This method has been shown to produce lower variability in lesion length than the method with mycelial suspensions for the pathogens Neof. parvum and Di. seriata . For inoculations, a power drill was used to wound the cutting, approximately 2 cm below the apical node. A 5-mm agar plug from a 7-day culture on PDA was aseptically inserted into the wound and sealed with Vaseline and parafilm to prevent inoculum desiccation. Cuttings were coated in melted paraffin wax and potted in a sterile potting mix amended with slow-release fertilizer . The plants were watered twice per week for 16 weeks. Plants were arranged in a completely randomized design in two separate greenhouses at the University of California Experiment Station in Davis from June 2016 to October 2016 [natural sunlight photo period, 25 ± 1 C , 18 ± 3 C ]. The second experiment was initiated 1 week after the first experiment. The length of internal wood discoloration extending out from the inoculation site up and down the stem was measured approximately 16 weeks after inoculation in October 2016. First, plants were inspected for foliar symptoms. Then the newly developed green shoots, roots, plastic square planter pots and bark of each plant were removed and discarded, and the woody stems were surface sterilized in 1% sodium hypochlorite for 2 min and rinsed with deionized water. The length of each stem was recorded and cut longitudinally to expose wood discoloration, the length of which was measured with a digital caliper.

From each plant, ten small wood pieces were cut from the margin of the lesion with flame-sterilized scalpels and plated onto PDA in an attempt to recover the inoculated fungi. Inoculated pathogens, identified based on colony morphology, were re-isolated from all samples. Lesion lengths were used as a measure of pathogenicity. Normality and homogeneity of variances were evaluated using Shapiro–Wilk’s and Levene’s tests, respectively. ANOVA was used to determine whether there were differences in lesion length among treatments. ANOVA was performed in R using the lesion size as a function of the inoculation treatment and the experiment. Means were compared for significant effects by Tukey’s HSD post hoc test.To expand the genomic information for Botryosphaeriaceae, we de novo assembled the genomes of fifteen species isolated from multiple hosts. We included the previously published genomes of Neof. parvum and Di. seriata in the comparative genomics analysis . For all seventeen species, pathogenicity was evaluated using inoculations of potted grapevines . All seventeen species produced dark necrotic lesions in the woody stems extending upward and downward from the point of inoculation at 15 weeks post inoculation. Overall, Lasiodiplodia and Neofusicoccum spp. were the most aggressive, while Diplodia and Dothiorella spp. caused the smallest lesions. All genomes were sequenced using Illumina technology at coverage 171 ± 10× . On average, sequencing Illumina reads were assembled into 1,066 ± 308 scaffolds . The total genome assembly size varied from 37 Mbp for Dothiorella viticola to 46 Mbp for B. dothidea with an average of 42.37 ± 0.69 Mbp. The expected and assembled genome size had a discrepancy of less than ten percent on average , which suggests near completeness of the assemblies .

This result was confirmed FIGURE 1 | Box plots illustrating the distribution of lesion sizes caused by individual isolates of the 17 Botryosphaeriaceae species after 4 months incubation in potted grapevine plants grown under greenhouse conditions. Bars with the same letter were not significantly different at P < 0.05 according to Tukey’s test. by CEGMA and BUSCOs analyses, which reported an average 98.3 ± 0.2% and 98.1 ± 0.1% completeness, respectively . Interspersed repeats only accounted for 1.87 ± 0.003% of the genome assemblies. Among the classified elements, long-terminal-repeats were the most abundant, ranging from a total of 315 kbp in B. dothidea to 26 kbp in Neoscytalidium dimidiatum . The predicted protein-coding genes in the seventeen genomes varied from 10,827 in Dothiorella iberica to 13,492 in Dothiorella sarmentorum. On average 12,193 ± 193 CDS were found per species . The predicted genes of the seventeen genomes were annotated using general databases for protein domains , gene ontology , as well as more specialized databases related to putative virulence factors. The last group included carbohydrateactive enzymes , cytochrome P450s, peroxidases, usually associated with host colonization and wood degradation, and secondary metabolism gene clusters, including toxins production, and cellular transporters . A total of 229,251 predicted protein-coding genes were annotated .A wide variety of monosaccharides can be linked to many different types of molecules and converting these glycoconjugates into one of the most structurally diverse substrates . CAZymes are the group of enzymes responsible for the assembly and breakdown of these diverse substrates . Not all CAZymes contribute to the pathogenicity of the microorganisms, however, predicting themin conjunction with signal peptides is widely used to obtain information about plant pathogen cell wall degrading enzymes .

An average of 20.9 ± 0.3% of the predicted secreted proteins among all seventeen Botryosphaeriaceae genomes shared similarity with the CAZymes in the dbCAN2 database . Glycoside Hydrolases and Auxiliary Activity CAZymes were the two groups with the most predicted proteins. GHs were especially abundant in Neofusicoccum spp. with an average of 336 ± 4 proteins compared to 303 ± 7 for the rest of the Botryosphaeriaceae species in this study . A total of 15 putative genes of GH3 were present in Neofusicoccum nonquaesitum and Lasiodiplodia missouriana, as well as 14 in Neof. parvum, Lasiodiplodia citricola, and Lasiodiplodia exigua. GH3 and GH43 families activities include β-glucosidases, β-xylosidases, glucanases, L-arabinofuranosidase, galactanase and others related to the hydrolysis of plant cell wall components into more simple sugars . Auxiliary activities were also more abundant in Neofusicoccum than the rest of the Botryosphaeriaceae species . The AA3 family was the most abundant with numerous copies in the genus Neofusicoccum , ranging from 21 to 26 predicted proteins in Neof. parvum . The genome of B. dothidea was predicted to possess 23 AA3 proteins. The AA3 and AA9 families include cellobiose dehydrogenases, alcohol oxidases, pyranose oxidase, acting over more complex substrates of the plant cell wall like cellulose and/or lignin .Cytochrome P450 enzyme evolution is thought to contribute to the adaptation of organisms to new ecological niches. The functions may vary from primary metabolism, detoxification of xenobiotic compounds, to producing a vast variety of secondary metabolites . These features sometimes play essential roles in pathogenesis . The P450s were classified in superfamilies as described by Fischer et al. . Neofusicoccum, Botryosphaeria, and Lasiodiplodia species encoded a larger number of predicted P450 compared to Diplodia and Dothiorella species . Neof. nonquaesitum and Neof. parvum showed the highest number of predicted P450s genes with 918 and 908, respectively . CYP53, CYP51, square pots for planting and CYP504 were the most abundant across all the species. CYP53 was especially numerous in Neofusicoccum , Lasiodiplodia , and Botryosphaeria species compared to the other genera . On the other hand, the CYP51 was very consistent in the Botryosphaeriaceae family . Other superfamilies like CYP706, CYP102, and CYP3 show the same pattern of higher representation in Neofusicoccum, Lasiodiplodia, and Botryosphaeria than in Diplodia and Dothiorella species.Fungal peroxidases are oxidoreductases that catalyze the oxidation of various compounds ranging from ligninolysis to the detoxification of host-derived reactive oxygen species and have been shown to contribute to virulence . The annotation of these peroxidases was based on the manually curated Fungal Peroxidases Database fPoxDB . Neofusicoccum species encoded the largest number of predicted peroxidases , followed by Lasiodiplodia, Botryosphaeria, and Diplodia with an average of 54 ± 1 annotated genes .

Dothiorella, with only 46 ± 3 was the genus with the least number of annotated peroxidases in the Botryosphaeriaceae . Hybrid Ascorbate-Cytochrome C peroxidases were more abundant in Neofusicoccum, Botryosphaeria, and Lasiodiplodia, ranging from 8 to 11 genes, while haloperoxidases were more abundant in the genus Neofusicoccum .Secondary metabolites play important roles in fungal development and interactions with other organisms, including plant hosts . Phytotoxic metabolites, e.g., melleins, produced by Neof. parvum both in vitro and in the wood of symptomatic grape are thought to be associated with pathogenesis . In fungi, the genes encoding the functions responsible for the biosynthesis of secondary metabolites are physically grouped in clusters of contiguous genes , which typically comprise a central biosynthetic gene as well as genes involved in post-synthesis modification of the metabolites and cellular transport. Using antiSMASH 5 , we detected an average of 43 ± 3 biosynthetic gene clusters in the seventeen Botryosphaeriaceae. The Type I Polyketide synthase cluster and the Non-ribosomal peptide synthetaselike fragment together accounted for 47% of all annotated BGCs. BGCs were most abundant in Botryosphaeria, Neofusicoccum, and Lasiodiplodia species with an average of 57 ± 8, 56 ± 1, and 49 ± 1 BGCs, respectively. In these genera, we also found the larger number of genes per BGC . In Neofusicoccum spp., 169 ± 7 genes were associated with T1PKS, 154 ± 12 in Lasiodiplodia and 175 ± 19 in Botryosphaeria . For these secondary metabolites as well as classes, we found fewer genes in the genomes of Diplodia and Dothiorella species . Toxins and other secondary metabolites are exported by cellular transporters . Homologies with the Transporter Classification Database were used to annotate hypothetical protein transporters. Overall, the Electrochemical Potential-driven Transporters was the most prominent group across all the species representing 31 ± 1% of the annotated transporters followed by the Primary Active Transporters and the Incompletely Characterized Transport Systems . More specifically, The Major Facilitator Superfamily represented the highest number in all the species but was especially abundantin Neofusicoccum, Lasiodiplodia, and Botryosphaeria from 455 to 514 predicted genes . The genome of Dothiorella sarmentorum encodes a higher number of genes in the ATP-binding Cassette superfamily compared to the other fungi analyzed . Both MFS and ABC transporters can be involved in toxin secretion and defense responses .We further evaluated the differences in putative virulence factors to identify gene families that have significantly expanded or contracted in specific lineages by statistical analysis of the evolution of the size of gene families using Computational Analysis of gene Family Evolution . CAFE estimates the global birth and death rate of gene families and identifies those families that have an accelerated rate of gain or loss . CAFE uses a clock-calibrated phylogenetic tree and gene family sizes in all the species’ genomes as input. We included F. mediterranea and St. hirsutum, two well-known wood decay basidiomycetes related to the white-rot symptom in Esca disease of grapevines, and Sa. cerevisiae. These additional species were used as calibration points for the estimated dates of monophyletic partition of Ascomycota and Dothideomycetes as in Floudas et al. and Beimforde et al. . To construct the phylogenetic tree, we identified twenty-one single-copy protein sequences that were previously used to study phylogenetic relationships across fungi . The phylogenetic tree was built using a multiple alignment comprising 12,066 amino acid positions. The topology of the clock-calibrated tree was confirmed independently using ITS and TEF , and was consistent with published ones . The gene families were computed using a Markov Cluster algorithm that groups putative orthologs and paralogs . In total, 237,976 proteins of the 20 fungal genomes were clustered into gene families . These family sizes and the clock calibrated tree produced byBEAST allowed CAFE to detect 666 families across all the species with a significantly higher than expected rate of gene gain/loss . The numbers of gene families expanded and contracted for each branch of the phylogeny are shown in Figure 3. The parent branches of the Neofusicoccum, Lasiodiplodia, and Botryosphaeria clades show a positive rate of gene gain/losses , which suggest an expansion of some set of proteins. On the other hand, the parent branches of Diplodia, Dothiorella, and Basidiomycetes clades present a negative rate of gain losses . Saccharomyces cerevisiae, as seen in previous studies , showed the lowest rate with −1.63. The 35,498 genes in the significantly expanded or contracted families were analyzed with a Fisher’s Exact test to identify functional enrichments within those families. We found that these enrichments were not always present in all the species of a clade . However, there are some patterns to highlight. The Lasiodiplodia clade represents an overall expansion of the transporter proteins. B. dothidea also shows a significant expansion of this family. On the other hand, the Diplodia and Neofusicoccum clades show an overall contraction of transporters.