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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.

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 Neofusicoccum parvum, one of the causal agents of Botryosphaeria dieback, is a destructive wood-infecting fungus and a serious threat to grape production worldwide. The capability to colonize woody tissue, combined with the secretion of phytotoxic compounds, is thought to underlie its pathogenicity and virulence. Here, we describe the repertoire of virulence factors and their transcriptional dynamics as the fungus feeds on different substrates and colonizes the woody stem. We assembled and annotated a highly contiguous genome using single-molecule real-time DNA sequencing. Transcriptome profiling by RNA sequencing determined the genome-wide patterns of expression of virulence factors both in vitro (potato dextrose agar or medium amended with grape wood as substrate) and in planta. Pairwise statistical testing of differential expression, followed by co-expression network analysis, revealed that physically clustered genes coding for putative virulence functions were induced depending on the substrate or stage of plant infection. Co-expressed gene clusters were significantly enriched not only in genes associated with secondary metabolism, but also in those associated with cell wall degradation, suggesting that dynamic co-regulation of transcriptional networks contributes to multiple aspects of N. parvum virulence. In most of the co-expressed clusters, all genes shared at least a common motif in their promoter region, indicative of co-regulation by the same transcription factor. Co-expression analysis also identified chromatin regulators with correlated expression with inducible clusters of virulence factors, suggesting a complex, multi-layered regulation of the virulence repertoire of N. parvum.

Grapevine trunk diseases cause important economic losses in vineyards worldwide. Neofusicoccum parvum, one of the most aggressive causal agents of the trunk disease Botryosphaeria dieback, colonizes cells and tissues of the grapevine wood, leading to the formation of an internal canker. Symptoms then extend to distal shoots, with wilting of leaves and bud mortality. Our aim was to characterize the transcriptional dynamics of grapevine genes in the woody stem and in the leaves during Neofusicoccum parvum colonization. Genome-wide transcriptional profiling at seven distinct time points (0, 3, and 24 hours; 2, 6, 8, and 12 weeks) showed that both stems and leaves undergo extensive transcriptomic reprogramming in response to infection of the stem. While most intense transcriptional responses were detected in the stems at 24 hours, strong responses were not detected in the leaves until the next sampling point at 2 weeks post-inoculation. Network co-expression analysis identified modules of co-expressed genes common to both organs and showed most of these genes were asynchronously modulated. The temporal shift between stem vs. leaf responses affected transcriptional modulation of genes involved in both signal perception and transduction, as well as downstream biological processes, including oxidative stress, cell wall rearrangement and cell death. Promoter analysis of the genes asynchronously modulated in stem and leaves during N. parvum colonization suggests that the temporal shift of transcriptional reprogramming between the two organs might be due to asynchronous co-regulation by common transcriptional regulators. Topology analysis of stem and leaf co-expression networks pointed to specific transcription factor-encoding genes, including WRKY and MYB, which may be associated with the observed transcriptional responses in the two organs.

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]

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]

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]

Fungi that cause grapevine trunk diseases, including Diplodia seriata , Eutypa lata , Neofusicoccum parvum , and Phaeomoniella chlamydospora , all colonize woody tissues, but lesions develop to different extents. Such variation is thought to correspond to fungal virulence, but it may also reflect host metabolic responses to limit colonization. From the woody stems of potted Vitis vinifera 'Cabernet-Sauvignon' in the greenhouse, we measured wood lesions and quantified metabolites at the lesion margins. Amino acids, sugars, phenolics, and terpenoids were quantified by appropriate chromatography techniques. At 3 months post-inoculation (MPI) lesions from P. chlamydospora and N. parvum were larger than those of D. seriata and E. lata. Among all pathogen treatments, compared to controls, lower concentrations of terpenoids at 0.5 MPI were followed at 2 MPI by higher concentrations of some phenolic compounds. Lesion length did not correlate with primary metabolites, but larger lesions did correlate with higher phenolic concentrations and lower terpenoid concentrations. • Lignicolous fungi infect grapevine pruning wounds to cause trunk diseases. • The metabolomic response to infection may help explain the wound-healing process. • In the greenhouse, we quantified metabolites in wood of inoculated grapevines. • High phenolic levels at 2 months may reflect compartmentalization of the fungi. [ABSTRACT FROM AUTHOR]