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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]

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]

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]