Phylogenomic analyses and comparative genomics of Pseudomonas syringae associated with almond (Prunus dulcis) in California.

We sequenced and comprehensively analysed the genomic architecture of 98 fluorescent pseudomonads isolated from different symptomatic and asymptomatic tissues of almond and a few other Prunus spp. Phylogenomic analyses, genome mining, field pathogenicity tests, and in vitro ice nucleation and antibiotic sensitivity tests were integrated to improve knowledge of the biology and management of bacterial blast and bacterial canker of almond. We identified Pseudomonas syringae pv. syringae, P. cerasi, and P. viridiflava as almond canker pathogens. P. syringae pv. syringae caused both canker and foliar (blast) symptoms. In contrast, P. cerasi and P. viridiflava only caused cankers, and P. viridiflava appeared to be a weak pathogen of almond. Isolates belonging to P. syringae pv. syringae were the most frequently isolated among the pathogenic species/pathovars, composing 75% of all pathogenic isolates. P. cerasi and P. viridiflava isolates composed 8.3 and 16.7% of the pathogenic isolates, respectively. Laboratory leaf infiltration bioassays produced results distinct from experiments in the field with both P. cerasi and P. syringae pv. syringae, causing significant necrosis and browning of detached leaves, whereas P. viridiflava conferred moderate effects. Genome mining revealed the absence of key epiphytic fitness-related genes in P. cerasi and P. viridiflava genomic sequences, which could explain the contrasting field and laboratory bioassay results. P. syringae pv. syringae and P. cerasi isolates harboured the ice nucleation protein, which correlated with the ice nucleation phenotype. Results of sensitivity tests to copper and kasugamycin showed a strong linkage to putative resistance genes. Isolates harbouring the ctpV gene showed resistance to copper up to 600 µg/ml. In contrast, isolates without the ctpV gene could not grow on nutrient agar amended with 200 µg/ml copper, suggesting ctpV can be used to phenotype copper resistance. All isolates were sensitive to kasugamycin at the label-recommended rate of 100µg/ml.

Identification and Pathogenicity of Fusarium Species Associated with Young Vine Decline in California.

Grapevine trunk diseases are caused by a broad diversity of fungal taxa that have serious impacts on the worldwide viticulture industry due to significant reductions in vineyards yield and lifespan. Field surveys carried out from 2018 to 2022 in California nurseries and young vineyards revealed a high incidence of Fusarium. Since Fusarium species are important pathogens of other perennial crops, the present study aimed to identify and determine the pathogenicity of the Fusarium species on grapevines. Morphology of the fungal colonies coupled with multilocus phylogenetic analyses using nucleotide sequences of the translation elongation factor 1-alpha (tef1) and the RNA polymerase II second largest subunit (rpb2) genes revealed the occurrence of 10 species clustering in six species complexes, namely F. fujikuroi (FFSC), F. oxysporum (FOSC), F. solani (FSSC), F. sambucinum (FSAMSC), F. incarnatum-equiseti (FIESC), and F. tricinctum (FTSC) species complexes. The species F. annulatum (FFSC) was the most prevalent in samples from both symptomatic young vineyards (73.5% incidence) and nursery propagation material (62.5% incidence). Pathogenicity of the 10 most frequent species was confirmed by fulfilling Koch's postulates on living woody tissue of 1103 Paulsen rootstocks. Our results suggest that Fusarium spp. are involved in the development of young vine decline, probably as opportunistic pathogens when grapevines are under stress conditions.

Comparative Pangenomic Insights into the Distinct Evolution of Virulence Factors Among Grapevine Trunk Pathogens.

The permanent organs of grapevines (Vitis vinifera L.), like those of other woody perennials, are colonized by various unrelated pathogenic ascomycete fungi secreting cell wall-degrading enzymes and phytotoxic secondary metabolites that contribute to host damage and disease symptoms. Trunk pathogens differ in the symptoms they induce and the extent and speed of damage. Isolates of the same species often display a wide virulence range, even within the same vineyard. This study focuses on Eutypa lata, Neofusicoccum parvum, and Phaeoacremonium minimum, causal agents of Eutypa dieback, Botryosphaeria dieback, and Esca, respectively. We sequenced 50 isolates from viticulture regions worldwide and built nucleotide-level, reference-free pangenomes for each species. Through examination of genomic diversity and pangenome structure, we analyzed intraspecific conservation and variability of putative virulence factors, focusing on functions under positive selection and recent gene family dynamics of contraction and expansion. Our findings reveal contrasting distributions of putative virulence factors in the core, dispensable, and private genomes of each pangenome. For example, carbohydrate active enzymes (CAZymes) were prevalent in the core genomes of each pangenome, whereas biosynthetic gene clusters were prevalent in the dispensable genomes of E. lata and P. minimum. The dispensable fractions were also enriched in Gypsy transposable elements and virulence factors under positive selection (polyketide synthase genes in E. lata and P. minimum, glycosyltransferases in N. parvum). Our findings underscore the complexity of the genomic architecture in each species and provide insights into their adaptive strategies, enhancing our understanding of the underlying mechanisms of virulence. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome Sequence and Assembly of 18 Fusarium Isolates from Florida Citrus under High Huanglongbing Disease Pressure and California Citrus under Low Huanglongbing Disease Pressure.

The genomes of eighteen Fusarium isolates cultured from diseased and healthy citrus trees were sequenced, assembled, and annotated. Isolate species identification was confirmed using single marker (TEF1-alpha) phylogenetic assessment. Studies of the traits and genotypes of plant-associated isolates are important to understanding the fungal contribution to phytobiomes of citrus.

Microbiome diversity, composition and assembly in a California citrus orchard.

The citrus root and rhizosphere microbiomes have been relatively well described in the literature, especially in the context of Huanglonbing disease. Yet questions addressing the assembly of root microbial endophytes have remained unanswered. In the above ground tree tissues, leaves and stems have been the research focus point, while flush and flower microbiomes, two important tissues in the vegetative and reproductive cycles of the tree, are not well described. In this study, the fungal and bacterial taxa in five biocompartments (bulk soil, rhizosphere, root endosphere, flower and flush) of citrus trees grown in a single California orchard were profiled using an amplicon-based metagenomic Illumina sequencing approach. Trees with no observable signs of abiotic or biotic stresses were sampled for two consecutive years during the floral development phase. The rhizosphere was the most biodiverse compartment compared to bulk soil, root endosphere, flower and flush microbiomes. In addition, the belowground bacteriome was more diverse than the mycobiome. Microbial richness decreased significantly from the root exosphere to the endosphere and was overall low in the above ground tissues. Root endophytic microbial community composition shared strong similarities to the rhizosphere but also contained few taxa from above ground tissues. Our data indicated compartmentalization of the microbiome with distinct profiles between above and below ground microbial communities. However, several taxa were present across all compartments suggesting the existence of a core citrus microbiota. These findings highlight key microbial taxa that could be engineered as biopesticides and biofertilizers for citriculture.

Microbial Turnover and Dispersal Events Occur in Synchrony with Plant Phenology in the Perennial Evergreen Tree Crop Citrus sinensis.

Emerging research indicates that plant-associated microbes can alter plant developmental timing. However, it is unclear if host phenology affects microbial community assembly. Microbiome studies in annual or deciduous perennial plants face challenges in separating effects of tissue age from phenological driven effects on the microbiome. In contrast, evergreen perennial trees, like Citrus sinensis, retain leaves for years, allowing for uniform sampling of similarly aged leaves from the same developmental cohort. This aids in separating phenological effects on the microbiome from impacts due to annual leaf maturation/senescence. Here, we used this system to test the hypothesis that host phenology acts as a driver of microbiome composition. Citrus sinensis leaves and roots were sampled during seven phenological stages. Using amplicon-based sequencing, followed by diversity, phylogenetic, differential abundance, and network analyses, we examined changes in bacterial and fungal communities. Host phenological stage is the main determinant of microbiome composition, particularly within the foliar bacteriome. Microbial enrichment/depletion patterns suggest that microbial turnover and dispersal were driving these shifts. Moreover, a subset of community shifts were phylogenetically conserved across bacterial clades, suggesting that inherited traits contribute to microbe-microbe and/or plant-microbe interactions during specific phenophases. Plant phenology influences microbial community composition. These findings enhance understanding of microbiome assembly and identify microbes that potentially influence plant development and reproduction. IMPORTANCE Research at the forefront of plant microbiome studies indicates that plant-associated microbes can alter the timing of plant development (phenology). However, it is unclear if host phenological stage affects microbial community assembly. Microbiome studies in annual or deciduous perennial plants can face difficulty in separating effects of tissue age from phenological driven effects on the microbiome. Evergreen perennial plants, like sweet orange, maintain mature leaves for multiple years, allowing for uniform sampling of similarly aged tissue across host reproductive stages. Using this system, multiyear sampling, and high-throughput sequencing, we identified plant phenology as a major driver of microbiome composition, particularly within the leaf-associated bacterial communities. Distinct changes in microbial patterns suggest that microbial turnover and dispersal are mechanisms driving these community shifts. Additionally, closely related bacteria have similar abundance patterns across plant stages, indicating that inherited microbial traits may influence how bacteria respond to host developmental changes. Overall, this study illustrates that plant phenology does indeed govern microbiome seasonal shifts and identifies microbial candidates that may affect plant reproduction and development.

Comparative Profiling of Wood Canker Pathogens from Spore Traps and Symptomatic Plant Samples Within California Almond and Walnut Orchards.

Fungi causing wood canker diseases are major factors limiting productivity and longevity of almond and walnut orchards. The goal of this study was to compare pathogen profiles from spore traps with those of plant samples collected from symptomatic almond and walnut trees and assess if profiles could be influenced by orchard type and age, rainfall amount and frequency, and/or neighboring trees. Three almond orchards and one walnut orchard with different characteristics were selected for this study. Fungal inoculum was captured weekly from nine trees per orchard using a passive spore-trapping device, during a 30-week period in the rainy season (October to April) and for two consecutive years. Fungal taxa identified from spore traps were compared with a collection of fungal isolates obtained from 61 symptomatic wood samples collected from the orchards. Using a culture-dependent approach coupled with molecular identification, we identified 18 known pathogenic species from 10 fungal genera (Ceratocystis destructans, Collophorina hispanica, Cytospora eucalypti, Diaporthe ampelina, Diaporthe chamaeropis/rhusicola, Diaporthe eres, Diaporthe novem, Diplodia corticola, Diplodia mutila, Diplodia seriata, Dothiorella iberica, Dothiorella sarmentorum, Dothiorella viticola, Eutypa lata, Neofusicoccum mediterraneum, Neofusicoccum parvum, Neoscytalidium dimidiatum, and Pleurostoma richardsiae), plus two unidentified Cytospora and Diaporthe species. However, only four species were identified with both methods (Diplodia mutila, Diplodia seriata, Dothiorella Iberica, and E. lata), albeit not consistently across orchards. Our results demonstrate a clear disparity between the two diagnostic methods and caution against using passive spore traps to predict disease risks. In particular, the spore trap approach failed to capture: insect-vectored pathogens such as Ceratocystis destructans that were often recovered from almond trunk and scaffold; Diaporthe chamaeropis/rhusicola commonly isolated from wood samples likely because Diaporthe species have a spatially restricted dispersal mechanism, as spores are exuded in a cirrus; and pathogenic species with low incidence in wood samples such as P. richardsiae and Collophorina hispanica. We propose that orchard inoculum is composed of both endemic taxa that are characterized by frequent and repeated trapping events from the same trees and isolated from plant samples, as well as immigrant taxa characterized by rare trapping events. We hypothesize that host type, orchard age, precipitation, and alternative hosts at the periphery of orchards are factors that could affect pathogen profile. We discuss the limitations and benefits of our methodology and experimental design to develop guidelines and prediction tools for fungal wood canker diseases in California orchards.

Biochemical and Histological Insights into the Interaction Between the Canker Pathogen Neofusicoccum parvum and Prunus dulcis.

The number of reports associated with wood dieback caused by fungi in the Botryosphaeriaceae in numerous perennial crops worldwide has significantly increased in the past years. In this study, we investigated the interactions between the canker pathogen Neofusicoccum parvum and the almond tree host (Prunus dulcis), with an emphasis on varietal resistance and host response at the cell wall biochemical and histological levels. Plant bioassays in a shaded house showed that among the four commonly planted commercial almond cultivars ('Butte', 'Carmel', 'Monterey', and 'Nonpareil'), there was no significant varietal difference with respect to resistance to the pathogen. Gummosis was triggered only by fungal infection, not by wounding. A two-dimensional nuclear magnetic resonance and liquid chromatography determination of cell wall polymers showed that infected almond trees differed significantly in their glycosyl and lignin composition compared with healthy, noninfected trees. Response to fungal infection involved a significant increase in lignin, a decrease in glucans, and an overall enrichment in other carbohydrates with a profile similar to those observed in gums. Histological observations revealed the presence of guaiacyl-rich cell wall reinforcements. Confocal microscopy suggested that N. parvum colonized mainly the lumina of xylem vessels and parenchyma cells, and to a lesser extent the gum ducts. We discuss the relevance of these findings in the context of the compartmentalization of decay in trees model in almond and its potential involvement in the vulnerability of the host toward fungal wood canker diseases.

Phylogenomics of Plant-Associated Botryosphaeriaceae Species.

The Botryosphaeriaceae is a fungal family that includes many destructive vascular pathogens of woody plants (e.g., Botryosphaeria dieback of grape, Panicle blight of pistachio). Species in the genera Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia, Neofusicoccum, and Neoscytalidium attack a range of horticultural crops, but they vary in virulence and their abilities to infect their hosts via different infection courts (flowers, green shoots, woody twigs). Isolates of seventeen species, originating from symptomatic apricot, grape, pistachio, and walnut were tested for pathogenicity on grapevine wood after 4 months of incubation in potted plants in the greenhouse. Results revealed significant variation in virulence in terms of the length of the internal wood lesions caused by these seventeen species. Phylogenomic comparisons of the seventeen species of wood-colonizing fungi revealed clade-specific expansion of gene families representing putative virulence factors involved in toxin production and mobilization, wood degradation, and nutrient uptake. Statistical analyses of the evolution of the size of gene families revealed expansions of secondary metabolism and transporter gene families in Lasiodiplodia and of secreted cell wall degrading enzymes (CAZymes) in Botryosphaeria and Neofusicoccum genomes. In contrast, Diplodia, Dothiorella, and Neoscytalidium generally showed a contraction in the number of members of these gene families. Overall, species with expansions of gene families, such as secreted CAZymes, secondary metabolism, and transporters, were the most virulent (i.e., were associated with the largest lesions), based on our pathogenicity tests and published reports. This study represents the first comparative phylogenomic investigation into the evolution of possible virulence factors from diverse, cosmopolitan members of the Botryosphaeriaceae.

Behind the curtain of the compartmentalization process: Exploring how xylem vessel diameter impacts vascular pathogen resistance.

A key determinant of plant resistance to vascular infections lies in the ability of the host to successfully compartmentalize invaders at the xylem level. Growing evidence supports that the structural properties of the vascular system impact host vulnerability towards vascular pathogens. The aim of this study was to provide further insight into the impact of xylem vessel diameter on compartmentalization efficiency and thus vascular pathogen movement, using the interaction between Vitis and Phaeomoniella chlamydospora as a model system. We showed experimentally that an increased number of xylem vessels above 100 µm of diameter resulted in a higher mean infection level of host tissue. This benchmark was validated within and across Vitis genotypes. Although the ability of genotypes to restore vascular cambium integrity upon infection was highly variable, this trait did not correlate with their ability to impede pathogen movement at the xylem level. The distribution of infection severity of cuttings across the range of genotype's susceptibility suggests that a risk-based mechanism is involved. We used this experimental data to calibrate a mechanistic stochastic model of the pathogen spread and we provide evidence that the efficiency of the compartmentalization process within a given xylem vessel is a function of its diameter.

Synthesis of Deoxyradicinin, an Inhibitor of Xylella fastidiosa and Liberibacter crescens, a Culturable Surrogate for Candidatus Liberibacter asiaticus.

Pierce's disease of grapevine and citrus huanglongbing are caused by the bacterial pathogens Xylella fastidiosa and Candidatus Liberibacter asiaticus (CLas), respectively. Both pathogens reside within the plant vascular system, occluding water and nutrient transport, leading to a decrease in productivity and fruit marketability and ultimately death of their hosts. Field observations of apparently healthy plants in disease-affected vineyards and groves led to the hypothesis that natural products from endophytes may inhibit these bacterial pathogens. Previously, we showed that the natural product radicinin from Cochliobolus sp. inhibits X. fastidiosa. Herein we describe a chemical synthesis of deoxyradicinin and establish it as an inhibitor of both X. fastidiosa and Liberibacter crescens, a culturable surrogate for CLas. The key to this three-step route is a zinc-mediated enolate C-acylation, which allows for direct introduction of the propenyl side chain without extraneous redox manipulations.

An In Vitro Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-"Candidatus Liberibacter asiaticus" Properties.

Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus plants, making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems, and leaves using traditional microbial culturing integrated with culture-independent methods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an in vitro agar diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against Liberibacter crescens, a culturable surrogate for the nonculturable "Candidatus Liberibacter asiaticus" bacterium associated with HLB. We identified microbes with robust inhibitory activity against L. crescens that include the fungi Cladosporium cladosporioides and Epicoccum nigrum and bacterial species of Pantoea, Bacillus, and Curtobacterium Purified bioactive natural products with anti-"Ca. Liberibacter asiaticus" activity were identified from the fungus C. cladosporioides Bioassay-guided fractionation of an organic extract of C. cladosporioides yielded the natural products cladosporols A, C, and D as the active agents against L. crescens This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand the functional roles of members of the microbiome, with the long-term goal of developing anti-"Ca Liberibacter asiaticus" bioinoculants that thrive in the citrus holosystem.IMPORTANCE Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens, a culturable surrogate for the unculturable HLB-associated bacterium "Candidatus Liberibacter asiaticus," we tested the hypothesis that members of the citrus microbiome produce potential anti-"Ca Liberibacter asiaticus" natural products with potential anti-"Ca Liberibacter asiaticus" activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens.

Temporal Dynamics of the Sap Microbiome of Grapevine Under High Pierce's Disease Pressure.

Grapevine is a pillar of the California state economy and agricultural identity. This study provides a comprehensive culture-independent microbiome analysis from the sap of grapevine overtime and in a context of a vascular disease. The vascular system plays a key role by transporting nutrient, water and signals throughout the plant. The negative pressure in the xylem conduits, and low oxygen and nutrient content of its sap make it a unique and underexplored microbial environment. We hypothesized that grapevine hosts in its sap, microbes that have a beneficial impact on plant health by protecting against pathogen attack and supporting key biological processes. To address this hypothesis, we chose a vineyard under high Pierce's disease (PD). PD is caused by the xylem-dwelling pathogenic bacterium Xylella fastidiosa. We selected ten grapevines within this vineyard with a range of disease phenotypes, and monitored them over 2 growing seasons. We sampled each vines at key phenological stages (bloom, veraison, and post-harvest) and used an amplicon metagenomics approach to profile the bacterial (16S -V4) and fungal (ITS) communities of the sap. We identified a core microbiome of the sap composed of seven bacterial (Streptococcus, Micrococcus, Pseudomonas, Bacteroides, Massilia, Acinetobacter and Bacillus) and five fungal (Cladosporium, Mycosphaerella, Alternaria, Aureobasidium, and Filobasidium) taxa that were present throughout the growing season. Overall, the sap microbial makeup collected from canes was more similar to the root microbial profile. Alpha diversity metrics indicated a microbial enrichment at bloom and in vines with moderate PD severity suggesting a host-driven microbial response to environmental cues. Beta diversity metrics demonstrated that disease condition and plant phenology impacted microbial community profiles. Our study identified several potential taxonomic targets with antimicrobial and plant growth promoting capabilities that inhabit the grapevine sap and that should be further tested as potential biological control or biofertilizer agents.

Modeling of xylem vessel occlusion in grapevine.

Morphological traits of the plant vascular system such as xylem vessel diameter have been implicated in many physiological processes including resistance to drought-induced xylem cavitation and vessel occlusion during infection with vascular wilt diseases. In both events, xylem vessels lose function because they become filled with air or tyloses and gels. Xylem cavitation has been well studied, whereas vessel occlusion remains purely descriptive even though it is a critical response to wounding injuries and compartmentalization of vascular pathogens. The timing of vessel occlusion is a key determinant to a successful compartmentalization of pathogens within the plant vascular system and we hypothesized that xylem vessel diameter is the driving variable. Using a dye injection method coupled with automated image analysis, we parameterized a model to investigate how xylem vessel diameter affects the speed of vessel occlusion in Vitis vinifera L. cv. Cabernet Sauvignon in response to wounding. Our dataset contains observations from 6,646 vessels at five kinetic points following stem pruning, over a time course of 1 week. Using this approach we provide evidence that the diameter of vessels is a key determinant of the timing of their occlusion. We discuss how these findings impact resistance to vascular wilt diseases in perennial woody hosts.

Profiling grapevine trunk pathogens in planta: a case for community-targeted DNA metabarcoding.

BACKGROUND: DNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Profiling of pathogen communities associated with grapevine trunk diseases is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the fungal kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology. RESULTS: We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock communities, (ii) time-course experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were over- or under-estimated with universal primers. CONCLUSIONS: Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.

Whole-Genome Resequencing and Pan-Transcriptome Reconstruction Highlight the Impact of Genomic Structural Variation on Secondary Metabolite Gene Clusters in the Grapevine Esca Pathogen Phaeoacremonium minimum.

The Ascomycete fungus Phaeoacremonium minimum is one of the primary causal agents of Esca, a widespread and damaging grapevine trunk disease. Variation in virulence among Pm. minimum isolates has been reported, but the underlying genetic basis of the phenotypic variability remains unknown. The goal of this study was to characterize intraspecific genetic diversity and explore its potential impact on virulence functions associated with secondary metabolism, cellular transport, and cell wall decomposition. We generated a chromosome-scale genome assembly, using single molecule real-time sequencing, and resequenced the genomes and transcriptomes of multiple isolates to identify sequence and structural polymorphisms. Numerous insertion and deletion events were found for a total of about 1 Mbp in each isolate. Structural variation in this extremely gene dense genome frequently caused presence/absence polymorphisms of multiple adjacent genes, mostly belonging to biosynthetic clusters associated with secondary metabolism. Because of the observed intraspecific diversity in gene content due to structural variation we concluded that a transcriptome reference developed from a single isolate is insufficient to represent the virulence factor repertoire of the species. We therefore compiled a pan-transcriptome reference of Pm. minimum comprising a non-redundant set of 15,245 protein-coding sequences. Using naturally infected field samples expressing Esca symptoms, we demonstrated that mapping of meta-transcriptomics data on a multi-species reference that included the Pm. minimum pan-transcriptome allows the profiling of an expanded set of virulence factors, including variable genes associated with secondary metabolism and cellular transport.

Closed-reference metatranscriptomics enables in planta profiling of putative virulence activities in the grapevine trunk disease complex.

Grapevines, like other perennial crops, are affected by so-called 'trunk diseases', which damage the trunk and other woody tissues. Mature grapevines typically contract more than one trunk disease and often multiple grapevine trunk pathogens (GTPs) are recovered from infected tissues. The co-existence of different GTP species in complex and dynamic microbial communities complicates the study of the molecular mechanisms underlying disease development, especially under vineyard conditions. The objective of this study was to develop and optimize a community-level transcriptomics (i.e. metatranscriptomics) approach that could monitor simultaneously the virulence activities of multiple GTPs in planta. The availability of annotated genomes for the most relevant co-infecting GTPs in diseased grapevine wood provided the unprecedented opportunity to generate a multi-species reference for the mapping and quantification of DNA and RNA sequencing reads. We first evaluated popular sequence read mappers using permutations of multiple simulated datasets. Alignment parameters of the selected mapper were optimized to increase the specificity and sensitivity for its application to metagenomics and metatranscriptomics analyses. Initial testing on grapevine wood experimentally inoculated with individual GTPs confirmed the validity of the method. Using naturally infected field samples expressing a variety of trunk disease symptoms, we show that our approach provides quantitative assessments of species composition, as well as genome-wide transcriptional profiling of potential virulence factors, namely cell wall degradation, secondary metabolism and nutrient uptake for all co-infecting GTPs.

Xylem Vessel Diameter Affects the Compartmentalization of the Vascular Pathogen Phaeomoniella chlamydospora in Grapevine.

Fungal wilt diseases are a threat to global food safety. Previous studies in perennial crops showed that xylem vessel diameter affects disease susceptibility. We tested the hypothesis that xylem vessel diameter impacts occlusion processes and pathogen compartmentalization in Vitis vinifera L. We studied the interaction between four grape commercial cultivars with the vascular wilt pathogen Phaeomoniella chlamydospora. We used qPCR and wood necrotic lesion length to measure fungal colonization coupled with histological studies to assess differences in xylem morphology, pathogen compartmentalization, and fungal colonization strategy. We provided evidence that grape cultivar with wide xylem vessel diameter showed increased susceptibility to P. chlamydospora. The host response to pathogen included vessel occlusion with tyloses and gels, deposition of non-structural phenolic compounds and suberin in vessel walls and depletion of starch in parenchyma cells. Pathogen compartmentalization was less efficient in wide xylem vessels than in narrow diameter vessels. Large vessels displayed higher number of tyloses and gel pockets, which provided substrate for P. chlamydospora growth and routes to escape occluded vessels. We discuss in which capacity xylem vessel diameter is a key determinant of the compartmentalization process and in turn grape cultivar resistance to disease caused by P. chlamydospora.

A Method to Detect and Quantify Eutypa lata and Diplodia seriata-Complex DNA in Grapevine Pruning Wounds.

Trunk diseases are factors that limit sustainability of vineyards worldwide. Botryosphaeria and Eutypa diebacks are caused by several fungi belonging to the Botryosphaeriaceae and Diatrypaceae, respectively, with Diplodia seriata and Eutypa lata being two of the most common species. Previous information indicated that the traditional isolation method used to detect these pathogens from plant samples could underestimate their incidence levels. In the present study, we designed two sets of primers that target the ß-tubulin gene and that are amenable for quantitative real-time PCR (qPCR) Sybr-Green assays for the detection and quantification of D. seriata-complex (DseCQF/R) and E. lata (ElQF/R) DNA. The design of a species-specific assay was achieved for E. lata. For D. seriata, a species-specific assay could not be designed. The low interspecific diversity across ß-tubulin genes resulted in an assay that could not discriminate D. seriata from some closely related species either not yet reported or presenting a low prevalence on grapevine, such as D. intermedia. We validated our technique on grapevine spur samples naturally and artificially infected with D. seriata and E. lata during the dormant season. Experimental grapevines were located in two counties of northern California where the incidence of both pathogens was previously reported. The qPCR assays revealed that a high frequency of pruning wound infections (65%) was achieved naturally by E. lata, while low infection frequency (less than 5%) was observed using the reisolation method. For D. seriata-complex, low (5%) to no natural infection frequencies were observed by the qPCR and the reisolation method, respectively. These results also provided evidence that our qPCR detection methods were more sensitive to assess the incidence of E. lata and D. seriata-complex in plant samples, than traditional isolation techniques. Benefits of molecular methods for the detection of canker pathogens in the field under natural conditions are discussed.