cause soft rot and blackleg diseases on numerous economically important crops. During sampling in 2018 in taro (Colocasia esculenta) fields in Hawaii, two bacterial strains PL152T and PL155 were isolated from taro. Peliminary molecular characterization placed both strains in the genus Pectobacterium. However, their taxanomic postions could not be resoved at the species level. A polyphasic taxonomic approach incorporating phenotypic characterization, 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA), whole genome based phylogeny, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) was therefore employed to determine their taxonomic status. Phylogenetic and phylogenomic analyses consistently placed PL152 and PL155 together in a distinct and well supported lineage within the genus Pectobacterium, separate from currently recognized species. The two strains exhibited high genomic relatedness to each other, whereas the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values, were lower than the accepted species delineation thresholds of 95% and 70%, respectively, supported the delineation of a novel bacterial species. The genomic characteristic of PL152 included a genome size of 5.02 Mb and a DNA G+C content of 52%. Collectively, based on a polyphasic approach including phylogenetic, phlyogenomic, biochemical, and phenotypic evidences supports the classification of strains PL152 and PL155 as representatives of a novel species within the genus Pectobacterium, for which the name Pectobacterium pacificum sp. nov. is proposed. Strain PL152 (ICMP 2436274, LMG 32536) is designated as the type strain.

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.

Sudden Death Syndrome (SDS), caused by Fusarium virguliforme, can significantly impact soybean yields. To advance the understanding of SDS resistance in plant introductions, we conducted genome-wide association studies (GWAS) on a set of 351 accessions from the USDA Soybean Germplasm Collection. The analysis revealed seven single nucleotide polymorphisms (SNPs) significantly associated with foliar SDS resistance; three significant QTL (on chromosomes 3, 9 and 11) were detected with Fixed and Random Model Circulating Probability Unification (FarmCPU) and four (on chromosomes 5, 6 and two on chromosome 7) with Bayesian-information and Linkage disequilibrium Iterative Nested Keyway (BLINK). Localized linkage disequilibrium blocks were estimated for these regions. Notably, all of them overlapped with regions where QTL associated with SDS resistance had been previously reported and where within, or close to, sequences annotated as implicated in plant defense mechanisms, such as toxin transporters and transcription factors. The architecture of SDS resistance appears quantitative and polygenic, with most loci explaining modest phenotypic variance. Six plant introductions in this study--PI 175188, PI 307889A, PI 323559, PI 578307C, PI 445683, and PI 323558--harbor favorable alleles at all seven loci, providing high-value resources for resistance introgression. This study underscores the value of leveraging the USDA Soybean Germplasm Collection for the discovery of resistance loci and provides a foundation for marker-assisted breeding to enhance SDS resistance in elite soybean lines.

Introduction. Histoplasma capsulatum is an important fungal pathogen of humans, especially in immunocompromised patients, and the World Health Organisation has listed Histoplasma as a High Priority fungal pathogen. The taxonomy of the genus Histoplasma is still under development, but its only species H. capsulatum has at least of seven lineages, and a range of sublineages. Gap Statement. Allelic multilocus sequence typing (MLST) has been very successfully applied for typing and epidemiology of pathogenic bacteria, but its adoption for fungal pathogens is more limited. Genome sequence-based MLST approaches promise much a higher resolution for comparative phylogenetics, but there are only a small number of genome assemblies available for H. capsulatum. Aim. To develop and validate genome-based MLST typing for H. capsulatum. Methodology. A total of 406 genome assemblies were generated from publicly available Illumina short-read sequencing samples. Selected genome assemblies were subsequently used to generate MLST schemes with chewBBACA, based on whole genome sequences (wgMLST), NCBI-obtained annotations (annMLST) and annotations obtained with Funannotate (fanMLST), and used for lineage identification, and comparative analyses of population structure. Results. Genome assemblies were successfully generated for 406 H. capsulatum samples, with BUSCO completeness scores of over 98%. A subset of 60 genome assemblies was used to generate and validate MLST schemes based on the 60 genome assemblies, 5 genome annotations obtained from Genbank, and 13 genome annotations generated using Funannotate, resulting in schemes with > 8,000 markers. Each of the MLST schemes allowed reproducible separation of all 7 lineages and 6 H. capsulatum Suramericanum sublineages, without the need for high-performance clusters or powerful computing equipment. Comparison of the different schemes using tanglegrams and scatter plots showed good reproducibility (r2 values of 0.8-1.0). Comparison of total sizes of genome assemblies showed significant lineage-dependent size differences, with the Suramericanum, LAmB, India, Capsulatum and Mississippiense lineages averaging a total genome size of 30-33 million bp, and the Africa and Ohiense lineages averaging at 39 million bp. Conclusion. Genome sequence- and especially genome annotation-based MLST analyses are a promising approach that can be adapted as a rapid, high-throughput method for genomic clustering and epidemiological tracing of pathogenic fungi.

Phakopsora pachyrhizi, the causal agent of soybean rust, is among the most aggressive and economically impactful pathogens affecting soybean (Glycine max) production worldwide. As a dikaryotic rust fungus, P. pachyrhizi maintains its genetic information as two discrete haplotypes that are sequestered within separate nuclei for most of its life cycle as it reproduces asexually via the production of infectious urediniospores. Previous genomes of three P. pachyrhizi isolates collected in South America revealed a large genome with a high transposable element content, measuring up to 1.25 Gb and 93%, respectively. Although these efforts provided valuable foundational resources, the complexity of the genome resulted in highly fragmented assemblies. Here, we generated a highly contiguous, substantially improved, annotated genome resource from the P. pachyrhizi isolate LA04-1, collected during the initial outbreak of soybean rust in the continental United States in 2004. The assembly is haplotype-phased, with each haplotype assembled into 18 chromosomes totaling 572 Mb and 569 Mb in size. The isolate contains unique mating-type genes and does not share haplotype genomes with any available isolates with whole-genome sequencing datasets. This resource will be valuable to future investigations into the underlying mechanisms of virulence in this important plant pathogen.

The potential of alternative agricultural systems, such as organic or conservation agriculture, for biodiversity restoration has been widely investigated. However, variability of practices within each of these two categories of agricultural system has rarely been accounted for. Furthermore, these systems have most often been studied separately, isolating either organic or conservation agriculture (itself often reduced to a soil tillage reduction), and rarely analyzing these systems side by side. This study attempts to disentangle these multiple interrelationships by sampling carabid beetles in a network of 48 commercial Belgian farms including organic agriculture (OA), conservation agriculture (CA), farms combining the two approaches (OCA) and farms applying conventional practices (CVA). In each farm, we sampled carabids using pitfall and emergence traps in one winter cereal field and collected a detailed description of crop management practices. We found that community composition was primarily structured by seasonality early in the season (March, April) followed by convergence (May, June) driven by the aggregation of the most dominant species. Based on agroecosystem classification, organic certification (i.e., the absence of synthetic pesticide use) exerted a twofold stronger effect than application of conservation agriculture principles (i.e., reduced or no soil tillage) on carabid beetle community composition. Notably, OCA supported carabid assemblages that were structurally distinct from those of CA and CVA. Furthermore, carabid communities associated with OA differed significantly in composition from those of CVA. Observed and estimated species richness were also significantly higher in OCA than in CA and CVA, while OA differed from CVA only for observed richness. Beyond differences among agricultural systems, community responses were associated with specific management practices, which explained nearly a quarter of the compositional variation with significant influence of the duration since organic conversion and the frequency of synthetic pesticide application. Carabid abundances were negatively affected by synthetic pesticides and moldboard ploughing, whereas richness was more strongly related to the time since conversion to OA, cover crops, and organic amendments. Overall, these results position synthetic inputs and time since conversion as the main filters over carabid communities. Our study highlights the importance of jointly considering system level analyses and detailed management practices to better understand the complex relationships between agricultural practices and carabid communities.

A native entomopathogenic nematode isolate, Heterorhabditis indica Che4.1, was recovered from soils in Valencia (Spain), and its symbiotic bacterium, Photorhabdus akhurstii Che4.1, was isolated and characterized. Nematode identification was confirmed by ITS and 28S rDNA sequencing, while genome-based analyses supported the taxonomic assignment of the bacterial symbiont. The nematode-bacterium complex exhibited insecticidal activity against Spodoptera exigua and Spodoptera littoralis. H. indica Che4.1 showed an LC of 53.7 infective juveniles (IJs)/mL against S. exigua, comparable to that of the commercial reference Larvanem (54.3 IJs/mL). Against S. littoralis, Che4.1 exhibited an approximately twofold lower LC than Larvanem (77.3 versus 155.9 IJs/mL), with non-overlapping 95% confidence intervals. The complete genome of P. akhurstii Che4.1 consists of a single circular chromosome of 5.66 Mb encoding a diverse repertoire of putative virulence factors, including toxin complex (Tc) components, YenTc-like proteins, Mcf toxins, App-family proteins, Pra/Prb proteins, and chitinases, together with several predicted secondary metabolite biosynthetic gene clusters. LC-MS/MS analyses of bacterial culture supernatants and infected insect cadavers detected multiple predicted virulence-associated proteins, including Tc-related, YenTc-like, App-family, and Pra/Prb proteins. In addition, bacterial cell-free supernatants caused 68.8-81.3% mortality in S. exigua neonates under laboratory conditions. Together, these findings reveal a diverse repertoire of potential virulence determinants in P. akhurstii Che4.1 and demonstrate the insecticidal potential of its native nematodebacterium complex. The performance of H. indica Che4.1 relative to a commercial reference supports further evaluation of this isolate for biological control of Spodoptera spp., particularly under greenhouse and field conditions.

Ralstonia pseudosolanacearum causes a lethal bacterial wilt disease in many plants. The pathogen enters through roots and systemically colonizes the xylem tissue, resulting in wilting and eventual death of the host. Using transposon mutagenesis followed by screening in tomato seedlings, we identified a mutant exhibiting virulence deficiency by root inoculation, but not by leaf inoculation. The mutation was recreated in the wild-type genome via natural transformation to prove the linkage of virulence deficiency with the transposon insertion. Whole genome sequencing revealed that the transposon insertion was in a cyp450 homologue, encoding cytochrome P450 monooxygenase. Two independent targeted mutations were generated in the cyp450 gene, and both the mutants recapitulated the root-specific virulence deficiency. The mutants rarely cause disease in mature plants following soil drenching but remained fully virulent upon petiole pricking inoculation. Complementation with the full length cyp450 gene amplified from the wild-type strain F1C1 restored virulence in the mutant. Compared to the wild-type F1C1, the cyp450 mutants exhibited greater sensitivity to tomato root extracts. HR-LCMS of tomato root extracts revealed the occurrence of several antimicrobial compounds. Cytochrome P450 enzymes are known to mediate the degradation or biosynthesis of diverse metabolites in bacteria. Considering the virulence difference due to the mode of inoculation, our findings indicate that cyp450 contributes to tissue-specific adaptation of the pathogen during infection has opened an avenue for integrating tissue metabolomics with bacterial wilt susceptibility research.

Double-stranded RNA (dsRNA) biopesticides that silence essential fungal genes by spray-induced gene silencing (SIGS) are advancing towards registration, underpinned by the claim that silencing is confined to sequence-matched target organisms, a claim that has never been tested systematically against beneficial fungi. We screened twelve dsRNA constructs (the whole transcripts of eleven canonical antifungal target genes of Fusarium graminearum: three CYP51 sterol 14-demethylases, six chitin synthases and two {beta}-tubulins, plus a concatenation of the three CYP51 transcripts modelling the flagship CYP3RNA design) for perfect 21-nucleotide (21-nt) identity, the primary match criterion of recent regulatory bioinformatics frameworks, against the transcriptomes of seven non-target fungi, including commercial biocontrol agents (Trichoderma harzianum, T. virens, Beauveria bassiana, Metarhizium anisopliae, M. brunneum), the arbuscular mycorrhizal fungus Rhizophagus irregularis and Saccharomyces cerevisiae. Eleven of the twelve constructs carried off-target hazard; only CYP51C was completely clean. TUB2b carried 580 perfect 21-nt matches and contained no hazard-free window of 250 consecutive 21-mer start positions, whereas 26.6-60.0% of screened windows were hazard-free in the designable genes. Hits landed overwhelmingly on orthologues of the targeted gene and concentrated in the Hypocreales; R. irregularis and S. cerevisiae were nearly clean. These are hazard findings, not risk findings: perfect siRNA-length matches to beneficial fungi are common in canonical antifungal dsRNA targets, and current regulatory screening panels contain no fungus that would detect them.

The bean thrips, Caliothrips phaseoli, is an emergent pest affecting peanut production. Thus, gaining a deeper understanding of the naturally occurring viral populations associated with this insect is of major importance, as such knowledge could support the design of novel pest management strategies that are both ecologically responsible and sustainable over the long term. In this study, we performed the first RNA high-throughput sequencing (HTS) analysis of C. phaseoli to unravel its virome, discovering a wide number of novel RNA viruses. Our HTS analysis revealed the presence of 31 viral segments, corresponding to 18 viruses, including double-stranded RNA viruses as well as positive- and negative-sense single-stranded RNA viruses. All these are novel viruses and cover multiple viral lineages belonging to diverse families. Overall, these results indicates that C. phaseoli hides a complex virome. The findings presented here provide an important framework for subsequent studies aimed at understanding the ecological significance, evolutionary patterns, and possible use of these viruses as components of biologically based management programs for this emerging peanut pest.