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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]

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]

Cytospora species are destructive canker and dieback pathogens of woody hosts in natural and agroecosystems around the world. In this genus, molecular identification has been limited due to the paucity of multi-locus sequence typing studies and the lack of sequence data from type specimens in public repositories, stalling robust phylogenetic reconstructions. In most cases a morphological species concept could not be applied due to the plasticity of characters and significant overlap of morphological features such as spore dimensions and fruiting body characters. In this study, we employed a molecular phylogenetic framework with the inclusion of four nuclear loci (ITS, translation elongation factor 1-alpha, actin, and beta-tubulin) to unveil the biodiversity and taxonomy of this understudied important genus of plant pathogens. Phylogenetic inferences based on 150 Californian isolates revealed 15 Cytospora species associated with branch and twig cankers and dieback of almond, apricot, cherry, cottonwood, olive, peach, pistachio, plum, pomegranate, and walnut trees in California. Of the 15 species recovered in this study, 10 are newly described and typified, in addition to one new combination. The pathogenic status of the newly described Cytospora species requires further investigation as most species were associated with severe dieback and decline of diverse and economically important fruit and nut crops in California. [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]

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]

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]