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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]

Ceratocystis canker caused by Ceratocystis destructans is a severe disease of almond, reducing the longevity and productivity of infected trees. Once the disease has established in an individual tree, there is no cure, and management efforts are often limited to removing the infected area of cankers. In this study, we present the genome assemblies of five C. destructans isolates isolated from symptomatic almond trees. The genomes were assembled into a genome size of 27.2 ± 0.9 Mbp with an average of 6924 ± 135 protein-coding genes and an average GC content of 48.8 ± 0.02%. We concentrated our efforts on identifying putative virulence factors of canker pathogens. Analysis of the secreted carbohydrate-active enzymes showed that the genomes harbored 83.4 ± 1.8 secreted CAZymes. The secreted CAZymes covered all the known categories of CAZymes. AntiSMASH revealed that the genomes had at least 7 biosynthetic gene clusters, with one of the non-ribosomal peptide synthases encoding dimethylcoprogen, a conserved virulence determinant of plant pathogenic ascomycetes. From the predicted proteome, we also annotated cytochrome P450 monooxygenases, and transporters, these are well-established virulence determinants of canker pathogens. Moreover, we managed to identify 57.4 ± 2.1 putative effector proteins. Gene Ontology (GO) annotation was applied to compare gene content with two closely related species C. fimbriata, and C. albifundus. This study provides the first genome assemblies for C. destructans, expanding genomic resources for an important almond canker pathogen. The acquired knowledge provides a foundation for further advanced studies, such as molecular interactions with the host, which is critical for breeding for resistance. [ABSTRACT FROM AUTHOR]

Ceratocystis canker caused by Ceratocystis destructans is a severe disease of almond, reducing the longevity and productivity of infected trees. Once the disease has established in an individual tree, there is no cure, and management efforts are often limited to removing the infected area of cankers. In this study, we present the genome assemblies of five C. destructans isolates isolated from symptomatic almond trees. The genomes were assembled into a genome size of 27.2 ± 0.9 Mbp with an average of 6924 ± 135 protein-coding genes and an average GC content of 48.8 ± 0.02%. We concentrated our efforts on identifying putative virulence factors of canker pathogens. Analysis of the secreted carbohydrate-active enzymes showed that the genomes harbored 83.4 ± 1.8 secreted CAZymes. The secreted CAZymes covered all the known categories of CAZymes. AntiSMASH revealed that the genomes had at least 7 biosynthetic gene clusters, with one of the non-ribosomal peptide synthases encoding dimethylcoprogen, a conserved virulence determinant of plant pathogenic ascomycetes. From the predicted proteome, we also annotated cytochrome P450 monooxygenases, and transporters, these are well-established virulence determinants of canker pathogens. Moreover, we managed to identify 57.4 ± 2.1 putative effector proteins. Gene Ontology (GO) annotation was applied to compare gene content with two closely related species C. fimbriata, and C. albifundus. This study provides the first genome assemblies for C. destructans, expanding genomic resources for an important almond canker pathogen. The acquired knowledge provides a foundation for further advanced studies, such as molecular interactions with the host, which is critical for breeding for resistance. [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]

• Biological control agents were tested in the field in almond, cherry, and grapevine. • Trichoderma atroviride SC1 protected efficiently grapevine pruning wounds. • Clonostachys rosea J1446 protected efficiently almond and cherry pruning wounds. The ascomycete fungi that cause wood canker diseases in perennial, horticultural crops are often generalist pathogens able to infect multiple host plants. These pathogens frequently infect pruning wounds, and the protection of such wounds with chemicals or biological control agents (BCAs) is an essential component of disease management. This study aimed at assessing four commercial BCAs for their ability to prevent almond, cherry, and grapevine pruning wound infections by three canker pathogens under field conditions for two growing seasons: Calosphaeria pulchella in cherry, Neofusicoccum parvum in almond and grapevine, and Eutypa lata in all three hosts. The two bacterial BCAs tested, Bacillus amyloliquefasciens MBI 600 and Streptomyces lydicus WYEC 108, performed poorly overall as infection rates in treated wounds were not significantly different than in the water-treated wounds for most pathogens in all crops. Trichoderma atroviride SC1 provided the best protection against E. lata and N. parvum in grapevine, achieving superior or equivalent protection than the standard conventional chemical thiophanate-methyl. Field trials also demonstrated that Clonostachys rosea J1446 provided satisfactory protection of pruning wounds against E. lata and C. pulchella in cherry and against E. lata and N. parvum in almond trees. The identification of effective BCAs for use in multiple crops and against multiple pathogens is a great step towards a sustainable management of fungal canker diseases by mitigating the negative impacts of chemical fungicides. [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]

Fatty acid methyl ester (FAME) analyses can be useful for distinguishing microbial species. This study conducted FAME analyses on 14 fungal species known to cause grapevine trunk diseases. FAME profiles were dominated by oleic acid, albeit profiles were characteristic enough to separate species. Discriminant analyses suggested that palmitoleic acid/sapienic acid, pentadecylic acid, and an unsaturated 17-carbon fatty acid (17:1ω8 c)could explain 79.8% of the variance in the profiles among species in the first three discriminant functions. FAME profile libraries were created for use in a commercialized software, which was able to accurately identify isolates to the species level, with a low rate (9.4%) of samples to be reassessed. Dendrograms created using neighbor-joining cluster analyses with data from FAME profiles were compared with those using internal transcribed spacer (ITS) region sequences. This revealed that FAME profiles, albeit useful for tentative species identification, should not be used for determining phylogenetic relationships because the dendrograms were significantly unconcordant. Regardless, these results demonstrated the potential of FAME analyses in quickly and initially identifying closely related fungal species or confirming conclusions from other species identification techniques that would require independent validation. [ABSTRACT FROM AUTHOR]