Molecular Plant Pathology

Plant pathogenic fungi secrete small proteins, termed effectors, to reprogram host metabolism and suppress immune responses during infection. Although transcriptional waves of effector expression have been described in several pathosystems, the cis-regulatory elements encoding infection-stage specificity remain largely unknown. Here, we investigate the temporal regulation of effector genes in the biotrophic smut fungus Ustilago maydis, a model organism for fungal plant pathogenesis. By integrating transcriptome reanalysis with comparative promoter motif enrichment across biotrophic fungi, we identify distinct promoter motifs associated with defined infection phases. In U. maydis, three candidate cis-regulatory elements correlate with early, proliferative, and late infection stages, respectively. Positional enrichment relative to transcription start sites supports their regulatory relevance. Functional promoter mutagenesis demonstrates that the early-phase motif GTGGG significantly contributes to effector gene expression in planta and is sufficient to drive stage-restricted gene expression in synthetic minimal promoters. Collectively, our findings demonstrate that temporal deployment of the effector repertoire is at least partially encoded at the promoter level. The identified cis-regulatory elements provide a framework for dissecting transcriptional control during biotrophic infection and offer tools for infection-stage-specific gene expression in synthetic biology applications.

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.

BackgroundThe cotton bollworm Helicoverpa armigera is a major global pest controlled by genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins, including Vip3Aa. While Vip3Aa is widely deployed, the genetic basis of resistance remains poorly understood. Previous work identified disruption of a thyroglobulin-like gene (HaVipR1) as one mechanism of resistance, suggesting additional loci may be involved. ResultsUsing linkage analysis, transcriptomics, long-read sequencing, and CRISPR-Cas9 gene editing, we identify a second thyroglobulin-like gene, HaVipR2, as a novel mediator of Vip3Aa resistance. Resistance in a field-derived H. armigera line was shown to be monogenic, recessive, and autosomal, mapping to chromosome 29. Long-read sequencing revealed a [~]16 kb transposable element insertion disrupting HaVipR2, which was undetectable using standard short-read approaches. CRISPR-Cas9 knockout of HaVipR2 conferred >900-fold resistance, confirming its causal role. Comparative analyses show that HaVipR1 and HaVipR2 share conserved domain architecture, indicating that thyroglobulin-domain proteins represent a recurrent target of resistance evolution. ConclusionsOur findings establish thyroglobulin-domain proteins as a new class of Bt resistance genes in Lepidoptera and demonstrate that transposable element insertions can drive adaptive resistance while evading detection by conventional methods. These results highlight the importance of long-read sequencing and accurate genome annotation for resistance monitoring and provide new insights into the molecular basis and evolution of Vip3Aa resistance.

Botrytis cinerea is a necrotrophic fungal pathogen that infects thousands of plant species. During infection, these diverse plant hosts produce different specialized metabolites that can inhibit pathogen growth and shape pathogen fitness. However, the genetic architecture of pathogen resistance toward individual host defense metabolites remains poorly understood. To address this question, we exposed 83 B. cinerea isolates to the metabolite linalool and quantified metabolic and structural responses. Exposure revealed extensive phenotypic diversity across isolates. Genome-wide association identified 101 genes of interest associated with membrane transport and stress response regulation. Genetic associations were stronger for morphological traits than for metabolic traits, suggesting that hyphal architecture may have a complex genetic architecture contributing to linalool resistance. Together, these results establish natural variation in linalool response and provide candidate loci for understanding how generalist pathogens respond to host-derived chemical defenses. Article SummaryTo understand how a generalist pathogen responds to host defenses, we asked how Botrytis cinerea responds to linalool, a widespread monoterpene involved in plant defense. We exposed 83 B. cinerea isolates to 1000 {micro}M of linalool for 72 hours and quantified metabolic traits (growth curves and growth dynamics over time) and morphological traits (hyphal network features). Using GWA, we linked phenotypic variation to genetic variants. Results indicate substantial natural variation in linalool resistance and distinct genetic architectures across trait classes: metabolic responses are driven by a relatively small number of loci with larger effects, whereas structural/morphological responses appear more polygenic.

Fungal pathogens are responsible for substantial crop losses worldwide. There is a pressing need to develop crops with improved disease resistance, especially given that climate change and human activities are exacerbating crop diseases. Our understanding of the molecular mechanisms by which fungi cause disease is incomplete. To address this limitation, we employed proteomics to identify candidate effector proteins from the pathogenic fungus Ustilago maydis that co-purified with the chloroplasts of maize host plants during infection. We specifically characterized the role of one putative chloroplast-associated effector, UmPce3, using heterologous expression in the non-host plant Arabidopsis thaliana. We discovered that UmPce3 interacts with the chloroplast DEAD-box RNA helicase, AtRH3. Phenotypes associated with the expression of UmPce3 in Arabidopsis mirrored those of plants with impaired AtRH3 function and included interference with chloroplast assembly, an impact on photosynthesis, and altered resistance to biotic and abiotic stresses. Support for RH3 as a bona fide effector target was obtained by identifying parallel phenotypic influences of UmPce3 in maize and by demonstrating an interaction between UmPce3 and maize ZmRH3b, an ortholog of AtRh3. Notably, UmPce3 contributes to biotrophy by promoting the virulence of U. maydis on maize seedlings and dampening virulence in plants challenged with salinity as an abiotic stress. Overall, this work highlights the chloroplast as a target of fungal pathogenesis and identifies RH3 as a potential hub for pathogen manipulation of organelle function to balance fungal proliferation and host health in support of biotrophy. Short summaryThe chloroplast plays a key role in plant immunity, in addition to its central contributions to photosynthesis, metabolism, and tolerance of abiotic stresses. The effector UmPce3 of the maize pathogen Ustilago maydis targets the DEAD-box RNA helicase RH3 in host plants to manipulate chloroplast function and enhance fungal pathogenesis. Unexpectedly, UmPce3 also influences host tolerance to salt stress thereby balancing the plant response to biotic and abiotic stressors in support of biotrophic development.

Cadophora luteo-olivacea is an ecologically versatile fungus associated with grapevine trunk diseases, yet the extent to which strains from different hosts and environments differ in genome composition, functional potential, and pathogenicity remains poorly understood. Here, we performed a comparative genomic analysis of 12 C. luteo-olivacea isolates recovered from grapevine, almond, apple, Crocus bulbs, soil, air, wastewater, and deep-sea sediment. Genome assemblies were highly complete (BUSCO >99%) and ranged from 46.94 to 50.70 Mbp. Pairwise average nucleotide identity (ANI) revealed a cohesive 11-strain group and one markedly divergent strain, CBS 266.93. Phylogenomic analysis based on 2,645 single-copy orthologs further showed that CBS 266.93 lies outside the main C. luteo-olivacea clade and forms a sister relationship with Cadophora malorum, indicating that its taxonomic placement warrants reassessment. Across the remaining strains, broad functional conservation was observed, including similar KOG profiles, extensive carbohydrate-active enzyme repertoires (798-849 genes per genome), and abundant biosynthetic gene clusters (26-35 per genome). Transposable element content varied substantially among strains (0.67-4.45% of genome), but this variation did not parallel overall functional profiles. All isolates colonized grapevine leaves in vitro, although lesion severity differed significantly among strains, indicating conserved plant-colonizing capacity with quantitative variation in aggressiveness. Small RNA profiling of inoculated grapevine leaves further revealed isolate-associated differences in host miRNA family expression, particularly for miR398, miR827, and miR156. Together, these results show that most C. luteo-olivacea strains share a conserved genomic framework compatible with plant colonization, while retaining lineage-and strain-level phenotypic and host-associated variation.

N-glycosylation is an essential post-translational modification required for proper protein folding, stability, trafficking, and secretion in eukaryotes. In such organisms, an efficient endoplasmic reticulum (ER) quality control, such as the ER-associated degradation (ERAD) pathway, is critical for maintaining cellular homeostasis. During ERAD, terminally misfolded glycoproteins undergo N-deglycosylation prior to proteasomal degradation, a process typically mediated by peptide N-glycanase (PNGase). However, in the filamentous fungi, the PNGase seems to be catalytically inactive, indicating evolutionary divergence from the canonical PNGase pathway. Filamentous fungi also encode endo-{beta}-N-acetylglucosaminidases (ENGases), particularly members of glycoside hydrolase family 18 (GH18), which may compensate for the loss of canonical PNGase activity. Here, we investigated the roles of the cytosolic GH18 ENGase and a putative acidic PNGase in N. crassa using transcriptomic and functional approaches. Our results demonstrate that the cytosolic GH18 ENGase is an active deglycosylating enzyme likely associated with the ERAD pathway, whereas no deglycosylation activity was detected for the acidic PNGase. Deletion of the ENGase severely compromises tolerance to diverse stress conditions and induces substantial transcriptomic reprogramming, including upregulation of a GH20 exo-{beta}-N-acetylhexosaminidase under ER stress. These findings identify cytosolic ENGase as a key component of fungal proteostasis and suggest that N. crassa activates alternative compensatory mechanisms to maintain protein quality control when canonical deglycosylation pathways are impaired.

Endosymbiotic bacteria can affect many ecological attributes of their insect hosts, including (in herbivorous insects) how insects interact with plants where they feed. This raises the issue of whether deliberate endosymbiont introductions could be used to decrease crop damage caused by insect pests. Here we investigate how transinfecting Rickettsiella viridis and Regiella insecticola endosymbionts into a novel pest aphid host, the Russian wheat aphid (Diuraphis noxia), influences population growth, alate production, dispersal ability and crop damage. Both the Rickettsiella (originating from pea aphids) and Regiella (from green peach aphids) were stably maintained in their new host where they had contrasting effects. Rickettsiella increased the severity of aphid damage on wheat and barley, resulting in greater leaf loss, chlorotic streaking, and higher aphid populations, whereas Regiella reduced aphid population growth and the severity of feeding damage by aphids. Their effects on dispersal morphology also differed: Regiella had no detectable impact on alate incidence, while Rickettsiella consistently suppressed wing formation in small cages, and in larger mesocosms with multiple wheat plants this endosymbiont suppressed dispersal. Endosymbiont-mediated changes in feeding damage did not involve the main plant immune response pathways: transinfected and wild type aphids induced similar levels of jasmonic acid, jasmonic acid-isoleucine, and salicylic acid in plant tissues, even though these plant defenses were strongly activated during aphid feeding. Novel endosymbionts can therefore modulate the severity of plant feeding damage by aphids as well as influencing aphid dispersal. Potential applications in controlling pest D. noxia populations are discussed. Significance statementEndosymbiotic bacteria that live within insect cells can have wide-ranging effects on the reproduction and fitness of their insect hosts in different environments. In herbivorous insects this includes effects on host plant use. Here we test if novel endosymbionts in a pest aphid, the Russian wheat aphid, might be used to decrease crop damage and dispersal. We show that the damage caused to wheat and barley plants from aphid feeding is modulated by novel but stably transmitted introduced endosymbionts. One endosymbiont (Rickettsiella) increased the severity of damage but decreased aphid dispersal, while another (Regiella) decreased damage severity without impacting dispersal. These contrasting effects may be associated with changes in aphid population growth and wing formation but were not linked to key plant immune response pathways. We discuss implications of these findings for using endosymbionts in agricultural pest management. Classification: Applied Biological Sciences, microbiology

Candida auris (Candidozyma auris) is an emerging multidrug-resistant fungal pathogen that poses a significant global health threat. However, the molecular mechanisms underlying its virulence remain incompletely understood. In this study, we performed in vivo transcriptome analysis using an immunosuppressed mouse gastrointestinal infection model to identify genes associated with host-adaptation and virulence during infection. By comparing fungal transcriptomes obtained from colonization and dissemination sites with those from in vitro cultures, we identified genes that were consistently upregulated during infection. Among these genes, the unfolded protein response regulator HAC1 was selected as a candidate virulence-associated gene for further analysis. RT-PCR and sequencing analyses revealed that HAC1 mRNA in C. auris undergoes an unconventional splicing event of 287 bp that is enhanced under ER stress conditions. The excised region spans the annotated open reading frame boundary, suggesting that the translated region of HAC1 may require re-evaluation. Notably, a proportion of HAC1 transcripts appeared to be spliced even under non-stress conditions, indicating a detectable basal level of UPR activation. Differences in splicing dynamics were also observed among clade strains. Functional analyses demonstrated that deletion of HAC1 increased sensitivity to ER stress and heat stress. The HAC1 deletion mutant also exhibited reduced virulence in both Galleria mellonella and immunosuppressed mouse infection models, as evidenced by delayed host mortality and decreased fungal burdens, respectively. These findings indicate that HAC1 contributes to ER stress adaptation, thermotolerance, and survival in the host environment, and identify HAC1 as a virulence-associated gene in C. auris.

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.

Fusarium oxysporum FO12 was originally isolated from cork oak (Quercus suber L.) and has been characterised as a highly effective biological control agent of wilt diseases on different crops. FO12 endophytically colonises roots and basal stems of plants, reducing the establishment of the soil-borne pathogen Verticillium dahliae and triggering plant defence-related genes. Here, we report a chromosome-level genome assembly of FO12 using Nanopore and Hi-C data. The 57.60 Mb assembly comprises 14 chromosome-scale scaffolds with centromeres resolved and telomeric repeats detected at 4 of 28 chromosome ends. This high-quality reference genome provides a valuable resource for further research into the use of FO12 in agriculture as a biocontrol agent.

Belowground, plants are exposed to a wide range of biotic stresses that vary in severity and nature, including tissue damage, disruption of vascular connectivity, and depletion of assimilates. How plants adapt their root systems to cope with different types of belowground biotic stresses is not well known. In this paper we compare above- and belowground plant adaptations to three nematode species with distinct tissue migration and feeding behaviours to study mechanisms underlying tolerance to different types of biotic stresses. We monitored both green canopy growth and changes in root system architecture of Arabidopsis inoculated with Pratylenchus penetrans, Heterodera schachtii, and Meloidogyne incognita. This revealed three distinct phases in aboveground plant responses: (i) initial growth inhibition associated with host invasion and tissue damage, (ii) persistent growth reduction associated with nematode sedentarism, and (iii) late growth stimulus in more advanced stages of infection. Specific adaptations in the root systems further revealed fundamentally different stress coping strategies. Tissue damage and intermittent feeding by P. penetrans in the root cortex did not induce significant changes in root system architecture. Tissue damage to the root cortex and prolonged feeding on host vascular cells by H. schachtii induced secondary root formation compensating for primary root growth inhibition. Prolonged feeding on host vascular cell by M. incognita alone did not induce secondary root formation, but was accompanied by typical local tissue swelling instead. Our data suggest that local secondary root formation and tissue swelling are two distinct compensatory mechanisms underlying tolerance to sedentarism by root-feeding nematodes. HighlightHow plants utilize root system plasticity to cope with different types of biotic stresses by root feeding nematodes remains largely unknown. Here, we report on specific adaptive growth responses in Arabidopsis roots to three nematode species, Pratylenchus penetrans, Heterodera schachtii, and Meloidogyne incognita, with fundamentally different strategies for host invasion, subsequent migration through host tissue, and feeding on host cells.

Apple scab, caused by Venturia inaequalis, remains one of the most damaging diseases in apple orchards, driving intensive pesticide use worldwide. Reducing this dependence requires the deployment of durable resistance, ideally through the combination of major resistance genes (R genes) with quantitative trait loci (QTL) that confer partial and potentially complementary protection. Yet, few apple scab QTLs have been functionally validated, and their underlying mechanisms remain largely unresolved. Here, we refined and functionally described, with transcriptomic data, five resistance QTLs in a biparental population of 1,970 individuals derived from the cross TN 10-8 x Fiesta. Using 43 newly developed KASP markers, QTL locations were substantially precised through high-resolution genotyping and phenotyping with two V. inaequalis isolates exhibiting contrasting virulence. Four QTL (qT1, qF11, qF17, qT13) were validated, while qF3 was not confirmed. Transcriptomic data comparison revealed the expression of candidate genes within the narrowed intervals, including receptor-like proteins in qT1, and RNAi- and signaling-related genes in qF11 and qF17, suggesting a diversified and complementary defense network. These findings refine the genetic architecture of apple scab resistance and suppose the existence of shared molecular pathways between major R gene, such as the well-described Rvi6 gene, and quantitative resistance, with for instance the QTL qT1. The identified loci and markers provide robust tools for marker-assisted and genomic breeding aimed at developing apple cultivars with complementary and potentially durable resistance pathways.

Beyond the significant impact of Cassava witches broom disease (CWBD), caused by the fungus Rhizoctonia (syn. Ceratobasidium) theobromae on cassava cultivation in French Guiana and Brazil, this disease also poses a potential threat to cacao trees in the region, since the fungus is responsible for Vascular Streak Dieback (VSD) of cacao in South East Asia. Cross-pathogenicity trials were conducted in several cassava fields in French Guiana by planting young cacao plants adjacent to diseased cassava plants. Vascular necrosis was observed in some cacao plants, and the presence of R. theobromae in the cacao tissues was confirmed through PCR diagnostics using primers specific to the fungus. Sequence analysis indicated 100% similarity between samples from both hosts and 97.53 to 99.74% identity with R. theobromae isolates previously reported from cassava in the Americas and Southeast Asia. Additionally, symptomatic cacao in a mixed cacao-cassava farm yielded R. theobromae-positive PCR results, suggesting a natural infection. Ongoing work includes artificial inoculations and controlled cross-pathogenicity trials under screenhouse conditions to attempt reproduction of the symptoms. While current data do not yet establish definitive causality, the findings indicate potential host jump and warrant rapid communication to researchers, policy makers, and farmers to safeguard cacao production and Theobroma biodiversity in the Amazon region.

Grain mold severely constrains sorghum [Sorghum bicolor (L.) Moench] productivity and grain quality in subhumid environments. Photoperiod-sensitive flowering plays a key role in mold avoidance and yield stability along north-south rainfall gradients. In response to the high susceptibility of elite cultivars in subhumid zones of Senegal, we developed and characterized a recombinant inbred line (RIL) population derived from Nganda (grain mold-susceptible) and Grinkan (photoperiod-sensitive) varieties. The population was evaluated across three distinct agro-ecological zones over two years. Environmental indices derived from genotype-environmental interactions, together with defined growth windows, strongly influenced flag leaf appearance (FLA), a photoperiodic flowering trait. Plasticity parameters (intercept and slope) for environmental indices, FLA, grain mold severity, and yield enabled identification of loci contributing to flowering response, mold resistance, and yield stability. The maturity gene Ma1 and two QTLs for FLA, qFLA6.2 and qFLA6.3, were identified, stable across environments, and colocalized with grain mold and yield QTLs. The wild-type Ma1 allele from Grinkan delayed FLA and reduced grain mold damage but was not associated with increased yield. The Ma1 effect was confirmed using the developed breeder-friendly KASP marker, Sbv3.1_06_40312464K, in 174 F3 three-way cross families. Photoperiod-sensitive lines with intermediate-to-late FLA alleles showed strong negative associations with mold damage. Overall, the identified stable loci and candidate lines provide foundations for effective molecular breeding of climate-resilient varieties. PLAIN LANGUAGE SUMMARYGrain mold is a fungal disease that reduces sorghum grain yield and quality, particularly in subhumid climates. With the limited number of resistant elite varieties, photoperiod-sensitive flowering to day length variation can contribute to grain mold escape at the end of rainy seasons. We characterized 286 sorghum recombinant inbred lines across three contrasting environments over two years along rainfall gradients in Senegal. Using flag leaf appearance (FLA), which is a photoperiodic flowering trait, strong genotype-environment interactions for FLA and genotypic plasticity were revealed. We identified and validated the common genomic locus associated with FLA variation and its plasticity across environments, the canonical maturity gene Ma1, which was influenced by temperature variation across environments. The presence of Ma1 in the background of photoperiod-sensitive lines enhances grain mold avoidance and yield stability along rainfall gradients in Senegal. CORE IDEASO_LIWe investigated photoperiodic flowering plasticity in sorghum as a contributor to grain mold resistance and yield stability along rainfall gradients. C_LIO_LIThe Maturity locus Ma1 (qFLA6.1) is the major contributor of photoperiodic flowering and its plasticity across semi-arid and subhumid environments. C_LIO_LIHybrid genotypes carrying two stable loci qFLA6.1 and qFLA6.2 sustain high grain mold avoidance in diverse environments. C_LIO_LIPhotoperiod-sensitive lines with medium to late flowering times are effective in avoiding grain mold, while maintaining yield stability in subhumid regions. C_LI

Recurrent epidemics of coffee leaf rust, caused by the fungal pathogen Hemileia vastatrix, have constrained production of Arabica coffee for over 150 years. Here, we present a pseudo-phased, chromosome-level genome resource for H. vastatrix, isolate Hv178a, to guide research into disease management. The Hv178a genome assembly is 665 and 638 Mbp for haplotype A and B respectively, localised to 18 chromosomes. We determined that the genomes are highly repetitive at [~]90%, with a GC content of [~]33%. We present the full annotation of 13,760 and 17,998 protein coding genes, and we predicted 452 and 496 effectors in haplotype A and B respectively. Depth-based comparisons with 11 additional H. vastatrix isolates revealed increased chromosome 17 (chr17) copy number in Hv178a. Validation with qPCR supports a chr17 trisomy in Hv178a absent from the ancestral lineage and potentially explaining the observed change in virulence.

Phytophthora species are globally significant soilborne oomycetes responsible for widespread ecosystem decline. Standard soil sampling protocols, originally developed for qualitative baiting assays, typically require collecting substantial soil volumes in order to capture viable propagules. While effective for culture-based detection, these protocols are labour-intensive and can damage the shallow root systems of sensitive host species such as New Zealand kauri (Agathis australis). Phytophthora agathidicida (PA), the pathogen associated with kauri dieback disease, is routinely surveyed using these methods. However, quantitative data describing the vertical distribution of PA in natural forest soils are lacking. Consequently, it remains unclear whether extensive depth sampling is necessary to ensure consistent molecular detection. In this study, we applied a quantitative oospore DNA (oDNA) qPCR assay to characterise the fine-scale vertical distribution of PA across four soil depth increments (0-5, 5-10, 10-15, 15-20 cm) from 12 kauri trees representing a range of disease stages. Results revealed distinct vertical stratification, with PA DNA concentrations peaking within the upper 0-10 cm of soil in non-symptomatic and possibly symptomatic trees. In symptomatic trees, the absolute peak occasionally reached 10-15 cm, while pathogen signals remained consistently detectable within the top 10 cm. Field validation from an additional eight trees confirmed that targeted 0-10 cm "shallow" sampling yielded higher PA concentrations than deeper sampling protocols. These findings provide a data-driven basis for refining soil sampling strategies, enabling more sensitive molecular detection while minimising disturbance and logistical effort in fragile ecosystems. IMPORTANCEPhytophthora species are among the most destructive soilborne pathogens globally, requiring robust diagnostic protocols for both agricultural and conservation settings. Traditional sampling frameworks were established to meet the biological requirements of baiting assays, which often necessitate collecting large soil volumes from broad depth profiles to ensure the capture of viable, infectious propagules. However, these extensive requirements are labour-intensive and can cause significant soil disturbance in sensitive forest ecosystems. Using P. agathidicida as a model, this study provides a high-resolution quantitative assessment of how pathogen DNA is distributed vertically across different disease stages. We demonstrate that while absolute peak abundance can shift within the 0-15 cm range as infection progresses, the pathogen signal remains consistently detectable within the top 10 cm. This evidence-based approach suggests that targeted, shallow sampling enhances sensitivity by reducing signal dilution, offering a lower-impact path for monitoring soilborne oomycetes worldwide.

The establishment of aphid-plant interaction involves the secretion of a salivary MIF protein. Morphological analyses revealed that aphid MpMIF1 prevents plant cell death, protects organelles from stress, and may promote plant cellular recovery. Co-expression of aphid MpMIF1 and the cell death inducer Npp1 revealed that MpMIF1 modulates autophagy-related genes ATG7/BECLIN1, impair plant senescence regulator ATAF1 and regulate apoptosis-like via Caspase-3-like activity. This effect on multiple-cell death pathways helps to maintain cellular homeostasis during aphid infection. Investigations on DNA Damage Response (DDR) signaling pathways demonstrated that aphid MpMIF1 reduces {gamma}H2A.X phosphorylation, maintains activity of the DNA repair protein RAD51 and stabilizes cell cycle checkpoint expression WEE1 under genotoxic stress. Therefore, MpMIF1 actively participates to the maintenance of a functional DDR. Finally, we showed that aphid MpMIF1 physically interacts with SOG1, a functional analog of animal p53 and central regulator of DDR, cell cycle arrest and programmed cell death in plants. These findings establish MpMIF1 as a key regulator of plant cell death during aphid-plant interactions and highlight its potential as a biotechnological tool for protecting major crops against aphid infection.

Plant-associated microbial communities play a critical role in plant health and disease resistance, but the mechanisms which reshape these communities during pathogen infection are poorly understood. In this study, we investigated how infection of maize by the smut fungus Ustilago maydis is functionally linked with the bacterial phyllosphere microbiome and explored the role of an antimicrobial effector GH25 in fungal infection. Using a combination of culture-dependent and culture-independent approaches, we compared the leaf microbiomes of infected and uninfected plants. We observed a significant increase in microbial abundance and pronounced shifts in community composition and identified distinct health-associated (HCom) and disease-associated (DCom) bacterial communities. To assess whether U. maydis directly manipulates the microbiome, we tested the antimicrobial activity of the antimicrobial effector GH25 against isolated strains. Notably, all HCom bacteria were sensitive to GH25 and co-inoculation of HCom bacteria with a U. maydis {Delta}gh25 knockout mutant significantly reduced fungal virulence. In contrast, DCom exhibited minimal sensitivity to U. maydis and did not affect the virulence of U. maydis {Delta}gh25. Functional profiling revealed infection-associated shifts in predicted metabolic potential, consistent with U. maydis induced leaf tumors being strong sink tissues. Together, the data shows that U. maydis infection reshapes the maize phyllosphere microbiome through a combination of effector-mediated antimicrobial activity and host metabolic reprogramming.

The common pine sawfly, Diprion pini, is a widespread defoliator of pine forests across Europe and Asia, with outbreaks causing substantial ecological and economic damages. However, genomic resources for this species have been limited, hindering advances in molecular ecology or pest management. Here, we present a near chromosome-level reference genome for D.pini, generated using PacBio HiFi reads, Oxford Nanopore MionION long reads, and 10x Genomics linked reads. The final assembly is organized into mostly chromosome-sized scaffolds. It spans a length of 268 Mb, comprises 81 scaffolds, and has a scaffold N50 of 18.7 Mb. BUSCO analysis (hymenoptera_odb10) indicates a high genome completeness of 97.2%. With 22,7 kb the mitochondrial genome is unusually large due to an extended non-coding control region (6,874 bp). Gene prediction identified 26,335 protein-coding genes, of which 12,769 were functionally annotated. Comparative analyses with other sawflies and Apocrita identified 2,472 proteins unique to D. pini, some of which are putatively associated with the processing of plant secondary metabolites. Notably, our genome assembly highlights that, when a closely related, high-quality reference genome is available, chromosome-scale assemblies can be generated without the need of Hi-C sequencing. The genome provides a valuable foundation for the development of improved monitoring and management strategies for D. pini outbreaks and contributes to advancing fundamental research on Hymenoptera evolution.