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Eutypa dieback of grapevine is a trunk disease that impacts vineyard productivity worldwide. Grape germplasm is typically evaluated for resistance to Eutypa dieback through controlled inoculations in the greenhouse, although the high level of replication required of this approach (40 plants per genotype) can limit the total number of genotypes evaluated. An alternative approach is to evaluate naturally infected genotypes in the field. We rated the incidence and severity of vines with the diagnostic leaf symptoms of Eutypa dieback and the incidence of mortality among such vines of 973 Vitis vinifera accessions (planted in duplicate) at the US Department of Agriculture, National Clonal Germplasm Repository in Davis, CA, USA, which is maintained as a living collection for grape research. Across 3 years and spanning a total of 5 years (2011, 2013, and 2015), 120 accessions had leaf symptoms in one or more years ("susceptible accessions"). Courbu blanc [Davis Vitis identification tag (DVIT) 2313], Frankenthal blanc (DVIT 2115), and Pinot gris (DVIT 0907) were the only accessions with leaf symptoms each year. Accessions with the most severe leaf symptoms (a rating of 5 points) were Chasselas Napoleon (DVIT 0375) and Queen of the Vineyard (DVIT 0496). We identified susceptible accessions--namely, those related to 'Chasselas' and 'Muscat'--with a shared genetic background, based on a previous single nucleotide polymorphism genotyping effort of the collection. Especially for grapevine, a long-lived perennial that is meant to produce a crop for decades, knowledge of susceptible accessions and their pedigrees can help inform breeding programs and studies on the host response to infection. [ABSTRACT FROM AUTHOR]

Eutypa dieback of grapevine is a trunk disease that affects vineyard productivity. Wood symptoms of this disease develop consistently in greenhouse-grown plants, after inoculation of woody stems with the causal fungus Eutypa lata. Wood symptoms are a common measure of host cultivar resistance and E. lata isolate virulence. Leaf symptoms of the disease also develop in the greenhouse, although reports of low correlations between severity of wood and leaf symptoms (for some cultivars and isolates) indicate that a definitive procedure is required for evaluating cultivar resistance. Three ‘phenotyping assays’, replicated with two E. lata isolates (BX1-10 and M14), were assessed for quantifying resistance of a set of Vitis vinifera cultivars (‘Black Corinth’, ‘Carignane’, ‘Husseine’, ‘Merlot’, ‘Muscat Hamburg’, ‘Palomino’, ‘Peloursin’, ‘Primitivo’, and ‘Thompson Seedless’). The methods were: Assay 1 (leaf and woodystem symptoms measured 1 year post-inoculation on plants propagated from rooted, dormant cuttings); Assay 2 (green stem symptoms measured 4 months post-inoculation on plants propagated from rooted, green cuttings); and Assay 3 (leaf symptoms measured 6 weeks post-inoculation on plants propagated from rooted, dormant cuttings). High rates of mortality among some cultivars (‘Merlot’) in Assay 3 confounded results based on leaf symptoms. Results from Assays 1 and 2 were more consistent with each other, especially for the most resistant cultivars [‘Merlot’ and ‘Primitivo’ (aka ‘Zinfandel’)]’, than they were for these cultivars in Assay 3. Compared to resistant cultivars, there was more variation in the most susceptible cultivar, including ‘Black Corinth’, ‘Carignane’, ‘Husseine’, and ‘Thompson Seedless’, regardless of the assay. Assay 1 with isolate BX1-10 was the most repeatable and provided data on wood and leaf symptoms for cultivar comparisons. Assay 2 was the most rapid, and gave results similar to those from Assay 1. Assay 2 also accommodated germplasm that can only be propagated from green cuttings. [ABSTRACT FROM AUTHOR]

Fungal canker pathogens of almond initiate infection in trees primarily through pruning wounds. Biological control agents (BCAs) have the potential to provide long-term protection of pruning wounds by colonizing the wound surfaces and underlying tissues. Laboratory and field tests were performed to assess the efficacy of various commercial and experimental BCAs as wound protectants against almond canker pathogens. Four Trichoderma-based BCAs were evaluated using detached almond stems in the laboratory against the canker pathogens Cytospora plurivora, Eutypa lata, Neofusicoccum parvum, and Neoscytalidium dimidiatum. Results indicated that Trichoderma atroviride SC1 and T. paratroviride RTFT014 significantly reduced infections by all four pathogens. The abilities of these four BCAs to protect almond pruning wounds against E. lata and N. parvum were further evaluated in field trials using two almond cultivars and during two consecutive years. Both T. atroviride SC 1 and T. paratroviride RTFT014 protected almond pruning wounds against E. lata and N. parvum as efficiently as thiophanate-methyl, the recommended fungicide for treatment of almond pruning wounds. Comparisons of different application timings of BCA in relation to pathogen inoculation revealed a significant improvement in wound protection when inoculations were conducted 7 days versus 24 h post-BCA application for N. parvum, but not for E. lata. T. atroviride SC 1 and T. paratroviride RTFT014 are promising candidates for the preventive protection of almond pruning wounds and for inclusion in integrated pest management programs and organic almond production systems. [ABSTRACT FROM AUTHOR]

Numerous fungal canker pathogens threaten almond production in California, causing high tree mortality and reducing orchard longevity. Pruning wounds are the main infection sites for these pathogens. This study aimed to identify the lowest risk period for pruning almond trees and to determine the duration of susceptibility of pruning wounds to fungal infections. During 2 years at two locations, pruning wounds were made monthly from September to January and inoculated with Botryosphaeria dothidea, Eutypa lata, Neofusicoccum parvum or Neoscytalidium dimidiatum, at 0, 1, 2, 3, 5 and 8 weeks after pruning. Pruning wounds were susceptible to infection by N. parvum and N. dimidiatum from September to December, with a significant decrease in susceptibility for wounds made in January relative to September. Fresh wounds had the highest probability of infection by N. dimidiatum, as wounds 1 week or older were less likely to be infected. Pruning month did not significantly impact wound susceptibility to B. dothidea; susceptibility decreased sharply with wound age. Wound susceptibility to E. lata significantly differed amongst pruning months, with pruning wounds of 1 week or older made in December and January being the least susceptible. These results indicated that delayed pruning in December and January can reduce the risks of canker pathogen infections compared to early pruning from September to November. This study showed that pruning wounds were most susceptible to infection during the first 2 weeks following pruning; thus, pruning wounds should be protected for at least 2 weeks shortly after pruning. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Seimatosporium spp. and closely related "pestalotioid fungi" have been isolated from vineyards worldwide, but their ecological status in grapevine wood is unclear. To determine their involvement in the grapevine trunkdisease complex, we tested the pathogenicity of Californian isolates obtained from vines with general symptoms of Botryosphaeria, Eutypa, and Phomopsis diebacks. Multilocus phylogenetic analyses revealed three species: Seimatosporium vitis and two newly described and typified species, S. luteosporum sp. nov. and S. vitifusiforme sp. nov. Inoculations to woody stems of potted grapevines of both isolates of S. vitis and one isolate of S. vitifusiforme, but not S. luteosporum, were associated with significantly larger lesions than those of noninoculated controls. Coinoculations with trunk pathogens (Cryptovalsa ampelina, Diaporthe ambigua, Diatrypella verruciformis, Dipladia seriata, and Eutypa lata), coisolated from the same wood cankers in the field, brought about increased lesion lengths for S. vitifusiforme paired with D. seriata, and S. luteosporum paired with Diaporthe ambigua. In contrast, there were no differences in lesion lengths of 5. vitis and Diatrypella verruciformis or 5. vitis and E. lata, inoculated alone or together. Our findings suggest that Seimatosporium spp. are involved in the grapevine trunk-disease complex, and their virulence may depend on or affect that of trank pathogens. [ABSTRACT FROM AUTHOR]

Key message: Rapid characterization of novel NB-LRR-associated resistance to Phomopsis cane spot on grapevine using high-throughput sampling and low-coverage sequencing for genotyping, locus mapping and transcriptome analysis provides insights into genetic resistance to a hemibiotrophic fungus.Abstract: Phomopsis cane and leaf spot, caused by the hemibiotrophic fungus Diaporthe ampelina (syn = Phomopsis viticola), reduces the productivity in grapevines. Host resistance was studied on three F1 families derived from crosses involving resistant genotypes ‘Horizon’, Illinois 547-1, Vitis cinerea B9 and V. vinifera ‘Chardonnay’. All families had progeny with extremely susceptible phenotypes, developing lesions on both dormant canes and maturing fruit clusters. Segregation of symptoms was observed under natural levels of inoculum in the field, while phenotypes on green shoots were confirmed under controlled inoculations in greenhouse. High-density genetic maps were used to localize novel qualitative resistance loci named Rda1 and Rda2 from V. cinerea B9 and ‘Horizon’, respectively. Co-linearity between reference genetic and physical maps allowed localization of Rda2 locus between 1.5 and 2.4 Mbp on chromosome 7, and Rda1 locus between 19.3 and 19.6 Mbp of chromosome 15, which spans a cluster of five NB-LRR genes. Further dissection of this locus was obtained by QTL mapping of gene expression values 14 h after inoculation across a subset of the ‘Chardonnay’ × V. cinerea B9 progeny. This provided evidence for the association between transcript levels of two of these NB-LRR genes with Rda1, with increased NB-LRR expression among susceptible progeny. In resistant parent V. cinerea B9, inoculation with D. ampelina was characterized by up-regulation of SA-associated genes and down-regulation of ethylene pathways, suggesting an R-gene-mediated response. With dominant effects associated with disease-free berries and minimal symptoms on canes, Rda1 and Rda2 are promising loci for grapevine genetic improvement. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

The grapevine trunk-disease complex limits vineyard longevity in all major grape-growing regions. Although trunk diseases have been distinguished based on etiologies (e.g., Botryosphaeria-, Eutypa-, and Phomopsis dieback, Esca) and causal agents, mixed infections of trunk pathogens and other wood-colonizing fungi are frequent in grapevines. These diverse fungal communities in grapevine span four classes in the Pezizomycotina (Ascomycota) and 10 genera in the Hymenochaetales (Basidiomycota). Traditional identification based on morphology is largely untenable because of overlap in colony characteristics or spore dimensions, or lack of sporulation in culture. When based on DNA sequencing, searches of uncurated, public molecular databases can lead to misidentifications. The new molecular database TrunkDiseaseID.org is populated with accurate rDNA ITS sequences from 250 + isolates (pathogens and saprobes) and secondary DNA barcodes for delineating closely related species. Currently, no such comprehensive database exists for grapevine wood-colonizing fungi. In addition to ITS and secondary barcode sequences, this database provides a scientific reference, origin, and ecological status for each isolate, to aid diagnosticians in communicating results and recommendations to growers. Outreach through the National Plant Diagnostic Network and the US Department of Agriculture's Identification Technology Program will connect diagnosticians to this new database for grape. [ABSTRACT FROM AUTHOR]

Grapevine wood hosts diverse fungal species, including pathogens that cause grapevine trunk diseases and wood decomposers, with detrimental effects on yields. This study focuses on the effects of two pruning systems, minimal (min-) or spur-pruning, on the community of trunk pathogens and other wood-colonizing fungi in the trunks of two cultivars, Mourvèdre and Syrah. Culture and DNA-based methods were used to describe the fungal communities. In both cultivars, especially Syrah, spur-pruned vines had more wood necrosis than min-pruned vines, and the community of spur-pruned Syrah was distinguished by its single-stranded conformational polymorphism (SSCP) profile. Diversity profiles of all 88 cultivated taxa and canonical correspondence analyses of the 15 most frequently isolated taxa revealed differences in community structure due to pruning system, trunk location, and/or wood type. Greater levels of wood necrosis may be due to the composition of the fungal community rather than to a greater diversity of taxa. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Some pathogenic species of the Botryosphaeriaceae have a latent phase, colonizing woody tissues while perennial hosts show no apparent symptoms until conditions for disease development become favorable. Detection of these pathogens is often limited to the later pathogenic phase. The latent phase is poorly characterized, despite the need for non-destructive detection tools and effective quarantine strategies, which would benefit from identification of host-based markers in leaves. Neofusicoccum parvum infects the wood of grapevines and other horticultural crops, killing the fruit-bearing shoots. We used light microscopy and high-resolution computed tomography (HRCT) to examine the spatio-temporal relationship between pathogen colonization and anatomical changes in stem sections. To identify differentially-expressed grape genes, leaves from inoculated and non-inoculated plants were examined using RNA-Seq. The latent phase occurred between 0 and 1.5 months post-inoculation (MPI), during which time the pathogen did not spread significantly beyond the inoculation site nor were there differences in lesion lengths between inoculated and non-inoculated plants. The pathogenic phase occurred between 1.5 and 2 MPI, when recovery beyond the inoculation site increased and lesion lengths of inoculated plants tripled. By 2 MPI, inoculated plants also had decreased starch content in xylem fibers and rays, and increased levels of gel-occluded xylem vessels, the latter of which HRCT revealed at a higher frequency than microscopy. RNA-Seq and screening of 21 grape expression datasets identified 20 candidate genes that were transcriptionally-activated by infection during the latent phase, and confirmed that the four best candidates (galactinol synthase, abscisic acid-induced wheat plasma membrane polypeptide-19 ortholog, embryonic cell protein 63, BURP domain-containing protein) were not affected by a range of common foliar and wood pathogens or abiotic stresses. Assuming such host responses are consistent among cultivars, and do not cross react with other trunk/foliar pathogens, these grape genes may serve as host-based markers of the latent phase of N. parvum infection. [ABSTRACT FROM AUTHOR]

Pathogen adaptation to different hosts can lead to specialization and, when coupled with reproductive isolation, genome-wide differentiation and ecological speciation. We tested the hypothesis of host specialization among California populations of Eutypa lata (causal fungus of Eutypa dieback of grapevine and apricot), which is reported from >90 species. Genetic analyses of nine microsatellite loci in 182 isolates from three hosts (grapevine, apricot, and willow) at three locations were complemented by cross-inoculations on cultivated hosts grapevine and apricot to reveal patterns of host specialization. The cultivated hosts are likely more important sources of inoculum than the wild host willow, based on our findings of higher pathogen prevalence and allelic richness in grapevine and apricot. High levels of gene flow among all three hosts and locations, and no grouping by clustering analyses, suggest neither host nor geographic differentiation. Cross-inoculations revealed diversified phenotypes harboring various performance levels in grapevine and apricot, with no apparent correlation with their host of origin. Such phenotypic diversity may enable this pathogen to persist and reproduce as a generalist. Regular genetic reshuffling through sexual recombination, frequent immigration among hosts, and the lack of habitat choice in this passively dispersed fungus may prevent fixation of alleles controlling host specialization. [ABSTRACT FROM AUTHOR]

Cadophora species are reported from grapevine ( Vitis vinifera L.) in California, South Africa, Spain, Uruguay, and Canada. Frequent isolation from vines co-infected with the Esca pathogens ( Togninia minima and Phaeomoniella chlamydospora ), and confirmation of its ability to cause wood lesions/discoloration in pathogenicity tests, suggest that C. luteo-olivacea is part of the trunk pathogen complex. In North America, little is known regarding the diversity, geographic distribution, and roles of Cadophora species as trunk pathogens. Accordingly, we characterized 37 Cadophora isolates from ten US states and two Canadian provinces, based on molecular and morphological comparisons, and pathogenicity. Phylogenetic analysis of three loci (ITS, translation elongation factor 1-alpha (TEF1-α) and beta-tubulin (BT)) distinguished two known species ( C. luteo-olivacea and Cadophora melinii ) and three newly-described species ( Cadophora orientoamericana , Cadophora novi-eboraci , and Cadophora spadicis ). C. orientoamericana , C. novi-eboraci , and C. spadicis were restricted to the northeastern US, whereas C. luteo-olivacea was only recovered from California. C. melinii was present in California and Ontario, Canada. Morphological characterization was less informative, due to significant overlap in dimensions of conidia, hyphae, conidiophores, and conidiogenous cells. Pathogenicity tests confirmed the presence of wood lesions after 24 m, suggesting that Cadophora species may have a role as grapevine trunk pathogens. [ABSTRACT FROM AUTHOR]

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', 'Caber-net 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 '111547-1', and Fennell 6 [V. aestivalis] × Malaga [V. vinifera] 'DVIT0166') to canker formation by seven trunk patho-gens (Neofusicoccum pan'um, Lasiodiplodia theobromae, Phaeomoni-ella 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. pan'um 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 rele-vant host resistance in grape-production systems where the dominant cultivars are very susceptible. [ABSTRACT FROM AUTHOR]

Grapevine trunk diseases threaten grape production worldwide. In California, preventative practices (delayed pruning, fungicide applications after pruning) that reduce infections of pruning wounds by pathogen spores produced with rain are timed during the dormant season, when high spore-trap counts of the causal fungi are reported from studies in mature, symptomatic vineyards. Similar studies in young, asymptomatic vineyards are lacking. Given infrequent usage by California growers of preventative practices in young vineyards, empirical data on spore detection may provide convincing evidence to start preventing trunk diseases before symptoms appear. Active and passive spore traps were examined after rain from December to March for four years (2013–2017) in six young, asymptomatic (<5-years-old in year 1) and six mature, symptomatic (13–18-years-old in year 1) vineyards, in Lodi and Napa, California, USA wine-grape regions. We compared detection of spores of causal fungi of Botryosphaeria-, Eutypa-, and Phomopsis diebacks, which are common and widespread trunk diseases in California. From 769 samples, we isolated 15 species in culture, with DNA sequencing of 2 bar-coding loci for species-level identification. Detections (defined as presence of a species at least once per timepoint per site) were sparse in years 1 and 2, and so statistical analyses of detections pooled across timepoints and sites were restricted to years 3 and 4. Most common among all 12 sites and both trap types were Diplodia seriata and Diaporthe chamaeropis. Our novel species-level detections of six Diaporthe species fill a gap in the knowledge of which species can spread during California's dormant season. Overall, detections in young sites in years 3 and 4 (10 and 29 detections, respectively) were significantly lower than those of mature sites (31 and 57 detections, respectively). The presence of spores in young sites, even with no symptomatic vines, suggests preventative practices are needed in young vineyards. Nonetheless, our findings of more detections in mature sites reinforces higher usage by growers of preventative practices in mature, symptomatic vineyards. [Display omitted] • Dispersal of trunk-disease spores has not been characterized in young vineyards. • Isolates of pathogenic fungi were identified by DNA sequencing of bar-coding loci. • Detection in young vineyards was a fraction of that in mature ones. • Detection in young vineyards has management implications. [ABSTRACT FROM AUTHOR]

Summary: •Investigating the dispersal of the root‐pathogenic fungus Armillaria mellea is necessary to understand its population biology. Such an investigation is complicated by both its subterranean habit and the persistence of genotypes over successive host generations. As such, host colonization by resident mycelia is thought to outcompete spore infections.•We evaluated the contributions of mycelium and spores to host colonization by examining a site in which hosts pre‐date A. mellea. Golden Gate Park (San Francisco, CA, USA) was established in 1872 primarily on sand dunes that supported no resident mycelia. Genotypes were identified by microsatellite markers and somatic incompatibility pairings. Spatial autocorrelation analyses of kinship coefficients were used to infer spore dispersal distance.•The largest genotypes measured 322 and 343 m in length, and 61 of the 90 total genotypes were recovered from only one tree. The absence of multilocus linkage disequilibrium and the high proportion of unique genotypes suggest that spore dispersal is an important part of the ecology and establishment of A. mellea in this ornamental landscape.•Spatial autocorrelations indicated a significant spatial population structure consistent with limited spore dispersal. This isolation‐by‐distance pattern suggests that most spores disperse over a few meters, which is consistent with recent, direct estimates based on spore trapping data. [ABSTRACT FROM AUTHOR]

Abstract: Outcomes of host-pathogen coevolution are influenced by migration rates of the interacting species. Reduced gene flow with increasing spatial distance between populations leads to spatial genetic structure, as predicted by the isolation by distance (IBD) model. In wind-dispersed plant-pathogenic fungi, a significant spatial genetic structure is theoretically expected if local spore dispersal is more frequent than long-distance dispersal, but this remains to be documented by empirical data. For 29 populations of the oilseed rape fungus Leptosphaeria maculans sampled from two French regions, genetic structure was determined using eight minisatellite markers. Gene diversity (H =0.62–0.70) and haplotypic richness (R =0.96–1) were high in all populations. No linkage disequilibrium was detected between loci, suggesting the prevalence of panmictic sexual reproduction. Analysis of molecular variance showed that >97% of genetic diversity was observed within populations. Genetic differentiation was low among populations (F st <0.05). Although direct methods previously revealed short-distance dispersal for L. maculans, our findings of no correlation between genetic and geographic distances among populations illustrate that the IBD model does not account for dispersal of the fungus at the spatial scale we examined. These results indicate high gene flow among French populations of L. maculans, suggesting high dispersal rates and/or large effective population sizes, two characteristics giving the pathogen high evolutionary potential against the deployment of resistant oilseed rape cultivars. [Copyright &y& Elsevier]

Armillaria mellea infects hundreds of plant species in natural and managed ecosystems throughout the Northern hemisphere. Previously reported nuclear genetic divergence between eastern and western U.S. isolates is consistent with the disjunct range of A. mellea in North America, which is restricted mainly to both coasts of the United States. We investigated patterns of population structure and genetic diversity of the eastern (northern and southern Appalachians, Ozarks, and western Great Lakes) and western (Berkeley, Los Angeles, St. Helena, and San Jose, CA) regions of the United States. In total, 156 diploid isolates were genotyped using 12 microsatellite loci. Absence of genetic differentiation within either eastern subpopulations (θST = -0.002, P = 0.5) or western subpopulations (θST = 0.004, P = 0.3) suggests that spore dispersal within each region is sufficient to prevent geographic differentiation. In contrast to the western United States, our finding of more than one genetic cluster of isolates within the eastern United States (K = 3), revealed by Bayesian assignment of multilocus genotypes in STRUCTURE and confirmed by genetic multivariate analyses, suggests that eastern subpopulations are derived from multiple founder sources. The existence of amplifiable and nonamplifiable loci and contrasting patterns of genetic diversity between the two regions demonstrate that there are two geographically isolated, divergent genetic pools of A. mellea in the United States. [ABSTRACT FROM AUTHOR]

Trunk and scaffold canker diseases (TSCDs) of almond cause significant yield and tree losses and reduce the lifespan of orchards. In California, several pathogens cause TSCDs, including Botryosphaeriaceae, Ceratocystis destructans, Eutypa lata, Collophorina hispanica, Pallidophorina paarla, Cytospora, Diaporthe, and Phytophthora spp. Field diagnosis of TSCDs is challenging because symptom delineation among the diseases is not clear. Accurate diagnosis of the causal species requires detailed examination of symptoms and subsequent isolation on medium and identification using morphological criteria and subsequent confirmation using molecular tools. The process is time-consuming and difficult, particularly as morphological characteristics are variable and overlap among species. To facilitate diagnosis of TSCD, we developed PCR assays using 23 species-specific primers designed by exploiting sequence differences in the translation elongation factor, ß-tubulin, or internal transcribed spacer gene. Using genomic DNA from pure cultures of each fungal and oomycete species, each primer pair successfully amplified a single DNA fragment from the target pathogen but not from selected nontarget pathogens or common endophytes. Although 10-fold serial dilution of fungal DNA extracted from either pure cultures or infected wood samples detected as little as 0.1 pg of DNA sample, consistent detection required 10 ng of pathogen DNA from mycelial samples or from wood chips or drill shavings from artificially or naturally infected almond wood samples with visible symptoms. The new PCR assay represents an improved tool for diagnostic laboratories and will be critical to implement effective disease surveillance and control measures. [ABSTRACT FROM AUTHOR]

In California's Mediterranean climate, management of grapevine trunk diseases (Botryosphaeria dieback, Eutypa dieback, Phomopsis dieback, Esca) focuses on protecting vines during the dormant season, when pruning coincides with rain-induced spore dispersal of the causal fungi ('trunk pathogens'). Applying protectants (fungicides, toxic barriers, sealants, biocontrol agents) after pruning can minimize infection of pruning wounds. For the dormant seasons of 2017–2019, in a 2-year-old Vitis vinifera 'Scarlet Royal' vineyard in the southern San Joaquin Valley, we evaluated tractor-spray applications of the following protectants labeled for table grapes: fluopyram + tebuconazole, pyraclostrobin + boscalid, thiophanate-methyl + myclobutanil, and Bacillus amyloliquefaciens. A comprehensive set of virulent and widespread trunk pathogens, including Diaporthe ampelina , Eutypa lata , Neofusicoccum parvum , and Phaeomoniella chlamydospora , were inoculated to pruning wounds post-application, with detection attempts after a 3 to 4-week incubation period (before budbreak). Consistently high efficacies of pyraclostrobin + boscalid (68–100% and 56–100%, respectively) and thiophanate-methyl + myclobutanil (64–77% and 91–100%, respectively) against Botryosphaeria-dieback pathogen N. parvum and Phomopsis-dieback pathogen D. ampelina suggest spray applications of these protectants could minimize infection by these common trunk pathogens. Low and variable detection rates of slow-growing Eutypa-dieback pathogen E. lata and Esca pathogen P. chlamydospora suggest that the incubation period should be longer or that more replication is needed. Low efficacy of B. amyloliquefaciens , especially with only 4 days in 2017 before pathogen inoculation, suggests this biological-control agent may need more time and/or repeated applications to become established on pruning wounds before being challenged by a pathogen. • Trunk diseases impact table grapes, but most research is done on wine grapes. • Pruning-wound protectants were tested in a California table-grape vineyard. • Two protectants were effective against Botryosphaeria and Phomopsis-dieback fungi. • A 1-month incubation may be too short for Phaeomoniella chlamydospora and Eutypa lata. • Biological-control agent Bacillus amyloliquefaciens may require repeated applications. [ABSTRACT FROM AUTHOR]

The purpose of this study was to isolate and identify Rhodococcus spp. strains from almond and pistachio rootstocks and trees in Tunisia. Twenty-eight strains were identified through 16S rDNA and vicA genes amplification and sequencing. Pea bioassay was performed to determine the pathogenicity of the strains. Representative 16S rDNA and vicA sequences of eight strains from pistachio and seven strains from almond were closely related (>98% similarity) to Rhodococcus spp. accessions in GenBank. Phylogenetic analysis based on 16S rDNA sequences revealed that the yellow-colored strains clustered with phytopathogenic Rhodococcusfascians. The red and orange-colored strains were separated into a different group with R. kroppenstedtii and R. corynebacteiroides isolates. Eleven strains affected the pea seedlings' growth and exhibited different levels of virulence. The number of shoots was significantly higher in seedlings inoculated with four Rhodococcus strains, whereas the other three strains caused up to 80% of plant height reduction and reduced root secondary growth compared to non-inoculated pea seedlings. These strains, most of which are epiphytes from asymptomatic hosts, showed strong pathogenicity during pea bioassay and were established endophytically in pea tissues. Ten att and five fas genes were detected in four strains and may represent a novel model of plant pathogenic Rhodococcus virulence. The results of our survey showed that Rhodococcus is present but not prevalent in all visited orchards of almond and pistachio rootstocks and trees. Our surveys complemented the investments being made on ornamental species in Tunisia and unveiled the presence of undocumented plant-associated Rhodococcus spp. on economically important crops. [ABSTRACT FROM AUTHOR]

It is commonly assumed that asexual lineages are short-lived evolutionarily, yet many asexual organisms can generate genetic and phenotypic variation, providing an avenue for further evolution. Previous work on the asexual plant pathogen Phytophthora ramorum NA1 revealed considerable genetic variation in the form of Structural Variants (SVs). To better understand how SVs arise and their significance to the California NA1 population, we studied the evolutionary histories of SVs and the forest conditions associated with their emergence. Ancestral state reconstruction suggests that SVs arose by somatic mutations among multiple independent lineages, rather than by recombination. We asked if this unusual phenomenon of parallel evolution between isolated populations is transmitted to extant lineages and found that SVs persist longer in a population if their genetic background had a lower mutation load. Genetic parallelism was also found in geographically distant demes where forest conditions such as host density, solar radiation, and temperature, were similar. Parallel SVs overlap with genes involved in pathogenicity such as RXLRs and have the potential to change the course of an epidemic. By combining genomics and environmental data, we identified an unexpected pattern of repeated evolution in an asexual population and identified environmental factors potentially driving this phenomenon. [ABSTRACT FROM AUTHOR]

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 x labruscana 'Concord') are primarily grown. However, the causal agents associated with the diseases in this region have not been conclusively identified. Examination of 48 vine-yards 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 in-fected by Eutypa spp. other than E. lata. Multigene phylogenies (inter- nal transcribed spacer ribosomal DNA, ß-tubulin, and RNA polymer-ase II) of isolates recovered from these vineyards indicated that Eutypa dieback is caused primarily by an undescribed Eutypa sp. and E. lae-vata. 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. [ABSTRACT FROM AUTHOR]

In eastern North America, Phomopsis cane and leaf spot, caused by Phomopsis viticola, is a foliar disease of grape but, in the Mediterra-nean climate of western North America, P. viticola is primarily associ-ated with wood cankers, along with other Diaporthe spp. To determine the identity of wood-infecting Diaporthe spp. in eastern North Amer-ica, 65 isolates were cultured from 190 wood-canker samples from 23 vineyards with a history of Phomopsis cane and leaf spot. Identifica-tion 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-a, and actin in comparison with those of type specimens. Three species were identified: P. viticola, P. fukushii, and Diaporthe eres. Inoc-ulations 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 Char-donnay in the field. Our finding of pathogenic, wood-infecting Di-aporthe spp. in all 23 vineyards suggests a frequent co-occurrence of the foliar symptoms of Phomopsis cane and leaf spot and wood can-kers, although the latter are not always due to P. viticola. [ABSTRACT FROM AUTHOR]

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 β-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 β-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. [ABSTRACT FROM AUTHOR]