Plant Disease

Vein clearing of zucchini (VCZ) and bacterial leaf spot (BLS) are caused by various strains of the Pseudomonas syringae species complex that infect zucchini (Cucurbita pepo) seeds. VCZ strains have a narrow host range of cucurbits and affect only seedlings, whereas BLS strains have a broader host range and cause symptoms on adult plants. A multiplex qPCR test showed that VCZ strains predominated in infected seed lots produced in different countries. We surveyed hybrid seed crops grown in parallel in two French regions to address inoculum sources. According to DNA-based approach, parental seed lots were positive to BLS strains, although no culturable bacteria were recovered. Hybrid seed lots produced in the Rhone Valley (southeastern France) showed higher infection rates than those produced in Limagne (central France), and VCZ strains were recovered only from the Rhone Valley. Two representative strains of VCZ and BLS colonized seeds through the vascular and floral pathways, whereas only the BLS strain was transmitted through the pericarp. These findings suggest that floral transmission, potentially mediated by pollinators, could explain the predominance of VCZ strains under favorable regional conditions, and that pericarp transmission in BLS strains could be linked to their capacity to cause disease on adult plants. Furthermore, some infections undetected in seeds became apparent after germination, indicating that testing germinated seeds rather than seeds could help seed industry to take in account only bacterial infections transmitted to the seedling. Together, these results provide valuable insights into the epidemiology of P. syringae transmission to zucchini seeds.

L-phenylalanine (Phe) is converted via the phenylpropanoid pathway into phenolic compounds, and elevated phenolic/flavonoid levels are generally associated with enhanced plant resistance. We previously showed that exogenous Phe suppresses the necrotrophic fungus Botrytis cinerea in petunia, chrysanthemum, determinant tomato, and several postharvest diseases. Here, we expanded evaluation to a broad range of pathosystems, including an indeterminate greenhouse tomato cultivar, multiple dicot species, and the monocot wheat. Phe, applied as spray or drench across concentrations, was consistently effective at [≥]4 mM. It reduced disease severity in diverse systems: Sclerotinia sclerotiorum (tomato, sweet basil, cucumber, lettuce), Leveillula taurica and Oidium neolycopersici (tomato), Podosphaera xanthii (cucumber), wheat foliar pathogens (Blumeria graminis f. sp. tritici, Zymoseptoria tritici, Puccinia triticina, P. striiformis f. sp. tritici), the oomycetes Pseudoperonospora cubensis (cucumber leaves) and Pythium aphanidermatum (roots), bacterial pathogens (Pseudomonas syringae pv. tomato and Clavibacter michiganensis subsp. michiganensis) and Tomato brown rugose fruit (ToBRFV) that belongs to the Tobamovirus genus. Phe was effective on both young and mature leaves and often performed comparably to chemical fungicides; combinations rarely improved control, except for enhanced activity with pyrimethanil against B. cinerea in tomato. Synergistic effects were observed when Phe was combined with an adjuvant against tomato powdery mildews. A formulated product (NaturaFend 550 SP) was more effective than non-formulated Phe in B. cinerea (tomato) and P. xanthii (cucumber). Application timing also influenced efficacy, with treatment 5 days before infection providing superior control compared with 0, 3, or 7 days. Overall, Phe effectively controlled biotrophic and necrotrophic fungi, oomycetes, bacterial pathogens and a virus across diverse crops under experimental greenhouses and commercial like conditions.

Tomato worldwide production is increasingly challenged by complex disease outbreaks involving multiple interacting pathogens. In Israel, recent years have seen a marked rise in vascular collapse symptoms in greenhouse-grown tomatoes, coinciding with the widespread emergence of tomato brown rugose fruit virus (ToBRFV) and pepino mosaic virus (PepMV). Here, we investigated the bacterial and viral agents associated with these outbreaks and examined how viral infection influences the development and severity of bacterial diseases that cause vascular collapse. Surveys conducted between 2021 and 2026 revealed that tomato pith necrosis outbreaks were associated with a diverse bacterial community dominated by members of the Pseudomonadales and Enterobacterales, while bacterial canker outbreaks were exclusively linked to Clavibacter michiganensis. Multilocus sequence analysis showed that pith necrosis-associated Pseudomonas isolates clustered primarily within the P. syringae, and P. corrugata phylogroups. Pathogenicity assays demonstrated that only a subset of pith necrosis-associated bacteria, P. mediterranea, P. capsici, P. viridiflava, and Xanthomonas euvesicatoria pv. perforans, induced pith necrosis under controlled conditions, with high variability in symptom severity. Co-inoculation experiments showed that ToBRFV- and PepMV-infected plants exhibited a 40-100% increase in lesion size following inoculation with pith necrosis-associated bacteria, without a corresponding increase in bacterial colonization, whereas the same viral infections attenuated wilt symptoms caused by C. michiganensis. Together, our findings demonstrate that endemic viral infections differentially modulate bacterial disease outcomes, either exacerbating or attenuating symptoms depending on the pathogen. These results highlight the importance of multi-pathogen interactions in disease severity and have important implications for tomato disease management.

Pseudocercospora fijiensis is the causal agent of Black Leaf Streak Disease (BLSD), also known as black Sigatoka, in banana. The disease affects many banana varieties, including the highly susceptible Cavendish banana that dominates global production and the export trade, and several cooking bananas that are a staple food for hundreds of millions of people worldwide. Currently, the disease is controlled using preventative fungicide treatments with up to 70 applications per year in Cavendish plantations, which accounts for approximately 30% of the production costs. Resistant cultivars are required for more sustainable production, but no resistance gene to BLSD has been identified. This is partly due to the poor genetic amenability of P. fijiensis and the lack of methods for functional gene analysis. To address these limitations, we developed a CRISPR/Cas9-mediated transformation system specifically optimized for P. fijiensis. We established a protocol to produce protoplasts, evaluated their capacity to regenerate into new colonies, and assessed antibiotic sensitivity. Subsequently, we confirmed the integration of foreign DNA, including resistance markers, using PEG-mediated transformation. We demonstrated targeted transformation using CRISPR-Cas9 to knockout the polyketide synthase gene PKS10-1, which is responsible for the production of the pigment melanin, and the mitogen-activated protein kinase (MAPK) gene Fus3. Following the successful generation of knockout mutants for these genes, achieving gene targeting efficiencies of respectively 96% and 58%, we subsequently generated knockout mutants of the renowned effector Avr4 in P. fijiensis. The resulting mutants exhibited no reduction in virulence on the susceptible banana cultivar Cavendish. In addition, we used the wild-type isolate and Avr4 knockout strains to test the resistant banana accession Calcutta 4. Contrary to a previous study, we demonstrate that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors. The established CRISPR/Cas9-mediated disruption system is highly efficient and enables routine functional gene characterization, which will help to elucidate genes involved in banana-P. fijiensis interaction, thereby supporting the discovery of resistance genes against BLSD.

Xylella fastidiosa is one of the most important plant pathogens worldwide, infecting more than 700 plant species and causing major economic losses in affected agricultural regions. In the absence effective curative treatments under field conditions, experimental research is essential. However, research efforts are severely constrained by strict phytosanitary regulations and the pathogen's restricted geographic distribution, as well as its demanding culture requirements. To the best of our knowledge, no studies have compared different host infection models under controlled growth-chamber conditions. To address these limitations, we developed and compared a microinjection-based inoculation protocol across herbaceous and woody hosts maintained in biosafety growth chambers. Stems of Vitis vinifera, Nicotiana tabacum, N. benthamiana, and Medicago sativa were inoculated with X. fastidiosa by microinjection and maintained under controlled growth-chamber conditions. Pathogen establishment was assessed by qPCR analysis of petiole tissue, together with disease progression, plant growth, and leaf scorch development. Approximately one month after inoculation, bacterial detection rates reached 100% in V. vinifera and 80% in N. tabacum. Both species subsequently exhibited measurable disease development, whereas bacterial establishment and phenotypic responses were less consistent in N. benthamiana and M. sativa.

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.

Cultivar susceptibility strongly influences the epidemiology of vector-borne plant diseases, and understanding cultivar-specific variation can inform management strategies. This is particularly relevant for flavescence doree, an incurable grapevine disease associated with a phytoplasma and transmitted by the leafhopper Scaphoideus titanus. In this study, we investigated the susceptibility of the main Swiss varieties, by combining controlled insect-mediated inoculation experiments with complementary field analyses conducted at progressively finer spatial scales. Together, these approaches allowed us to compare both infection probability and phytoplasma relative titre under standardised transmission conditions with disease incidence and relative titre under natural epidemiological conditions. For most cultivars, laboratory results were broadly consistent with field observations. However, a marked discrepancy emerged in the relative infection pattern between the two main grapevine cultivars grown in Switzerland: Chasselas and Pinot Noir. Under controlled conditions, they did not differ significantly in either their probability of infection or the phytoplasma relative titre, indicating no detectable difference in susceptibility to phytoplasma infection. In contrast, Pinot Noir consistently showed higher disease incidence than Chasselas under natural conditions. This pattern was observed across all spatial scales examined, from regional surveys to neighbouring vineyard plots, and was mirrored by higher phytoplasma relative titres. Importantly, under controlled conditions, S. titanus mortality during the one-week inoculation period was significantly higher on Chasselas than on Pinot Noir, indicating that Chasselas may provide a less favourable host for S. titanus. Together, these findings support the hypothesis that differences in field disease incidence between these cultivars may arise from differences in vector performance rather than intrinsic susceptibility to phytoplasma infection. This highlights the importance of considering plant-vector interactions, alongside susceptibility to infection, when assessing cultivar-specific vulnerability to vector-borne plant diseases.

BACKGROUNDBlackleg, caused by Leptosphaeria maculans, is a major disease limiting oilseed rape production worldwide, and its management increasingly requires sustainable alternatives to chemical fungicides. In this study, we evaluated the antagonistic activity and plant growth-promoting potential of three endophytic bacteria, Bacillus velezensis Bro5, Bacillus subtilis Bro11, and Pantoea agglomerans Bru13, against a geographically diverse collection of 139 L. maculans isolates from five oilseed rape-producing regions of Argentina. RESULTSDual culture assays revealed strong inhibitory activity by Bro5 and Bro11, with mean inhibition rates of [~]80% across isolates, while Bru13 showed variable inhibition (<75% for most isolates). Greenhouse and growth chamber assays confirmed the protective potential of these strains. At the cotyledon stage, Bro11 and Bro5 reduced lesion size by 47% and 28%, respectively, while their combination achieved a 51% reduction. In greenhouse trials, combined application of Bro5 and Bro11 reduced stem base necrosis by 45% and increased the proportion of plants with [&le;]50% damage to 98%, compared to only 70% in controls. Key disease metrics, including disease index, incidence, and severity, decreased by 60%, 23%, and 26%, respectively. Beyond pathogen suppression, inoculation with the Bro5-Bro11 consortium enhanced plant growth, increasing shoot biomass by 89% at early stages, and improving stem dry weight and diameter by 10% and 35%, respectively, at maturity. CONCLUSIONThese findings highlight the robustness of Bacillus endophytes as biocontrol agents, their capacity to suppress diverse pathogen isolates, and their dual role in plant growth promotion, supporting their potential integration into sustainable blackleg management programs.

Calonectria ilicicola, an emerging and economically important soilborne pathogen in the U.S. Midwest, causes red crown rot (RCR) disease of soybean. It is essential to have an early and accurate detection method for effective management of RCR, since disease symptoms are nearly identical to those of other soybean diseases. In this study, we developed and validated a high resolution melting (HRM) assay targeting the translation elongation factor 1&alpha (TEF-1&alpha) gene for the specific detection of C. ilicicola. The assay was evaluated using a specificity panel of 78 DNA samples, including 67 fungal and oomycete DNA samples and 11 soybean host-DNA samples, as well as DNA from 56 field-collected symptomatic and asymptomatic soybean tissues. The HRM assay generated distinct melting profiles and, where applicable, absence or delayed amplification that reliably differentiated C. ilicicola from non target fungi and oomycetes. Additionally, the marker distinguished three haplotypes among C. ilicicola isolates, consistent with nucleotide polymorphisms in the TEF-1&alpha target region. The assay demonstrated strong quantitative performance, exhibiting high linearity (R^2= 0.9992) and acceptable amplification efficiency (94.05%) across a broad DNA concentration range. In field samples, HRM based detection aligned with disease status, accurately identifying C. ilicicola in symptomatic plants and no detection in asymptomatic tissues. Overall, this HRM assay provides a rapid, sensitive, and reliable diagnostic tool for routine molecular detection of C. ilicicola.

Black pod disease is a major threat to global cacao (Theobroma cacao L.) production. It is caused by multiple Phytophthora species with geographic ranges from regionally restricted to pantropical, and developing resistant cultivars requires screening against the diversity of these pathogens. This study evaluated the response of 25 cacao clones to 13 isolates of P. palmivora (eight), P. megakarya (two), and P. theobromicola (three) by nonwounding inoculation of detached pods. Lesion size and lesion expansion status were evaluated 4 days after inoculation. The linear mixed model included 178 combinations among 17 clones and 12 isolates, each represented by at least two biological replicate pods. Lesion size differed among clones (F(16, 449.2) = 25.16) and among isolates (F(11, 416.0) = 32.90), and the clone by isolate interaction was significant (F(150, 465.4) = 1.72; all P < 0.001). PENTAGONA-17 had the smallest estimated marginal lesion size (9.3 mm) and fitted values of 3.8 to 16.9 mm across 11 isolates representing all three species. ARF-31, ICS-47, POUND-7, and PMCT-73 had the next four smallest estimates, whereas NA-246 had the largest (36.7 mm). Isolate estimates varied more than fourfold, from 9.6 to 42.0 mm. Despite a marginal estimate of 12.9 mm, POUND-7 had a fitted estimate of 49.2 mm with P. megakarya GH34. The probability of lesion expansion also differed among clones and isolates (both P < 0.001). Together, these findings identify candidates for further resistance evaluation and show that multi-isolate screening provides a stronger basis for selecting materials for black pod resistance breeding.

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.

Red crown rot of soybean (RCR), caused by Calonectria ilicicola, is an emerging soilborne disease whose quantification is challenging due to its complex symptom development across root and foliage levels. This study developed and evaluated a multi-scale framework to improve the assessment of RCR severity from controlled environments to field conditions using root imaging and standardized visual scales. Under controlled conditions, a standard area diagram (SAD) for root necrosis was developed and validated, and SAD-assisted evaluations significantly improved accuracy, precision, and inter-rater agreement compared with unaided assessments. In field conditions, a diagrammatic symptom scale (DSS) was developed using consensus-rated images from experts and showed high reliability, repeatability, and reproducibility across 18 raters, with strong intra- and inter-rater agreement. This study developed and evaluated complementary methods to improve the assessment of RCR severity from controlled environments to field conditions using root imaging and standardized visual scales.

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

Peanut smut, caused by Thecaphora frezzii, is an important constraint to peanut production in Argentina, but quantitative estimates of yield losses across environments remain limited. We quantified the relationship between disease incidence and kernel yield using 922 observations from 26 field studies conducted in Cordoba, Argentina, between 2021 and 2025. Study-specific incidence-yield relationships were analyzed using linear regression, random-effects meta-analysis, and linear mixed-effects models. Peanut smut incidence was consistently associated with yield reduction across studies. The estimated damage coefficient ranged from 24.2 to 28.7 kg ha-{superscript 1} per 1% increase in disease incidence, corresponding to a relative yield reduction of 0.74-0.87% of attainable yield. In contrast, attainable yield varied markedly among studies, ranging from 1,370 to 5,409 kg ha-{superscript 1}. Although an exploratory segmented analysis suggested a breakpoint near 12% incidence, subsequent moderator analyses, study- specific regressions, and normalized response curves provided no evidence of a biologically meaningful change in the damage coefficient across incidence or yield classes. These results indicate that differences among environments were primarily associated with attainable yield rather than with changes in the magnitude of disease-associated yield loss. The resulting damage function provides a quantitative basis for yield-loss assessment and disease management in peanut.

Leptosphaeria maculans is a plant-pathogenic fungus that infects Brassica species, including Brassica napus (oilseed rape). Breeding oilseed rape varieties with genetic resistance is an efficient way to control the disease; however, L. maculans can adapt and overcome these resistances. Understanding the mechanisms that enable L. maculans to adapt is crucial for managing the emergence of better-adapted isolates. Brassica carinata, the Ethiopian Mustard, although closely related to B. napus, is considered a nonhost species of L. maculans because this fungus cannot infect it. Despite the extreme resistance of B. carinata, one natural L. maculans isolate has been identified as unable to infect B. napus, causing moderate and atypical symptoms on this species. We performed a cross between this isolate and an isolate adapted to B. napus, followed by a QTL analysis, which identified seven QTL, each encompassing candidate genes involved in L. maculans adaptation to B. carinata or B. napus. Additionally, we observed transgression in the progeny, wherein a few strains caused significantly more or less aggressive symptoms on both species of Brassica. We found that epistasis within the L. maculans genome contributes to the observed transgression. These initial findings provide further opportunities to study the adaptive capacities of L. maculans, as well as data to initiate analysis of the extreme resistance of B. carinata to L. maculans. HighlightsO_LISeven pathogenicity QTL identified, carrying several interesting candidate genes C_LIO_LITransgression of some progeny isolates on B. napus and B. carinata was reported C_LIO_LIEpistasis plays a significant role in the adaptation of L. maculans toward host and nonhost Brassica species C_LI

Beech leaf disease (BLD), caused by the migratory plant pathogenic nematode, Litylenchus crenatae (LC, Anguinidae) is presently one of the most concerning forest diseases in the United States. BLD has spread rapidly across the landscape and is currently found in 15 U.S. states and Ontario, Canadian, making monitoring a top priority. Here, we report on the development and testing of a probe-based qPCR detection assay that integrates previously reported LC-specific primers with a newly designed probe, enabling sensitive and reproducible quantification of nematodes in plant and non-plant matrices. This assay exhibited strong in silico and in planta specificity and high quantitative performance (R2 = 0.988-0.996), with amplification efficiencies of 90.0-96.8%. The assay also performed consistently on LC-spiked spore trap membranes used to monitor the fungal aerobiome, despite matrix-associated shifts in amplification efficiency. These results support the utility of the assay for monitoring and surveillance. The reliability was further evaluated by two independent diagnostic laboratories, demonstrating high inter-laboratory concordance in detection and strong agreement in relative quantification. The analytical sensitivity and reproducible performance of the assay align with key validation principles outlined by the APS Diagnostic Assay Validation Network (DAVN) and support its application as a molecular tool in experimental, diagnostic, and surveillance contexts, with important implications for national and international LC monitoring and disease management.

Background: Rvi6, introgressed from Malus floribunda 821, is the most widely deployed apple scab resistance gene, but Venturia inaequalis races overcoming it are widespread. Transcriptomic differences between Rvi6-effective and Rvi6-breaking interactions in one host genotype remain unresolved. Aims: To compare these interactions in cv. Ametyst and nominate genes for subsequent validation. Methods: Own-rooted Ametyst plants were mock-inoculated or inoculated with an Rvi6-avirulent isolate (Rin; incompatible) or an Rvi6-virulent isolate (Rola; compatible) and harvested at 4 days post-inoculation (21 mRNA libraries). Expression was quantified using GDDH13 reference-based and de novo Trinity strategies, followed by DESeq2, WGCNA and GSEA. Results: Rin vs Rola yielded 1,339 candidate genes (more than two-fold change, FDR < 0.01) in the reference-based analysis. Curation of Rin-induced defense candidates retained 151 genes, 92 of which concentrated in a single co-expression module. Thirty-one also passed the Rin-vs-Rola thresholded test and were prioritized for genetic validation. Conclusions: The incompatible interaction was associated with a coherent defense program and the compatible interaction with a distinct transcriptional state. With one isolate per class, isolate identity and Rvi6 virulence cannot be separated.

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.