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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.

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.

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.

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.

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.

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.

BackgroundTrunk 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.ResultsThe 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.ConclusionsThis 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.

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]

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]

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]

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]