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BACKGROUND: Capturing the genetic diversity of wild relatives is crucial for improving crops because wild species are valuable sources of agronomic traits that are essential to enhance the sustainability and adaptability of domesticated cultivars. Genetic diversity across a genus can be captured in super-pangenomes, which provide a framework for interpreting genomic variations. RESULTS: Here we report the sequencing, assembly, and annotation of nine wild North American grape genomes, which are phased and scaffolded at chromosome scale. We generate a reference-unbiased super-pangenome using pairwise whole-genome alignment methods, revealing the extent of the genomic diversity among wild grape species from sequence to gene level. The pangenome graph captures genomic variation between haplotypes within a species and across the different species, and it accurately assesses the similarity of hybrids to their parents. The species selected to build the pangenome are a great representation of the genus, as illustrated by capturing known allelic variants in the sex-determining region and for Pierces disease resistance loci. Using pangenome-wide association analysis, we demonstrate the utility of the super-pangenome by effectively mapping short reads from genus-wide samples and identifying loci associated with salt tolerance in natural populations of grapes. CONCLUSIONS: This study highlights how a reference-unbiased super-pangenome can reveal the genetic basis of adaptive traits from wild relatives and accelerate crop breeding research.

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 basidiomycete Moniliophthora roreri causes frosty pod rot of cacao (Theobroma cacao) in the western hemisphere. Moniliophthora roreri is considered asexual and haploid throughout its hemibiotrophic life cycle. To understand the processes driving genome modification, using long-read sequencing technology, we sequenced and assembled 5 high-quality M. roreri genomes out of a collection of 99 isolates collected throughout the pathogens range. We obtained chromosome-scale assemblies composed of 11 scaffolds. We used short-read technology to sequence the genomes of 22 similarly chosen isolates. Alignments among the 5 reference assemblies revealed inversions, translocations, and duplications between and within scaffolds. Isolates at the front of the pathogens expanding range tend to share lineage-specific structural variants, as confirmed by short-read sequencing. We identified, for the first time, 3 new mating type A locus alleles (5 in total) and 1 new potential mating type B locus allele (3 in total). Currently, only 2 mating type combinations, A1B1 and A2B2, are known to exist outside of Colombia. A systematic survey of the M. roreri transcriptome across 2 isolates identified an expanded candidate effector pool and provided evidence that effector candidate genes unique to the Moniliophthoras are preferentially expressed during the biotrophic phase of disease. Notably, M. roreri isolates in Costa Rica carry a chromosome segment duplication that has doubled the associated gene complement and includes secreted proteins and candidate effectors. Clonal reproduction of the haploid M. roreri genome has allowed lineages with unique genome structures and compositions to dominate as it expands its range, displaying a significant founder effect.

Wine cultivars are available to growers in multiple clonal selections with agronomic and enological differences. Phenotypic differences between clones originated from somatic mutations that accrued over thousands of asexual propagation cycles. Genetic diversity between grape cultivars remains unexplored, and tools to discriminate unequivocally clones have been lacking. This study aimed to uncover genetic variations among a group of clonal selections of 4 important Vitis vinifera cultivars: Cabernet sauvignon, Sauvignon blanc, Chardonnay, and Merlot, and use this information to develop genetic markers to discriminate the clones of these cultivars. We sequenced with short-read sequencing technology the genomes of 18 clones, including biological replicates for a total of 46 genomes. Sequences were aligned to their respective cultivars reference genome for variant calling. We used reference genomes of Cabernet sauvignon, Chardonnay, and Merlot and developed a de novo genome assembly of Sauvignon blanc using long-read sequencing. On average, 4 million variants were detected for each clone, with 74.2% being single nucleotide variants and 25.8% being small insertions or deletions (InDel). The frequency of these variants was consistent across all clones. From these variants, we validated 46 clonal markers using high-throughput amplicon sequencing for 77.7% of the evaluated clones, most of them small InDel. These results represent an advance in grapevine genotyping strategies and will benefit the viticulture industry for the characterization and identification of the plant material.

Xylella fastidiosa is a bacterium that infects crops like grapevines, coffee, almonds, citrus and olives. There is little understanding of the genes that contribute to plant resistance, the genomic architecture of resistance, and the potential role of climate in shaping resistance, in part because major crops like grapevines (Vitis vinifera) are not resistant to the bacterium. Here we study a wild grapevine species, V. arizonica, that segregates for resistance. Using genome-wide association, we identify candidate resistance genes. Resistance-associated kmers are shared with a sister species of V. arizonica but not with more distant species, suggesting that resistance evolved more than once. Finally, resistance is climate dependent, because individuals from low (

The domestication history of the avocado (Persea americana) remains unclear. We created a reference genome from the Gwen varietal, which is closely related to the economically dominant Hass varietal. Our genome assembly had an N50 of 3.37 megabases, a BUSCO score of 91%, and was scaffolded with a genetic map, producing 12 pseudo-chromosomes with 49,450 genes. We used the Gwen genome as a reference to investigate population genomics, based on a sample of 34 resequenced accessions that represented the 3 botanical groups of P. americana. Our analyses were consistent with 3 separate domestication events; we estimated that the Mexican group diverged from the Lowland (formerly known as "West Indian") and Guatemalan groups >1 million years ago. We also identified putative targets of selective sweeps in domestication events; within the Guatemalan group, putative candidate genes were enriched for fruit development and ripening. We also investigated divergence between heterodichogamous flowering types, providing preliminary evidence for potential candidate genes involved in pollination and floral development.

Postharvest fungal pathogens benefit from the increased host susceptibility that occurs during fruit ripening. In unripe fruit, pathogens often remain quiescent and unable to cause disease until ripening begins, emerging at this point into destructive necrotrophic lifestyles that quickly result in fruit decay. Here, we demonstrate that one such pathogen, Botrytis cinerea, actively induces ripening processes to facilitate infections and promote disease in tomato (Solanum lycopersicum). Assessments of ripening progression revealed that B. cinerea accelerated external coloration, ethylene production, and softening in unripe fruit, while mRNA sequencing of inoculated unripe fruit confirmed the corresponding upregulation of host genes involved in ripening processes, such as ethylene biosynthesis and cell wall degradation. Furthermore, an enzyme-linked immunosorbent assay (ELISA)-based glycomics technique used to assess fruit cell wall polysaccharides revealed remarkable similarities in the cell wall polysaccharide changes caused by both infections of unripe fruit and ripening of healthy fruit, particularly in the increased accessibility of pectic polysaccharides. Virulence and additional ripening assessment experiments with B. cinerea knockout mutants showed that induction of ripening depends on the ability to infect the host and break down pectin. The B. cinerea double knockout Δbc polygalacturonase1 Δbc polygalacturonase2 lacking two critical pectin degrading enzymes was incapable of emerging from quiescence even long after the fruit had ripened at its own pace, suggesting that the failure to accelerate ripening severely inhibits fungal survival on unripe fruit. These findings demonstrate that active induction of ripening in unripe tomato fruit is an important infection strategy for B. cinerea.

Background'Nebbiolo' is a grapevine cultivar typical of north-western Italy, appreciated for producing high-quality red wines. Grapevine cultivars are characterized by possessing highly heterozygous genomes, including a great incidence of genomic rearrangements larger than 50 bp, so called structural variations (SVs). Even though abundant, SVs are an under-explored source of genetic variation mainly due to methodological limitations at their detection.ResultsWe employed a multiple platform approach to produce long-range genomic data for two different 'Nebbiolo' clones, namely: optical mapping, long-reads and linked-reads. We performed a haplotype-resolved de novo assembly for cultivar 'Nebbiolo' (clone CVT 71) and used an ab-initio strategy to annotate it. The annotated assembly enhanced our ability to detect SVs, enabling the study of genomic regions not present in the grapevines' reference genome and accounting for their functional implications. We performed variant calling analyses at three different organizational levels: i) between haplotypes of clone CVT 71 (primary assembly vs haplotigs), ii) between 'Nebbiolo' and 'Cabernet Sauvignon' assemblies and iii) between clones CVT 71 and CVT 185, representing different 'Nebbiolo' biotypes. The cumulative size of non-redundant merged SVs indicated a total of 79.6 Mbp for the first comparison and 136.1 Mbp for the second one, while no SVs were detected for the third comparison. Interestingly, SVs differentiating cultivars and haplotypes affected similar numbers of coding genes.ConclusionsOur results suggest that SVs accumulation rate and their functional implications in 'Nebbiolo' genome are highly-dependent on the organizational level under study. SVs are abundant when comparing 'Nebbiolo' to a different cultivar or the two haplotypes of the same individual, while they turned absent between the two analysed clones.

De novo genome assembly is essential for genomic research. High-quality genomes assembled into phased pseudomolecules are challenging to produce and often contain assembly errors because of repeats, heterozygosity, or the chosen assembly strategy. Although algorithms that produce partially phased assemblies exist, haploid draft assemblies that may lack biological information remain favored because they are easier to generate and use. We developed HaploSync, a suite of tools that produces fully phased, chromosome-scale diploid genome assemblies, and performs extensive quality control to limit assembly artifacts. HaploSync scaffolds sequences from a draft diploid assembly into phased pseudomolecules guided by a genetic map and/or the genome of a closely related species. HaploSync generates a report that visualizes the relationships between current and legacy sequences, for both haplotypes, and displays their gene and marker content. This quality control helps the user identify misassemblies and guides Haplosync's correction of scaffolding errors. Finally, HaploSync fills assembly gaps with unplaced sequences and resolves collapsed homozygous regions. In a series of plant, fungal, and animal kingdom case studies, we demonstrate that HaploSync efficiently increases the assembly contiguity of phased chromosomes, improves completeness by filling gaps, corrects scaffolding, and correctly phases highly heterozygous, complex regions.

Muscadinia rotundifolia cv. Trayshed is a valuable source of resistance to grape powdery mildew. It carries 2 powdery mildew resistance-associated genetic loci, Run1.2 on chromosome 12 and Run2.2 on chromosome 18. The purpose of this study was to identify candidate resistance genes associated with each haplotype of the 2 loci. Both haplotypes of each resistance-associated locus were identified, phased, and reconstructed. Haplotype phasing allowed the identification of several structural variation events between haplotypes of both loci. Combined with a manual refinement of the gene models, we found that the heterozygous structural variants affected the gene content, with some resulting in duplicated or hemizygous nucleotide-binding leucine-rich repeat genes. Heterozygous structural variations were also found to impact the domain composition of some nucleotide-binding leucine-rich repeat proteins. By comparing the nucleotide-binding leucine-rich repeat proteins at Run1.2 and Run2.2 loci, we discovered that the 2 loci include different numbers and classes of nucleotide-binding leucine-rich repeat genes. To identify powdery mildew resistance-associated genes, we performed a gene expression profiling of the nucleotide-binding leucine-rich repeat genes at Run1.2b and Run2.2 loci with or without powdery mildew present. Several nucleotide-binding leucine-rich repeat genes were constitutively expressed, suggesting a role in powdery mildew resistance. These first complete, haplotype-resolved resistance-associated loci and the candidate nucleotide-binding leucine-rich repeat genes identified by this study are new resources that can aid the development of powdery mildew-resistant grape cultivars.

Cultivated grapevines are commonly grafted on closely related species to cope with specific biotic and abiotic stress conditions. The three North American Vitis species V. riparia, V. rupestris, and V. berlandieri, are the main species used for breeding grape rootstocks. Here, we report the diploid chromosome-scale assembly of three widely used rootstocks derived from these species: Richter 110 (110R), Kober 5BB, and 101-14 Millardet et de Grasset (Mgt). Draft genomes of the three hybrids were assembled using PacBio HiFi sequences at an average coverage of 53.1 X-fold. Using the tool suite HaploSync, we reconstructed the two sets of nineteen chromosome-scale pseudomolecules for each genome with an average haploid genome size of 494.5 Mbp. Residual haplotype switches were resolved using shared-haplotype information. These three reference genomes represent a valuable resource for studying the genetic basis of grape adaption to biotic and abiotic stresses, and designing trait-associated markers for rootstock breeding programs.

Multiple grape powdery mildew (PM) genetic resistance (R) loci have been found in wild grape species. Little is known about the defense responses associated with each R locus. In this study, we compare the defense mechanisms associated with PM resistance in interspecific crosses segregating for a single R locus from Muscadinia rotundifolia (Run1, Run1.2b, Run2.1, Run2.2), Vitis cinerea (Ren2), V. romanetii (Ren4D and Ren4U), and the interspecific hybrid Villard blanc (Ren3). By combining optical microscopy, visual scoring, and biomass estimation, we show that the eight R loci confer resistance by limiting infection at different stages. We assessed the defense mechanisms triggered in response to PM at 1 and 5 days post-inoculation (dpi) via RNA sequencing. To account for the genetic differences between species, we developed for each accession a diploid synthetic reference transcriptome by incorporating into the PN40024 reference homozygous and heterozygous sequence variants and de novo assembled transcripts. Most of the R loci exhibited a higher number of differentially expressed genes (DEGs) associated with PM resistance at 1 dpi compared to 5 dpi, suggesting that PM resistance is mostly associated with an early transcriptional reprogramming. Comparison of the PM resistance-associated DEGs showed a limited overlap between pairs of R loci, and nearly half of the DEGs were specific to a single R locus. The largest overlap of PM resistance-associated DEGs was found between Ren3 +, Ren4D +, and Ren4U + genotypes at 1 dpi, and between Ren4U + and Run1 + accessions at 5 dpi. The Ren3 +, Ren4D +, and Ren4U + were also found to have the highest number of R locus-specific DEGs in response to PM. Both shared and R locus-specific DEGs included genes from different defense-related categories, indicating that the presence of E. necator triggered distinct transcriptional responses in the eight R loci.

BackgroundIntrogressive hybridization can reassort genetic variants into beneficial combinations, permitting adaptation to new ecological niches. To evaluate evolutionary patterns and dynamics that contribute to introgression, we investigate six wild Vitis species that are native to the Southwestern United States and useful for breeding grapevine (V. vinifera) rootstocks.ResultsBy creating a reference genome assembly from one wild species, V. arizonica, and by resequencing 130 accessions, we focus on identifying putatively introgressed regions (pIRs) between species. We find six species pairs with signals of introgression between them, comprising up to ~ 8% of the extant genome for some pairs. The pIRs tend to be gene poor, located in regions of high recombination and enriched for genes implicated in disease resistance functions. To assess potential pIR function, we explore SNP associations to bioclimatic variables and to bacterial levels after infection with the causative agent of Pierce's disease (Xylella fastidiosa). pIRs are enriched for SNPs associated with both climate and bacterial levels, suggesting that introgression is driven by adaptation to biotic and abiotic stressors.ConclusionsAltogether, this study yields insights into the genomic extent of introgression, potential pressures that shape adaptive introgression, and the evolutionary history of economically important wild relatives of a critical crop.

Grapevine leafroll-associated virus (GLRaV) infections are accompanied by symptoms influenced by host genotype, rootstock, environment, and which individual or combination of GLRaVs is present. Using a dedicated experimental vineyard, we studied the responses to GLRaVs in ripening berries from Cabernet Franc grapevines grafted to different rootstocks and with zero, one, or pairs of leafroll infection(s). RNA sequencing data were mapped to a high-quality Cabernet Franc genome reference assembled to carry out this study and integrated with hormone and metabolite abundance data. This study characterized conserved and condition-dependent responses to GLRaV infection(s). Common responses to GLRaVs were reproduced in two consecutive years and occurred in plants grafted to different rootstocks in more than one infection condition. Though different infections were inconsistently distinguishable from one another, the effects of infections in plants grafted to different rootstocks were distinct at each developmental stage. Conserved responses included the modulation of genes related to pathogen detection, abscisic acid (ABA) signalling, phenylpropanoid biosynthesis, and cytoskeleton remodelling. ABA, ABA glucose ester, ABA and hormone signalling-related gene expression, and the expression of genes in several transcription factor families differentiated the effects of GLRaVs in berries from Cabernet Franc grapevines grafted to different rootstocks. These results support that ABA participates in the shared responses to GLRaV infection and differentiates the responses observed in grapevines grafted to different rootstocks.

Ribosomal DNA amplicon sequencing of grape musts has demonstrated that microorganisms occur nonrandomly and are associated with the vineyard of origin, suggesting a role for the vineyard, grape, and wine microbiome in shaping wine fermentation outcomes. Here, ribosomal DNA amplicon sequencing from grape musts and RNA sequencing of eukaryotic transcripts from primary fermentations inoculated with the wine yeast Saccharomyces cerevisiae RC212 were used to profile fermentations from 15 vineyards in California and Oregon across two vintages. These data demonstrate that the relative abundance of fungal organisms detected by ribosomal DNA amplicon sequencing correlated with neither transcript abundance from those same organisms within the RNA sequencing data nor gene expression of the inoculated RC212 yeast strain. These data suggest that the majority of the fungi detected in must by ribosomal DNA amplicon sequencing were not active during the primary stage of these inoculated fermentations and were not a major factor in determining RC212 gene expression. However, unique genetic signatures were detected within the ribosomal DNA amplicon and eukaryotic transcriptomic sequencing that were predictive of vineyard site and region. These signatures included S. cerevisiae gene expression patterns linked to nitrogen, sulfur, and thiamine metabolism. These genetic signatures of site offer insight into specific environmental factors to consider with respect to fermentation outcomes and vineyard site and regional wine characteristics.IMPORTANCE The wine industry generates billions of dollars of revenue annually, and economic productivity is in part associated with regional distinctiveness of wine sensory attributes. Microorganisms associated with grapes and wineries are influenced by region of origin, and given that some microorganisms play a role in fermentation, it is thought that microbes may contribute to the regional distinctiveness of wine. In this work, as in previous studies, it is demonstrated that specific bacteria and fungi are associated with individual wine regions and vineyard sites. However, this work further shows that their presence is not associated with detectable fungal gene expression during the primary fermentation or the expression of specific genes by the inoculate Saccharomyces cerevisiae strain RC212. The detected RC212 gene expression signatures associated with region and vineyard site also allowed the identification of flavor-associated metabolic processes and environmental factors that could impact primary fermentation outcomes. These data offer novel insights into the complexities and subtleties of vineyard-specific inoculated wine fermentation and starting points for future investigations into factors that contribute to regional wine distinctiveness.

Hermaphroditic (perfect) flowers were a key trait in grapevine domestication, enabling a drastic increase in yields due to the efficiency of self-pollination in the domesticated grapevine (Vitis vinifera L. ssp. vinifera). In contrast, all extant wild Vitis species are dioecious, each plant having only male or female flowers. In this study, we identified the male (M) and female (f) haplotypes of the sex-determining region (SDR) in the wild grapevine species V. cinerea and confirmed the boundaries of the SDR. We also demonstrated that the SDR and its boundaries are precisely conserved across the Vitis genus using shotgun resequencing data of 556 wild and domesticated accessions from North America, East Asia, and Europe. A high linkage disequilibrium was found at the SDR in all wild grape species, while different recombination signatures were observed along the hermaphrodite (H) haplotype of 363 cultivated accessions, revealing two distinct H haplotypes, named H1 and H2. To further examine the H2 haplotype, we sequenced the genome of two grapevine cultivars, 'Riesling' and 'Chardonnay'. By reconstructing the first two H2 haplotypes, we estimated the divergence time between H1 and H2 haplotypes at ∼6 million years ago, which predates the domestication of grapevine (∼8,000 y ago). Our findings emphasize the important role of recombination suppression in maintaining dioecy in wild grape species and lend additional support to the hypothesis that at least two independent recombination events led to the reversion to hermaphroditism in grapevine.

Muscadinia rotundifolia, the muscadine grape, has been cultivated for centuries in the southeastern United States. M. rotundifolia is resistant to many of the pathogens that detrimentally affect Vitis vinifera, the grape species commonly used for winemaking. For this reason, M. rotundifolia is a valuable genetic resource for breeding. Single-molecule real-time reads were combined with optical maps to reconstruct the two haplotypes of each of the 20 M. rotundifolia cv. Trayshed chromosomes. The completeness and accuracy of the assembly were confirmed using a high-density linkage map. Protein-coding genes were annotated using an integrated and comprehensive approach. This included using full-length cDNA sequencing (Iso-Seq) to improve gene structure and hypothetical spliced variant predictions. Our data strongly support that Muscadinia chromosomes 7 and 20 are fused in Vitis and pinpoint the location of the fusion in Cabernet Sauvignon and PN40024 chromosome 7. Disease-related gene numbers in Trayshed and Cabernet Sauvignon were similar, but their clustering locations were different. A dramatic expansion of the Toll/Interleukin-1 Receptor-like Nucleotide-Binding Site Leucine-Rich Repeat (TIR-NBS-LRR) class was detected on Trayshed chromosome 12 at the Resistance to Uncinula necator 1 (RUN1)/Resistance to Plasmopara viticola 1 (RPV1) locus, which confers strong dominant resistance to powdery and downy mildews. A genome browser, annotation, and Blast tool for Trayshed are available at www.grapegenomics.com.

Pierce's disease (PD) in grapevine (Vitis vinifera) is caused by the bacterial pathogen Xylella fastidiosa. X. fastidiosa is limited to the xylem tissue and following infection induces extensive plant-derived xylem blockages, primarily in the form of tyloses. Tylose-mediated vessel occlusions are a hallmark of PD, particularly in susceptible V. vinifera. We temporally monitored tylose development over the course of the disease to link symptom severity to the level of tylose occlusion and the presence/absence of the bacterial pathogen at fine-scale resolution. The majority of vessels containing tyloses were devoid of bacterial cells, indicating that direct, localized perception of X. fastidiosa was not a primary cause of tylose formation. In addition, we used X-ray computed microtomography and machine-learning to determine that X. fastidiosa induces significant starch depletion in xylem ray parenchyma cells. This suggests that a signalling mechanism emanating from the vessels colonized by bacteria enables a systemic response to X. fastidiosa infection. To understand the transcriptional changes underlying these phenotypes, we integrated global transcriptomics into the phenotypes we tracked over the disease spectrum. Differential gene expression analysis revealed that considerable transcriptomic reprogramming occurred during early PD before symptom appearance. Specifically, we determined that many genes associated with tylose formation (ethylene signalling and cell wall biogenesis) and drought stress were up-regulated during both Phase I and Phase II of PD. On the contrary, several genes related to photosynthesis and carbon fixation were down-regulated during both phases. These responses correlate with significant starch depletion observed in ray cells and tylose synthesis in vessels.

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.

Grapevine Trunk Diseases (GTDs) are a major challenge to the grape industry worldwide. GTDs are responsible for considerable loss of quality, production, and vineyard longevity. Seventy-five percent of Chilean vineyards are estimated to be affected by GTDs. GTDs are complex diseases caused by several fungi species, including members of the Botryosphaeriaceae family and Phaeomoniella chlamydospora, considered some of the most important causal agents for these diseases in Chile. In this study, we isolated 169 endophytic and 209 rhizospheric fungi from grapevines grown under organic and conventional farming in Chile. Multiple isolates of Chaetomium sp., Cladosporium sp., Clonostachys rosea, Epicoccum nigrum, Purpureocillium lilacinum, and Trichoderma sp. were evaluated for their potential of biocontrol activity against Diplodia seriata, Neofusicoccum parvum, and Pa. chlamydospora. Tests of antagonism were carried out using two dual-culture-plate methods with multiple media types, including agar containing grapevine wood extract to simulate in planta nutrient conditions. Significant pathogen growth inhibition was observed by all isolates tested. Clonostachys rosea showed 98.2% inhibition of all pathogens in the presence of grapevine wood extract. We observed 100% pathogen growth inhibition when autoclaved lignified grapevine shoots were pre-inoculated with either C. rosea strains or Trichoderma sp. Overall, these results show that C. rosea strains isolated from grapevines are promising biocontrol agents against GTDs.

Microbiomes are integral to viticulture and winemaking - collectively termed winegrowing - where diverse fungi and bacteria can exert positive and negative effects on grape health and wine quality. Wine is a fermented natural product, and the vineyard serves as a key point of entry for quality-modulating microbiota, particularly in wine fermentations that are conducted without the addition of exogenous yeasts. Thus, the sources and persistence of wine-relevant microbiota in vineyards critically impact its quality. Site-specific variations in microbiota within and between vineyards may contribute to regional wine characteristics. This includes distinctions in microbiomes and microbiota at the strain level, which can contribute to wine flavor and aroma, supporting the role of microbes in the accepted notion of terroir as a biological phenomenon. Little is known about the factors driving microbial biodiversity within and between vineyards, or those that influence annual assembly of the fruit microbiome. Fruit is a seasonally ephemeral, yet annually recurrent product of vineyards, and as such, understanding the sources of microbiota in vineyards is critical to the assessment of whether or not microbial terroir persists with inter-annual stability, and is a key factor in regional wine character, as stable as the geographic distances between vineyards. This review examines the potential sources and vectors of microbiota within vineyards, general rules governing plant microbiome assembly, and how these factors combine to influence plant-microbe interactions relevant to winemaking.

BackgroundThe geographic and temporal distributions of bacterial and fungal populations are poorly understood within the same wine grape cultivar. In this work, we describe the microbial composition from 'Pinot noir' must with respect to vintage, growing region, climate, and must chemistry across the states of California and Oregon, USA.Materials and methodsWe sampled 'Pinot noir' clone 667 clusters from 15 vineyards existing in a latitudinal gradient spanning nearly 1,200 km in California and Oregon for two vintages (2016 and 2017). Regions included five American Viticultural Areas (AVA). In order from southern California to Oregon, these AVAs were Santa Barbara, Monterey, Sonoma, Mendocino, and Willamette Valley. Uninoculated grape musts were subjected to 16S rRNA gene and ITS-1 amplicon sequencing to assess composition of microbial communities. We also measured grape maturity metrics. Finally, to describe regions by precipitation and growing degree days, we queried the Parameter-elevation Regressions on Independent Slopes Model (PRISM) spatial climate dataset.ResultsMost of the dominant bacterial taxa in must samples were in the family Enterobacteriaceae, notably the lactic acid bacteria or the acetic acid bacteria groups, but some, like the betaproteobacterial genus Massilia, belonged to groups not commonly found in grape musts. Fungal communities were dominated by Hanseniaspora uvarum (Saccharomycetaceae). We detected relationships between covariates (e.g., vintage, precipitation during the growing season, pH, titratable acidity, and total soluble solids) and bacterial genera Gluconobacter and Tatumella in the family Enterobacteraceae, Sphingomonas (Sphingomonodaceae), Lactobacillus (Lactobacillaceae), and Massilia (Oxalobacteraceae), as well as fungal genera in Hanseniaspora, Kazachstania, Lachancea, Torulaspora in the family Saccharomycetaceae, as well as Alternaria (Pleosporaceae), Erysiphe (Erysiphaceae), and Udeniomyces (Cystofilobasidiaceae). Fungal community distances were significantly correlated with geographic distances, but this was not observed for bacterial communities. Climate varied across regions and vintages, with growing season precipitation ranging from 11 mm to 285 mm and growing degree days ranging from 1,245 to 1,846.DiscussionWe determined that (1) bacterial beta diversity is structured by growing season precipitation, (2) fungal beta diversity reflects growing season precipitation and growing degree days, and (3) microbial differential abundances of specific genera vary with vintage, growing season precipitation, and fruit maturity metrics. Further, the correlation between fungal community dissimilarities and geographic distance suggests dispersal limitation and the vineyard as a source for abundant fungal taxa. Contrasting this observation, the lack of correlation between bacterial community dissimilarity and geographic distance suggests that environmental filtering is shaping these communities.

Flavor is a key attribute defining melon fruit quality and driving consumer preferences. We characterized and compared fruit ripening patterns (ethylene, respiration), physicochemical properties (rind/flesh color, firmness, soluble solids, acidity), aroma volatiles, and flavor-related sensory attributes in seven melon genotypes differing in shelf life capacity. Fruits were evaluated at optimal maturity and after storage for six days at 5 °C plus one day at room temperature. Total volatile content increased after storage in all genotypes, with esters being dominant. Shorter shelf-life genotypes, displaying a sharper climacteric phase, correlated with fruity/floral/sweet flavor-related descriptors, and with esters, sulfur-containing compounds and a terpenoid. Longer shelf-life types were associated with firmness, green and grassy aroma/flavor and aldehydes. Multivariate regression identified key volatiles that predict flavor sensory perception, which could accelerate breeding of longer shelf-life melons with improved flavor characteristics.

Flavescence dorée (FD) is a destructive phytoplasma disease of European grapevines. Spontaneous and cultivar-dependent recovery (REC) may occur in the field in FD-infected vines starting the year following the first symptoms. However, the biological underpinnings of this process are still largely unexplored. In this study, transcriptome sequencing (RNAseq), whole-genome bisulphite sequencing (WGBS) and metabolite analysis were combined to dissect molecular and metabolic changes associated to FD and REC in leaf veins collected in the field from healthy (H), FD and REC plants of the highly susceptible Vitis vinifera 'Barbera'. Genes involved in flavonoid biosynthesis, carbohydrate metabolism and stress responses were overexpressed in FD conditions, whereas transcripts linked to hormone and stilbene metabolisms were upregulated in REC vines. Accumulation patterns of abscisic acid and stilbenoid compounds analysed in the same samples confirmed the RNAseq data. In recovery conditions, we also observed the persistence of some FD-induced expression changes concerning inhibition of photosynthetic processes and stress responses. Several differentially expressed genes tied to those pathways also underwent post-transcriptional regulation by microRNAs, as outlined by merging our transcriptomic data set with a previously conducted smallRNAseq analysis. Investigations by WGBS analysis also revealed different DNA methylation marks between REC and H leaves, occurring within the promoters of genes tied to photosynthesis and secondary metabolism. The results allowed us to advance the existence of a "molecular memory" of FDp infection, involving alterations in the DNA methylation status of REC plants potentially related to transcriptional reprogramming events, in turn triggering changes in hormonal and secondary metabolite profiles.

Phytophthora megakarya and P. palmivora are oomycete pathogens that cause black pod rot of cacao (Theobroma cacao), the most economically important disease on cacao globally. While P. palmivora is a cosmopolitan pathogen, P. megakarya, which is more aggressive on cacao than P. palmivora, has been reported only in West and Central Africa where it has been spreading and devastating cacao farms since the 1950s. In this study, we reconstructed the complete diploid genomes of multiple isolates of both species using single-molecule real-time sequencing. Thirty-one additional genotypes were sequenced to analyze inter- and intra-species genomic diversity. The P. megakarya genome is exceptionally large (222 Mbp) and nearly twice the size of P. palmivora (135 Mbp) and most known Phytophthora species (∼100 Mbp on average). Previous reports pointed toward a whole-genome duplication (WGD) in P. palmivora In this study, we demonstrate that both species underwent independent and relatively recent WGD events. In P. megakarya we identified a unique combination of WGD and large-scale transposable element driven genome expansion, which places this genome in the upper range of Phytophthora genome sizes, as well as effector pools with 1,382 predicted RxLR effectors. Finally, this study provides evidence of adaptive evolution of effectors like RxLRs and Crinklers, and discusses the implications of effector expansion and diversification.

It remains a major challenge to identify the genes and mutations that lead to plant sexual differentiation. Here, we study the structure and evolution of the sex-determining region (SDR) in Vitis species. We report an improved, chromosome-scale Cabernet Sauvignon genome sequence and the phased assembly of nine wild and cultivated grape genomes. By resolving twenty Vitis SDR haplotypes, we compare male, female, and hermaphrodite haplotype structures and identify sex-linked regions. Coupled with gene expression data, we identify a candidate male-sterility mutation in the VviINP1 gene and potential female-sterility function associated with the transcription factor VviYABBY3. Our data suggest that dioecy has been lost during domestication through a rare recombination event between male and female haplotypes. This work significantly advances the understanding of the genetic basis of sex determination in Vitis and provides the information necessary to rapidly identify sex types in grape breeding programs.

BACKGROUND: Vegetatively propagated clones accumulate somatic mutations. The purpose of this study was to better appreciate clone diversity and involved defining the nature of somatic mutations throughout the genome. Fifteen Zinfandel winegrape clone genomes were sequenced and compared to one another using a highly contiguous genome reference produced from one of the clones, Zinfandel 03. RESULTS: Though most heterozygous variants were shared, somatic mutations accumulated in individual and subsets of clones. Overall, heterozygous mutations were most frequent in intergenic space and more frequent in introns than exons. A significantly larger percentage of CpG, CHG, and CHH sites in repetitive intergenic space experienced transition mutations than in genic and non-repetitive intergenic spaces, likely because of higher levels of methylation in the region and because methylated cytosines often spontaneously deaminate. Of the minority of mutations that occurred in exons, larger proportions of these were putatively deleterious when they occurred in relatively few clones. CONCLUSIONS: These data support three major conclusions. First, repetitive intergenic space is a major driver of clone genome diversification. Second, clones accumulate putatively deleterious mutations. Third, the data suggest selection against deleterious variants in coding regions or some mechanism by which mutations are less frequent in coding than noncoding regions of the genome.

The grape is one of the oldest and most important horticultural crops. Grape and wine aroma has long been of cultural and scientific interest. The diverse compound classes comprising aroma result from multiple biosynthetic pathways. Only fairly recently have researchers begun to elucidate the genetic mechanisms behind the biosynthesis and metabolism of grape volatile compounds. This review summarizes current findings regarding the genetic bases of grape and wine aroma with an aim towards highlighting areas in need of further study. From the literature, we compiled a list of functionally characterized genes involved in berry aroma biosynthesis and present them with their corresponding annotation in the grape reference genome.

In this genome report, we describe the sequencing and annotation of the genome of the wine grape Carménère (clone 02, VCR-702). Long considered extinct, this old French wine grape variety is now cultivated mostly in Chile where it was imported in the 1850s just before the European phylloxera epidemic. Genomic DNA was sequenced using Single Molecule Real Time technology and assembled with FALCON-Unzip, a diploid-aware assembly pipeline. To optimize the contiguity and completeness of the assembly, we tested about a thousand combinations of assembly parameters, sequencing coverage, error correction and repeat masking methods. The final scaffolds provide a complete and phased representation of the diploid genome of this wine grape. Comparison of the two haplotypes revealed numerous heterozygous variants, including loss-of-function ones, some of which in genes associated with polyphenol biosynthesis. Comparisons with other publicly available grape genomes and transcriptomes showed the impact of structural variation on gene content differences between Carménère and other wine grape cultivars. Among the putative cultivar-specific genes, we identified genes potentially involved in aroma production and stress responses. The genome assembly of Carménère expands the representation of the genomic variability in grapes and will enable studies that aim to understand its distinctive organoleptic and agronomical features and assess its still elusive extant genetic variability. A genome browser for Carménère, its annotation, and an associated blast tool are available at http://cantulab.github.io/data.

Transcriptomics has been widely applied to study grape berry development. With few exceptions, transcriptomic studies in grape are performed using the available genome sequence, PN40024, as reference. However, differences in gene content among grape accessions, which contribute to phenotypic differences among cultivars, suggest that a single reference genome does not represent the species' entire gene space. Though whole genome assembly and annotation can reveal the relatively unique or "private" gene space of any particular cultivar, transcriptome reconstruction is a more rapid, less costly, and less computationally intensive strategy to accomplish the same goal. In this study, we used single molecule-real time sequencing (SMRT) to sequence full-length cDNA (Iso-Seq) and reconstruct the transcriptome of Cabernet Sauvignon berries during berry ripening. In addition, short reads from ripening berries were used to error-correct low-expression isoforms and to profile isoform expression. By comparing the annotated gene space of Cabernet Sauvignon to other grape cultivars, we demonstrate that the transcriptome reference built with Iso-Seq data represents most of the expressed genes in the grape berries and includes 1,501 cultivar-specific genes. Iso-Seq produced transcriptome profiles similar to those obtained after mapping on a complete genome reference. Together, these results justify the application of Iso-Seq to identify cultivar-specific genes and build a comprehensive reference for transcriptional profiling that circumvents the necessity of a genome reference with its associated costs and computational weight.

BACKGROUND:DNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Profiling of pathogen communities associated with grapevine trunk diseases is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the fungal kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology. RESULTS:We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock communities, (ii) time-course experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were over- or under-estimated with universal primers. CONCLUSIONS:Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.

Summary: Grapevine downy mildew, caused by the oomycete Plasmopara viticola (P. viticola, Berk. & M. A. Curtis; Berl. & De Toni), is a global threat to Eurasian wine grapes Vitis vinifera. Although resistant grapevine varieties are becoming more accessible, P. viticola populations are rapidly evolving to overcome these resistances. We aimed to uncover avirulence genes related to Rpv3.1‐mediated grapevine resistance.We sequenced the genomes and characterized the development of 136 P. viticola strains on resistant and sensitive grapevine cultivars. A genome‐wide association study was conducted to identify genomic variations associated with resistant‐breaking phenotypes.We identified a genomic region associated with the breakdown of Rpv3.1 grapevine resistance (avrRpv3.1 locus). A diploid‐aware reassembly of the P. viticola INRA‐Pv221 genome revealed structural variations in this locus, including a 30 kbp deletion. Virulent P. viticola strains displayed multiple deletions on both haplotypes at the avrRpv3.1 locus. These deletions involve two paralog genes coding for proteins with 800–900 amino acids and signal peptides. These proteins exhibited a structure featuring LWY‐fold structural modules, common among oomycete effectors. When transiently expressed, these proteins induced cell death in grapevines carrying Rpv3.1 resistance, confirming their avirulence nature.This discovery sheds light on the genetic mechanisms enabling P. viticola to adapt to grapevine resistance, laying a foundation for developing strategies to manage this destructive crop pathogen. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa is a Gram-negative bacterial plant pathogen with an extremely wide host range. This species has recently been resolved into subspecies that correlate with host specificity. This review focuses on the status of X. fastidiosa pathogenic associations in plant hosts in which the bacterium is either endemic or has been recently introduced. Plant diseases associated with X. fastidiosa have been documented for over a century, and much about what is known in the context of host-pathogen interactions is based on these hosts, such as grape and citrus, in which this pathogen has been well described. Recent attention has focused on newly emerging X. fastidiosa diseases, such as in olives.TaxonomyBacteria; Gammaproteobacteria; family Xanthomonadaceae; genus Xylella; species fastidiosa.Microbiological propertiesGram-negative rod (0.25-0.35 × 0.9-3.5 μm), non-flagellate, motile via Type IV pili-mediated twitching, fastidious.Host rangeXylella fastidiosa has a broad host range that includes ornamental, ecological and agricultural plants belonging to over 300 different species in 63 different families. To date, X. fastidiosa has been found to be pathogenic in over 100 plant species. In addition, it can establish non-symptomatic associations with many plants as a commensal endophyte. Here, we list the four distinct subspecies of X. fastidiosa and some of the agriculturally relevant diseases caused by them: X. fastidiosa ssp. fastidiosa causes Pierce's disease (PD) of grapevine (Vitis vinifera); X. fastidiosa ssp. multiplex causes almond leaf scorch (ALS) and diseases on other nut and shade tree crops; X. fastidiosa ssp. pauca causes citrus variegated chlorosis (CVC) (Citrus spp.), coffee leaf scorch and olive quick decline syndrome (OQDS) (Olea europaea); X. fastidiosa ssp. sandyi causes oleander leaf scorch (OLS) (Nerium oleander). Significant host specificity seemingly exists for some of the subspecies, although this could be a result of technical biases based on the limited number of plants tested, whereas some subspecies are not as stringent in their host range and can infect several plant hosts.Disease symptomsMost X. fastidiosa-related diseases appear as marginal leaf necrosis and scorching of the leaves. In the case of PD, X. fastidiosa can also cause desiccation of berries (termed 'raisining'), irregular periderm development and abnormal abscission of petioles. In olive trees affected with OQDS, leaves exhibit marginal necrosis and defoliation, and overall tree decline occurs. Plants with ALS and OLS also exhibit the characteristic leaf scorch symptoms. Not all X. fastidiosa-related diseases exhibit the typical leaf scorch symptoms. These include CVC and Phony Peach disease, amongst others. In the case of CVC, symptoms include foliar wilt and interveinal chlorosis on the upper surfaces of the leaves (similar to zinc deficiency), which correspond to necrotic, gum-like regions on the undersides of the leaves. Additional symptoms of CVC include defoliation, dieback and hardening of fruits. Plants infected with Phony Peach disease exhibit a denser, more compact canopy (as a result of shortened internodes, darker green leaves and delayed leaf senescence), premature bloom and reduced fruit size. Some occlusions occur in the xylem vessels, but there are no foliar wilting, chlorosis or necrosis symptoms . USEFUL WEBSITES: http://www.piercesdisease.org/; https://pubmlst.org/xfastidiosa/; http://www.xylella.lncc.br/; https://nature.berkeley.edu/xylella/; https://ec.europa.eu/food/plant/plant_health_biosecurity/legislation/emergency_measures/xylella-fastidiosa_en.

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.

Lipopolysaccharides (LPS) are among the known pathogen-associated molecular patterns (PAMPs). LPSs are potent elicitors of PAMP-triggered immunity (PTI), and bacteria have evolved intricate mechanisms to dampen PTI. Here we demonstrate that Xylella fastidiosa (Xf), a hemibiotrophic plant pathogenic bacterium, possesses a long chain O-antigen that enables it to delay initial plant recognition, thereby allowing it to effectively skirt initial elicitation of innate immunity and establish itself in the host. Lack of the O-antigen modifies plant perception of Xf and enables elicitation of hallmarks of PTI, such as ROS production specifically in the plant xylem tissue compartment, a tissue not traditionally considered a spatial location of PTI. To explore translational applications of our findings, we demonstrate that pre-treatment of plants with Xf LPS primes grapevine defenses to confer tolerance to Xf challenge.

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.

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.

Pierce's disease is a major threat to grapevines caused by the bacterium Xylella fastidiosa. Although devoid of a type 3 secretion system commonly employed by bacterial pathogens to deliver effectors inside host cells, this pathogen is able to influence host parenchymal cells from the xylem lumen by secreting a battery of hydrolytic enzymes. Defining the cellular and biochemical changes induced during disease can foster the development of novel therapeutic strategies aimed at reducing the pathogen fitness and increasing plant health. To this end, we investigated the transcriptional, proteomic, and metabolomic responses of diseased Vitis vinifera compared to healthy plants. We found that several antioxidant strategies were induced, including the accumulation of gamma-aminobutyric acid (GABA) and polyamine metabolism, as well as iron and copper chelation, but these were insufficient to protect the plant from chronic oxidative stress and disease symptom development. Notable upregulation of phytoalexins, pathogenesis-related proteins, and various aromatic acid metabolites was part of the host responses observed. Moreover, upregulation of various cell wall modification enzymes followed the proliferation of the pathogen within xylem vessels, consistent with the intensive thickening of vessels' secondary walls observed by magnetic resonance imaging. By interpreting the molecular profile changes taking place in symptomatic tissues, we report a set of molecular markers that can be further explored to aid in disease detection, breeding for resistance, and developing therapeutics.

Hybrid (Vitis vinifera ×Vitis labrusca) table grape cultivars grown in the subtropics often fail to accumulate sufficient anthocyanins to achieve good uniform berry color. Growers of V. vinifera table grapes in temperate regions generally use ethephon and, more recently, (S)-cis-abscisic acid (S-ABA) to overcome this problem. The objective of this study was to determine if S-ABA applications at different timings and concentrations have an effect on anthocyanin regulatory and biosynthetic genes, pigment accumulation, and berry color of the Selection 21 cultivar, a new V. vinifera ×V. labrusca hybrid seedless grape that presents lack of red color when grown in subtropical areas. Applications of S-ABA 400 mg/L resulted in a higher accumulation of total anthocyanins and of the individual anthocyaninsanthocyanins: delphinidin-3-glucoside, cyanidin-3-glucoside, peonidin-3-glucoside, and malvidin-3-glucoside in the berry skin and improved the color attributes of the berries. Treatment with two applications at 7 days after véraison (DAV) and 21 DAV of S-ABA 400 mg/L resulted in a higher accumulation of total anthocyanins in the skin of berries and increased the gene expression of CHI, F3H, DFR, and UFGT and of the VvMYBA1 and VvMYBA2 transcription factors in the seedless grape cultivar.

We gathered genomic data from grapes (Vitis vinifera ssp. vinifera), a clonally propagated perennial crop, to address three ongoing mysteries about plant domestication. The first is the duration of domestication; archaeological evidence suggests that domestication occurs over millennia, but genetic evidence indicates that it can occur rapidly. We estimated that our wild and cultivated grape samples diverged ∼22,000 years ago and that the cultivated lineage experienced a steady decline in population size (Ne ) thereafter. The long decline may reflect low-intensity management by humans before domestication. The second mystery is the identification of genes that contribute to domestication phenotypes. In cultivated grapes, we identified candidate-selected genes that function in sugar metabolism, flower development, and stress responses. In contrast, candidate-selected genes in the wild sample were limited to abiotic and biotic stress responses. A genomic region of high divergence corresponded to the sex determination region and included a candidate male sterility factor and additional genes with sex-specific expression. The third mystery concerns the cost of domestication. Annual crops accumulate putatively deleterious variants, in part due to strong domestication bottlenecks. The domestication of perennial crops differs from that of annuals in several ways, including the intensity of bottlenecks, and it is not yet clear if they accumulate deleterious variants. We found that grape accessions contained 5.2% more deleterious variants than wild individuals, and these were more often in a heterozygous state. Using forward simulations, we confirm that clonal propagation leads to the accumulation of recessive deleterious mutations but without decreasing fitness.

Grapevine red blotch-associated virus (GRBaV) is a major threat to the wine industry in the USA. GRBaV infections (aka red blotch disease) compromise crop yield and berry chemical composition, affecting the flavor and aroma properties of must and wine. In this study, we combined genome-wide transcriptional profiling with targeted metabolite analyses and biochemical assays to characterize the impact of the disease on red-skinned berry ripening and metabolism. Using naturally infected berries collected from two vineyards, we were able to identify consistent berry responses to GRBaV across different environmental and cultural conditions. Specific alterations of both primary and secondary metabolism occurred in GRBaV-infected berries during ripening. Notably, GRBaV infections of post-véraison berries resulted in the induction of primary metabolic pathways normally associated with early berry development (e.g. thylakoid electron transfer and the Calvin cycle), while inhibiting ripening-associated pathways, such as a reduced metabolic flux in the central and peripheral phenylpropanoid pathways. We show that this metabolic reprogramming correlates with perturbations at multiple regulatory levels of berry development. Red blotch caused the abnormal expression of transcription factors (e.g. NACs, MYBs, and AP2-ERFs) and elements of the post-transcriptional machinery that function during red-skinned berry ripening. Abscisic acid, ethylene, and auxin pathways, which control both the initiation of ripening and stress responses, were also compromised. We conclude that GRBaV infections disrupt normal berry development and stress responses by altering transcription factors and hormone networks, which result in the inhibition of ripening pathways involved in the generation of color, flavor, and aroma compounds.

Little is known about the hierarchical effects of management practices, soil attributes and location factors on structure of vineyard soil microbiota. A hierarchical effect occurs when the specific influence of an experimental factor (e.g. cover crop type, compost application) on soil-borne bacterial communities is greater within a subset composing the larger set but not across the entire set (e.g. bacterial communities only respond to a management practice within a subset of soil types but not across the entire set composed of all soil types). To address this concept, we measured differences in soil bacterial and archaeal diversity in wine-grape vineyard soils throughout Napa Valley, California. We describe how vineyard management practices influence soil resources, which in turn determine shifts in soil-borne bacterial communities. Soil bacterial communities were structured with respect to management practices, specifically cover crop presence and cover crop mix, tillage, and agricultural system designation, i.e. conventional, organic and biodynamic production systems. Distinctions with respect to management were associated with differences in pH and soil resource pools: total carbon and total nitrogen of the

While genome assembly projects have been successful in many haploid and inbred species, the assembly of noninbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F1 hybrid of Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata, samples that have challenged short-read assembly approaches. The FALCON-based assemblies are substantially more contiguous and complete than alternate short- or long-read approaches. The phased diploid assembly enabled the study of haplotype structure and heterozygosities between homologous chromosomes, including the identification of widespread heterozygous structural variation within coding sequences.