The Dothiorella clade and Neos. dimidiatum show no specific pattern. The expanded secondary metabolite proteins were specifically abundant in L. missouriana, L. exigua, B. dothidea, Do. sarmentorum, and Neos. dimidiatum. The P450 family was expanded mostly in Lasiodiplodia species and B. dothidea but contracted in Diplodia species and Neos. dimidiatum. Neofusicoccum species and B. dothidea have an important representation of expanded secreted CAZymes, Diplodia and Dothiorella represent several expanded proteins, however, the numbers in Lasiodiplodia are extremely low. Transporter related genes in the Major Facilitator Superfamily were the most enriched in all the clades analyzed . The secondary metabolite related proteins type 1 Polyketide Synthases were expanded in Neos. dimidiatum, L. missouriana, L. exigua, and Do. sarmentorum, whereas Non-Ribosomal Peptide Synthetases were expanded in B. dothidea and Neos. dimidiatum. For the secreted CAZymes, Neof. nonquaesitum, Neofusicoccum hellenicum, and B. dothidea show an enrichment of the Auxiliary Activity family 3. Also, Do. iberica and Diplodia mutila show an enrichment of the Glycoside Hydrolase Family 3.To identify similarities between species in the Botryosphaeriaceae family, a phylogenetically informed-principal component analysis was applied to the significantly expanded families of virulence functions. These gene families were grouped into the functional categories based on the specialized databases, square pots plastic and the PCA was carried out using the Phyl.PCA . Phyl.PCA considers correlations among species due to phylogenetic relatedness, while correcting the matrices for nonindependence among observations .
Two separate analyses were conducted using the clock-calibrated tree presented previously and the tables of the number of genes classified as secreted CAZymes and Secondary Metabolism . Due to the close phylogenetic relationship of the Botryosphaeriaceae family, the set of secreted CAZymes is remarkably similar. However, there is a clear separation of the species that are considered to be more virulent , those belonging to the genera Neofusicoccum, Lasiodiplodia, and Botryosphaeria . At the same time, we observe a close cluster of Neofusicoccum species which are separated from the other groups mostly by the abundance of AA1, AA3, and GH5. In addition, the genus Lasiodiplodia is tightly clustered together with B. dothidea. This is driven by the abundance of AA9, GH28, and GH3, with the last family being more abundant in Lasiodiplodia species. The close clustering of Neofusicoccum, Botryosphaeria, and Lasiodiplodia is driven mostly by their similar profile of GH16 and AA3. Neoscytalidium dimidiatum is well separated from the rest of the species by the higher presence of GH76 and PL3 proteins. The PCA on secondary metabolite genes shows a similar separation of the most virulent genera from the others . Lasiodiplodia species are grouped together by similarly high profiles of T1PKS, Beta-lactone and T1PKS/NRPS clusters. Neofusicoccum species are grouped due to high numbers of terpene synthases and NRPS-like clusters. Botryosphaeria dothidea is separated because of its high abundance of NRPS, T1PKS, Terpenes, Beta-lactone, and NRPS-like clusters.In this study, we describe the genome sequences of seventeen well-known canker-causing fungal species in the Botryosphaeriaceae. The genomes assembled coupled with in-planta experiments allowed us to start analyzing the pathogenicity levels and the virulence factor profiles within this important fungal family. The level of completeness of the assembled genomes is consistent across all the drafts based on the expected and assembled genome sizes. This behavior is also confirmed by the high representation of conserved genes .
The completeness of the genomes, as well as the protein-coding genes and the repetitive DNA content, are similar to those of other woodcolonizing fungi of grape, such as Diaporthe ampelina DA912 , Di. seriata DS831 , and L. theobromae LA-SOL3 . Apart from the estimated completeness of the genomes, it is necessary to understand some of the limitations of the short reads technology, like copy number errors, chimeric contigs, and under-representation of repetitive regions . The functional annotation of the seventeen Botryosphaeriaceae species presents a broad and variableprofile of virulence factors that are used in different ways by fungi to colonize and survive in their hosts . The results show a great variation in the number of genes identified with a functional category, and these differences were usually associated with the genus of each species like those observed by Baroncelli et al. in Colletotrichum and Morales-Cruz et al. in other grapevine trunk pathogens. Researchers are inclined to think that the gene content is associated with the lifestyle and the variety of hosts . The expansion or contraction of a gene family usually occurs on functions that are under positive or negative selection. For instance, the genes related to host colonization and defense are under high pressure, therefore, it is common to encounter duplications or even losses. On the other hand, genes related to growth are more conserved and usually selected against these changes . Gene duplication events are crucial as they are considered to be one of the main processes that generate functional innovation . This process plays one of the most important roles in fungal adaptation and divergence . Host colonization during infection is mostly driven by gene expression of some groups of well-known proteins, namely, the secreted CAZymes, cytochrome P450 monooxygenases, peroxidases, and secondary metabolite-producing proteins .
The Botryosphaeriaceae family has a variable profile of these sets of genes, with the most virulent and aggressive species having, on average, greater numbers of annotated genes in these categories . In grape and pistachio, species in the genera Neofusicoccum and Lasiodiplodia, are typically more virulent than species in the genera Diplodia and Dothiorella . GH functions of β-glucosidases, β-xylosidases, glucanases, L-arabinofuranosidase, and galactanase were present in all the pathogens in this study and significantly more in Neofusicoccum and Lasiodiplodia. In the same way as the GH, AA functions like cellobiose dehydrogenases, alcohol oxidases, pyranoseoxidase were more abundant among Neofusicoccum species and B. dothidea. GH and AA play a critical role in the degradation of the host cell wall compounds , which is involved with the degree of pathogenicity within these genera, albeit on grape, the host we examined. Marsberg et al. ; Massonnet et al. , and Félix et al. , found similar numbers of CAZymes in Neof. parvum, L. theobromae, and B. dothidea, respectively. P450s are instrumental to the development of all organisms. These enzymes are involved in many aspects of primary and secondary metabolisms and are responsible for xenobiotic detoxification and degradation . Virulence may in part reflect the ability of some species to better tolerate and, further, to metabolize phenolic compounds produced by the host. Both Neof. parvum and Di. seriata can eliminate the stilbene piceid and its derivative resveratrol in vitro , but the former is better able to tolerate resveratrol derivatives ampelopsin A, hopeaphenol, isohopeaphenol, miyabenol C, and ε-viniferin, which are produced at higher levels in planta in response to Neof. parvum versus Di. seriata infection . Therefore, it is not unexpected to see a variable profile amongst genera in the Botryosphaeriaceae family and even within a single genus. As presented in Figure 2, some superfamilies are abundant in Neofusicoccum, Lasiodiplodia and Botryosphaeria genera, but other superfamilies are especially more numerous in the Basidiomycetes species included in this study. On the other hand, for most of the superfamilies presented, Sa. cerevisiae shows a considerable lack of such annotated genes, but CYP53 and CYP578 the counts are comparable with the rest of the species. This variation is sourced by the constant evolution and adaptation of the microorganism and hosts to their specific environment . As plants evolve new defense mechanisms and compounds against pathogens, plastic grow pots the fungi diversify their methods to degrade these compounds or generate new metabolites to attack their hosts . The Botryosphaeriaceae species in this study and the two Basidiomycetes present a set of fungal peroxidases that range from 41 to 62. As for the previous putative virulence factors, Neofusicoccum, Lasiodiplodia, and Botryosphaeria genera have the most annotated peroxidases, however, in this case, Diplodia also showed a comparable amount. Manganese peroxidase was only found in the two basidiomycetes. This enzyme has a critical role in the degradation of lignocellulose compounds by basidiomycetes , therefore it is very common in white-rot fungi such as F. mediterranea and St. hirsutum . Ascomycetes that rot wood are characterized as soft-rot fungi, which do not degrade lignin by producing manganese peroxidase, but instead “alter” lignin by producing lignin peroxidases, peroxidases, polyphenol oxidases, and laccases .
The former enzyme was found in higher numbers in the genus Neofusicoccum compared to other genera within the family. The hybrid ascorbate-cytochrome C peroxidase was over represented in the genera Neofusicoccum, Lasiodiplodia, and Botryosphaeria and is associated directly with the detoxification of ROS . The wide array of transporters annotated in this study suggests a high adaptation to toxic compounds, either produced by other microorganisms, the host, or potentially chemical synthesized fungicides . The number of proteins in the Major Facilitator Superfamily and Superfamily in Neofusicoccum, Lasiodiplodia, and Botryosphaeria were more numerous than the other Botryosphaeriaceae species. Protein members of the MFS family may have different functions in the influx/efflux of molecules between cells and the exterior environment, and several cases of fungicide resistances have been associated with the overexpression of certain MFS channels . The former genera have been reported to have lower sensitivities to almost full resistance to different synthetic fungicides . Similar behavior was observed in Do. sarmentorum, were the ATP-binding Cassette ishighly represented. The ABC superfamily plays different roles in fungicide resistance, mycelial growth, and overall pathogenicity . In addition, the array of secondary metabolite gene clusters is more expanded in the Botryosphaeriaceae family than in the Basidiomycetes except for terpene synthase gene clusters. T1PKS, NRPS, and hybrids of T1PKS-NRPS produce toxic polyketides and toxic polypeptides, which kill host cells and leads to disease development . To evaluate the potential differences in virulence within the Botryosphaeriaceae family in more detail, we executed a Computational Analysis of gene Family Evolution . By identifying species and gene families with higher rates of gain and loss can help us to better understand the differences in pathogenicity as it relates to the numbers of copies of virulence genes . Six hundred and sixty-six gene families of the proteins analyzed in this study have a significantly higher than expected rate of gain/loss. The annotation of putative virulence factors in Neofusicoccum, Lasiodiplodia, and Botryosphaeria shows an average expansion of these gene families, even if some of the species shows a contraction, the overall clade rate is positive. Among those expanded or contracted families there is a set of functions that are over represented. The secreted CAZymes seem to be expanding in Neof. hellenicum, Neof. nonquaesitum, B. dothidea, Di. mutila, Do. iberica, and Do. sarmentorum, whereas the Dothiorella species show contractions in some families. However, almost no significant gain/loss of secreted CAZymes appears to be occurring in the genomes of Lasiodiplodia species. The opposite scenario is observed for the P450s, where Lasiodiplodia appears to be actively evolving, showing major expansions in three of the four species in this study. Also, B. dothidea and three Neofusicoccum species show an expansion of these families. On the other side, Neos. dimidiatum, B. dothidea, Do. sarmentorum, L. exigua, and L. missouriana are actively expanding their secondary metabolite gene clusters. Finally, the wide variety of transporters present in fungi, is the result of the positive selection pressure over them. The need of the fungi to adapt to new environments and hosts had selected for multiple mutations that diversifies the transporters functions . The MFS displays the largest effect of expansion and contraction among all the species. Botryosphaeria dothidea, L. missouriana, L. exigua, and Di. mutila appear to be actively expanding the MFS transporters. However, Neos. dimidiatum, Di. seriata, Neofusicoccum vitifusiforme, Neof. australe, and Neof. mediterraneum are contracting MFS transporters. Phylo PCAs results support the idea that within the Botryosphaeriaceae family, Neofusicoccum, Lasiodiplodia, and Botryosphaeria genera are the most virulent . There was a very clear separation of these species from the Diplodia, Dothiorella, and Neoscytalidium. The secreted CAZymes that cause the clustering of the Neofusicoccum species are usually associated with laccases, cellobiose dehydrogenases, and cellulase activities. These enzymes usually target components of the plant cell wall such as lignin, cellulose, cellobiose . Among the functions driving the clustering of Lasiodiplodia and Botryosphaeria, the lytic polysaccharide monooxygenases are one of the most important. They have a role in the oxidative degradation of various biopolymers such as cellulose and chitin.