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.

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.

First Report of Binucleate Rhizoctonia AG-G Causing Grapevine (Vitis Vinifera) Trunk Diseases in California Nurseries.

Grapevine Trunk Diseases (GTD) are caused by a consortium of fungal pathogens that affect the biological functions of the vascular system of mature and young grapevines (Gramaje et al. 2018). We conducted surveys to profile GTD pathogens in California grapevine nurseries and collected 784 cuttings of cvs. Cabernet Sauvignon and Chardonnay grafted on 1103P rootstock. Several vines exhibited wood necrotic lesions and cankers at the graft union and the root ball (Figure 1A). Symptomatic wood tissues were cultured on PDA medium and after two weeks of incubation at room temperature (22°C), several known GTD pathogens were recovered. We also identified Rhizoctonia from 42 of the 784 vines (5.3% incidence) based on the morphological characteristics of a brown pigmented mycelium (Figure 1B), hyphae branched at a right angle with constrictions at the branch point (Figure 1C) and absence of spores (González García et al., 2006). A subsample of four isolates (DCHG2B, DCSG22R, JCSG9B, and JCHG12B) were randomly selected for further DNA-based taxonomic identification and pathogenicity evaluation to grapevine. The ITS and beta tubulin regions were amplified using the ITS1/ITS4 and B36F/B12R primer sets, respectively (González et al. 2006), and sequences were deposited in the NCBI database (Accession numbers: OR052655, OR052656, OR052657, OR052658 and OR059207, OR059208, OR059209, OR059210). Sequences displayed >99% and >96% identity with the respective ITS and beta tubulin sequences of the binucleate Rhizoctonia AG-G specimen C-653 (González et al. 2006). A phylogenetic tree constructed using the Neighbor-Joining method indicated a 100% bootstrap support with the binucleate Rhizoctonia AG-G (Figure 2). Pathogenicity of the binucleate AG-G Rhizoctonia were confirmed on two separate technical replicates using standard methods. For each replicate, one-year-old rootstock 1103P were wounded with sterile drill bits and inoculated with a single 5 mm diameter agar plug collected from Rhizoctonia growing cultures, while control vines were inoculated with sterile agar. The first replicate lasted 28 weeks with (DCHG2B, DCSG22R) inoculated on seven vines. The second bioassay lasted 24 weeks with two additional isolates (JCSG9B, JCHG12B) inoculated on twelve vines. Rhizoctonia-inoculated vines developed wood symptoms similar to those observed on cuttings in nurseries, with necrotic lesions lengths significantly longer than the controls (First replicate: 3.5  0.4 cm vs. 1.3  0.6 cm; Second replicate: 6.8  0.8 cm vs. 1.1  0.2 cm), based on one-way ANOVA statistical test (P value < 0.05). Rhizoctonia isolates recovery from wood necrotic lesions were confirmed by ITS sequencing, thereby fulfilling Koch's postulate. Several binucleate Rhizoctonia anastomosis groups, including AG-G, have been found to cause root rot and stem necrosis in plant nurseries (Aiello et al., 2017; Rinehart et al., 2007). Rhizoctonia has also been reported to be associated with grapevine nurseries in Europe (Pintos et al., 2018), South Africa (Halleen et al., 2003) and Australia (Walker, 1992). However, the multinucleate Rhizoctonia solani was the only species confirmed to cause root rot on grapevine (Walker, 1992). Our data suggests that the binucleate Rhizoctonia from the AG-G anastomosis group also cause wood necrosis in grapevine. Those findings warrant further studies on the complexity of Rhizoctonia anastomosis groups in nursery and their aggressiveness to grapevine.

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.

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.

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.

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.

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.

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.

Global transcriptional analysis suggests Lasiodiplodia theobromae pathogenicity factors involved in modulation of grapevine defensive response.

BACKGROUND: Lasiodiplodia theobromae is a fungus of the Botryosphaeriaceae that causes grapevine vascular disease, especially in regions with hot climates. Fungi in this group often remain latent within their host and become virulent under abiotic stress. Transcriptional regulation analysis of L. theobromae exposed to heat stress (HS) was first carried out in vitro in the presence of grapevine wood (GW) to identify potential pathogenicity genes that were later evaluated for in planta expression. RESULTS: A total of 19,860 de novo assembled transcripts were obtained, forty-nine per cent of which showed homology to the Botryosphaeriaceae fungi, Neofusicoccum parvum or Macrophomina phaseolina. Three hundred ninety-nine have homology with genes involved in pathogenic processes and several belonged to expanded gene families in others fungal grapevine vascular pathogens. Gene expression analysis showed changes in fungal metabolism of phenolic compounds; where genes encoding for enzymes, with the ability to degrade salicylic acid (SA) and plant phenylpropanoid precursors, were up-regulated during in vitro HS response, in the presence of GW. These results suggest that the fungal L-tyrosine catabolism pathway could help the fungus to remove phenylpropanoid precursors thereby evading the host defense response. The in planta up-regulation of salicylate hydroxylase, intradiol ring cleavage dioxygenase and fumarylacetoacetase encoding genes, further supported this hypothesis. Those genes were even more up-regulated in HS-stressed plants, suggesting that fungus takes advantage of the increased phenylpropanoid precursors produced under stress. Pectate lyase was up-regulated while a putative amylase was down-regulated in planta, this could be associated with an intercellular growth strategy during the first stages of colonization. CONCLUSIONS: L. theobromae transcriptome was established and validated. Its usefulness was demonstrated through the identification of genes expressed during the infection process. Our results support the hypothesis that heat stress facilitates fungal colonization, because of the fungus ability to use the phenylpropanoid precursors and SA, both compounds known to control host defense.

Distinctive expansion of gene families associated with plant cell wall degradation, secondary metabolism, and nutrient uptake in the genomes of grapevine trunk pathogens.

BACKGROUND: Trunk diseases threaten the longevity and productivity of grapevines in all viticulture production systems. They are caused by distantly-related fungi that form chronic wood infections. Variation in wood-decay abilities and production of phytotoxic compounds are thought to contribute to their unique disease symptoms. We recently released the draft sequences of Eutypa lata, Neofusicoccum parvum and Togninia minima, causal agents of Eutypa dieback, Botryosphaeria dieback and Esca, respectively. In this work, we first expanded genomic resources to three important trunk pathogens, Diaporthe ampelina, Diplodia seriata, and Phaeomoniella chlamydospora, causal agents of Phomopsis dieback, Botryosphaeria dieback, and Esca, respectively. Then we integrated all currently-available information into a genome-wide comparative study to identify gene families potentially associated with host colonization and disease development. RESULTS: The integration of RNA-seq, comparative and ab initio approaches improved the protein-coding gene prediction in T. minima, whereas shotgun sequencing yielded nearly complete genome drafts of Dia. ampelina, Dip. seriata, and P. chlamydospora. The predicted proteomes of all sequenced trunk pathogens were annotated with a focus on functions likely associated with pathogenesis and virulence, namely (i) wood degradation, (ii) nutrient uptake, and (iii) toxin production. Specific patterns of gene family expansion were described using Computational Analysis of gene Family Evolution, which revealed lineage-specific evolution of distinct mechanisms of virulence, such as specific cell wall oxidative functions and secondary metabolic pathways in N. parvum, Dia. ampelina, and E. lata. Phylogenetically-informed principal component analysis revealed more similar repertoires of expanded functions among species that cause similar symptoms, which in some cases did not reflect phylogenetic relationships, thereby suggesting patterns of convergent evolution. CONCLUSIONS: This study describes the repertoires of putative virulence functions in the genomes of ubiquitous grapevine trunk pathogens. Gene families with significantly faster rates of gene gain can now provide a basis for further studies of in planta gene expression, diversity by genome re-sequencing, and targeted reverse genetic approaches. The functional validation of potential virulence factors will lead to a more comprehensive understanding of the mechanisms of pathogenesis and virulence, which ultimately will enable the development of accurate diagnostic tools and effective disease management.

Identification of Eutypa spp. Causing Eutypa Dieback of Grapevine in Eastern North America.

Eutypa dieback of grapevine is caused by Eutypa lata in production areas with Mediterranean climates in California, Australasia, Europe, and South Africa. Eutypa dieback has also been described in the colder, eastern North American vineyards where cultivars adapted from native Vitis spp. (e.g., Vitis × labruscana 'Concord') are primarily grown. However, the causal agents associated with the diseases in this region have not been conclusively identified. Examination of 48 vineyards showing symptoms of dieback in the northeastern United States (Connecticut, Massachusetts, Michigan, New York, Ohio, and Rhode Island) and Ontario, Canada revealed that vineyards were mainly infected by Eutypa spp. other than E. lata. Multigene phylogenies (internal transcribed spacer ribosomal DNA, ß-tubulin, and RNA polymerase II) of isolates recovered from these vineyards indicated that Eutypa dieback is caused primarily by an undescribed Eutypa sp. and E. laevata. Eutypa sp. was recovered from 56% of the vineyards examined, whereas E. laevata and E. lata were less far common (17 and 6%, respectively). Fruiting body morphology and spore dimensions supported phylogenetic separation of the three taxa. Pathogenicity tests conducted on Vitis vinifera 'Chardonnay' in the greenhouse and in the field verified that all three species were able to cause wood canker and to infect pruning wounds, respectively.

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.

Characterization of Species of Diaporthe from Wood Cankers of Grape in Eastern North American Vineyards.

In eastern North America, Phomopsis cane and leaf spot, caused by Phomopsis viticola, is a foliar disease of grape but, in the Mediterranean climate of western North America, P. viticola is primarily associated with wood cankers, along with other Diaporthe spp. To determine the identity of wood-infecting Diaporthe spp. in eastern North America, 65 isolates were cultured from 190 wood-canker samples from 23 vineyards with a history of Phomopsis cane and leaf spot. Identification of 29 representative isolates was based initially on morphology, followed by phylogenetic analyses of DNA sequences of the ribosomal DNA internal transcribed spacer region, elongation factor subunit 1-α, and actin in comparison with those of type specimens. Three species were identified: P. viticola, P. fukushii, and Diaporthe eres. Inoculations onto woody stems of potted Vitis labruscana 'Concord' and V. vinifera 'Chardonnay' showed that D. eres and P. fukushii were pathogenic (mean lesion lengths of 7.4 and 7.1 mm, respectively, compared with 3.5 mm for noninoculated controls) but significantly less so than wood-canker and leaf-spot isolates of P. viticola (13.5 mm). All three species infected pruning wounds of Concord and Chardonnay in the field. Our finding of pathogenic, wood-infecting Diaporthe spp. in all 23 vineyards suggests a frequent co-occurrence of the foliar symptoms of Phomopsis cane and leaf spot and wood cankers, although the latter are not always due to P. viticola.

Susceptibility of Cultivated and Wild Vitis spp. to Wood Infection by Fungal Trunk Pathogens.

Cultivars of European grapevine, Vitis vinifera, show varying levels of susceptibility to Eutypa dieback and Esca, in terms of foliar symptoms. However, little is known regarding cultivar susceptibility of their woody tissues to canker formation. Accordingly, we evaluated the relative susceptibility of V. vinifera cultivars ('Cabernet Franc', 'Cabernet Sauvignon', 'Chardonnay', 'Merlot', 'Riesling', 'Petite Syrah', and 'Thompson Seedless') and species or interspecific hybrids of North American Vitis (Vitis hybrid 'Concord', V. arizonica 'b42-26', V. rupestris × V. cinerea 'Ill547-1', and Fennell 6 [V. aestivalis] × Malaga [V. vinifera] 'DVIT0166') to canker formation by seven trunk pathogens (Neofusicoccum parvum, Lasiodiplodia theobromae, Phaeomoniella chlamydospora, Togninia minima, Phomopsis viticola, Eutypa lata, and an undescribed Eutypa sp.). Susceptibility was based on the length of wood discoloration (LWD) in the woody stems of rooted plants in duplicate greenhouse experiments. Cultivars of V. vinifera and Concord did not vary significantly in susceptibility to N. parvum or L. theobromae (LWD of 21 to 88 mm at 14 weeks post inoculation; P > 0.16), suggesting that they are similarly susceptible to Botryosphaeria dieback. The table-grape Thompson Seedless was most susceptible to P. viticola (mean LWD of 61 mm at 11 months post inoculation; P < 0.0001). V. vinifera cultivars and Concord showed similar susceptibility to the Esca pathogens, Phaeomoniella chlamydospora and T. minima. Susceptibility to E. lata was greatest in V. arizonica b42-26 (mean LWD of 96 mm at 11 months post inoculation; P < 0.03). In fact, all four American Vitis spp. were more susceptible to Eutypa dieback than the V. vinifera cultivars. Our findings suggest that no one cultivar is likely to provide resistance to the range of trunk pathogens but that certain cultivars may be promising candidates for commercially relevant host resistance in grape-production systems where the dominant cultivars are very susceptible.

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.

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.

Fungi associated with the potato taste defect in coffee beans from Rwanda.

BACKGROUND: Potato taste defect (PTD) of coffee is characterized by a raw potato like smell that leads to a lower quality taste in the brewed coffee, and harms the commercial value of some East African coffees. Although several causes for PTD have been proposed, none of them have been confirmed. Recently, high throughput sequencing techniques and bioinformatic analysis have shown great potential for identifying putative causal agents of plant diseases. Toward the goal of determining the cause of PTD, we examined raw coffee beans from Rwanda exhibiting varying PTD scores using an Illumina-based sequence analysis of the fungal rRNA ITS region. RESULTS: Six fungal amplicon sequence variants (ASVs) with high relative abundances correlated with coffee taste scores. Four of these ASVs exhibited negative correlations - Aspergillus versicolor, Penicillium cinnamopurpureum, Talaromyces radicus, and Thermomyces lanuginosus - indicating that they might be causing PTD. Two of these fungi exhibited positive correlations - Kazachstania humilis and Clavispora lusitaniae - indicating that they might be inhibiting organisms that cause PTD. CONCLUSIONS: This study addressed PTD causality from a new angle by examining fungi with high throughput sequencing. To our knowledge, this is the first study characterizing fungi associated with PTD, providing candidates for both causality and biocontrol.