Phenotyping grapevine resistance to downy mildew: deep learning as a promising tool to assess sporulation and necrosis.

BACKGROUND: Downy mildew is a plant disease that affects all cultivated European grapevine varieties. The disease is caused by the oomycete Plasmopara viticola. The current strategy to control this threat relies on repeated applications of fungicides. The most eco-friendly and sustainable alternative solution would be to use bred-resistant varieties. During breeding programs, some wild Vitis species have been used as resistance sources to introduce resistance loci in Vitis vinifera varieties. To ensure the durability of resistance, resistant varieties are built on combinations of these loci, some of which are unfortunately already overcome by virulent pathogen strains. The development of a high-throughput machine learning phenotyping method is now essential for identifying new resistance loci. RESULTS: Images of grapevine leaf discs infected with P. viticola were annotated with OIV 452-1 values, a standard scale, traditionally used by experts to assess resistance visually. This descriptor takes two variables into account the complete phenotype of the symptom: sporulation and necrosis. This annotated dataset was used to train neural networks. Various encoders were used to incorporate prior knowledge of the scale's ordinality. The best results were obtained with the Swin transformer encoder which achieved an accuracy of 81.7%. Finally, from a biological point of view, the model described the studied trait and identified differences between genotypes in agreement with human observers, with an accuracy of 97% but at a high-throughput 650% faster than that of humans. CONCLUSION: This work provides a fast, full pipeline for image processing, including machine learning, to describe the symptoms of grapevine leaf discs infected with P. viticola using the OIV 452-1, a two-symptom standard scale that considers sporulation and necrosis. If symptoms are frequently assessed by visual observation, which is time-consuming, low-throughput, tedious, and expert dependent, the method developed sweeps away all these constraints. This method could be extended to other pathosystems studied on leaf discs where disease symptoms are scored with ordinal scales.

The pivotal role of high-resolution mass spectrometry in the study of grape glycosidic volatile precursors for the selection of grapevines resistant to mildews.

A breeding program to produce new grape varieties tolerant to main vine fungal pathogens (Plasmopara viticola and Erysiphe necator) is carrying out by crossing Vitis vinifera cv. "Glera" with resistant genotypes such as "Solaris," "Bronner," and "Kunleany." Firstly, resistance gene-based markers analyses allowed the identification of five genotypes, which have inherited the resistance loci against mildews. To select those that also inherited the phenotype as close as possible to 'Glera' suitable to be introduced in the Prosecco wine production protocols, the grape glycosidic derivatives were studied by UHPLC/QTOF mass spectrometry. Targeted identification of the metabolites was performed using a database expressly constructed by including the glycosidic volatile precursors previously identified in grape and wine. A total of 77 glycosidic derivatives including many aroma precursors and some variety markers, were identified. Original resistant genotypes had distinct metabolomic profiles and different to 'Glera', while the crossings showed varying similarity degrees to V. vinifera parent. Findings demonstrated the Glera × Bronner and Glera × Solaris crossings are more suitable to produce high-sustainable Prosecco wines. Coupling of glycosidic volatile precursors profiling to multivariate statistical analysis was effective for phenotypic characterization of grapes and to evaluate their enological potential.

Phenotyping for QTL identification: A case study of resistance to Plasmopara viticola and Erysiphe necator in grapevine.

Vitis vinifera is the most widely cultivated grapevine species. It is highly susceptible to Plasmopara viticola and Erysiphe necator, the causal agents of downy mildew (DM) and powdery mildew (PM), respectively. Current strategies to control DM and PM mainly rely on agrochemical applications that are potentially harmful to humans and the environment. Breeding for resistance to DM and PM in wine grape cultivars by introgressing resistance loci from wild Vitis spp. is a complementary and more sustainable solution to manage these two diseases. During the last two decades, 33 loci of resistance to P. viticola (Rpv) and 15 loci of resistance to E. necator (Ren and Run) have been identified. Phenotyping is salient for QTL characterization and understanding the genetic basis of resistant traits. However, phenotyping remains a major bottleneck for research on Rpv and Ren/Run loci and disease resistance evaluation. A thorough analysis of the literature on phenotyping methods used for DM and PM resistance evaluation highlighted phenotyping performed in the vineyard, greenhouse or laboratory with major sources of variation, such as environmental conditions, plant material (organ physiology and age), pathogen inoculum (genetic and origin), pathogen inoculation (natural or controlled), and disease assessment method (date, frequency, and method of scoring). All these factors affect resistance assessment and the quality of phenotyping data. We argue that the use of new technologies for disease symptom assessment, and the production and adoption of standardized experimental guidelines should enhance the accuracy and reliability of phenotyping data. This should contribute to a better replicability of resistance evaluation outputs, facilitate QTL identification, and contribute to streamline disease resistance breeding programs.

First investigation on polyphenols and glycosidic aroma precursors in a spontaneous colour mutant of 'Glera', the principal grape variety of Prosecco sparkling wine.

BACKGROUND: Somatic mutations in Vitis spp. are relatively frequent and can generate new agronomically interesting phenotypes. We report the discovery, genetic and chemical characterization of 'Glera rosa', a mutant for the berry skin colour of 'Glera', the main white cultivar used to produce Prosecco wine. RESULTS: We ascertained the relationship between the skin colour of 'Glera rosa' and the polymorphisms in the Myb-gene transcription factors involved in polyphenol biosynthesis. We established that VvMybA1 was homozygous (VvMybA1a/VvMybA1a) in 'Glera' but heterozygous (VvmybA1a/VvmybA1b) in the 'Glera rosa' mutant. We verified that the VvMybA1a non-functional allele contained Grapevine Retrotransposon 1 (Gret1), while in the VvmybA1b allele Gret1 was missing, and the gene function was partially restored. The effects of mutation on 'Glera rosa' grape metabolites were studied by high-resolution mass spectrometry and gas chromatography/mass spectrometry analysis. Fifteen anthocyanins and five unique flavonols were found in the 'Glera rosa' mutant. The mutation also increased the contents of trans-resveratrol and its derivatives (i.e., piceatannol, E-ε-viniferin, cis- and trans-piceid) and of some flavonols in grape. Finally, the mutation did not significantly affect the typical aroma precursors of Glera grape such as glycosidic monoterpenes, norisoprenoids and benzenoids. CONCLUSION: 'Glera rosa' could be an interesting genetic source for the wine industry to produce Prosecco DOC rosé typology (made by adding up to 15% of 'Pinot Noir'), which was introduced to the market in 2020 with a worldwide massive success. © 2022 Society of Chemical Industry.

Construction of a high-density genetic map and detection of a major QTL of resistance to powdery mildew (Erysiphe necator Sch.) in Caucasian grapes (Vitis vinifera L.).

BACKGROUND: Vitis vinifera L. is the most cultivated grapevine species worldwide. Erysiphe necator Sch., the causal agent of grape powdery mildew, is one of the main pathogens affecting viticulture. V. vinifera has little or no genetic resistances against E. necator and the grape industry is highly dependent on agrochemicals. Some Caucasian V. vinifera accessions have been reported to be resistant to E. necator and to have no genetic relationships to known sources of resistance to powdery mildew. The main purpose of this work was the study and mapping of the resistance to E. necator in the Caucasian grapes 'Shavtsitska' and 'Tskhvedianis tetra'. RESULTS: The Caucasian varieties 'Shavtsitska' and 'Tskhvedianis tetra' showed a strong partial resistance to E. necator which segregated in two cross populations: the resistant genotypes delayed and limited the pathogen mycelium growth, sporulation intensity and number of conidia generated. A total of 184 seedlings of 'Shavtsitska' x 'Glera' population were genotyped through the Genotyping by Sequencing (GBS) technology and two high-density linkage maps were developed for the cross parents. The QTL analysis revealed a major resistance locus, explaining up to 80.15% of the phenotypic variance, on 'Shavtsitska' linkage group 13, which was associated with a reduced pathogen infection as well as an enhanced plant necrotic response. The genotyping of 105 Caucasian accessions with SSR markers flanking the QTL revealed that the resistant haplotype of 'Shavtsitska' was shared by 'Tskhvedianis tetra' and a total of 25 Caucasian grape varieties, suggesting a widespread presence of this resistance in the surveyed germplasm. The uncovered QTL was mapped in the region where the Ren1 locus of resistance to E. necator, identified in the V. vinifera 'Kishmish vatkana' and related grapes of Central Asia, is located. The genetic analysis conducted revealed that the Caucasian grapes in this study exhibit a resistant haplotype different from that of Central Asian grape accessions. CONCLUSIONS: The QTL isolated in 'Shavtsitska' and present in the Caucasian V. vinifera varieties could be a new candidate gene of resistance to E. necator to use in breeding programmes. It co-localizes with the Ren1 locus but shows a different haplotype from that of grapevines of Central Asia. We therefore consider that the Caucasian resistance locus, named Ren1.2, contains a member of a cluster of R-genes, of which the region is rich, and to be linked with, or possibly allelic, to Ren1.

Rpv Mediated Defense Responses in Grapevine Offspring Resistant to Plasmopara viticola.

Downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most serious grapevine diseases. The development of new varieties, showing partial resistance to downy mildew, through traditional breeding provides a sustainable and effective solution for disease management. Marker-assisted-selection (MAS) provide fast and cost-effective genotyping methods, but phenotyping remains necessary to characterize the host-pathogen interaction and assess the effective resistance level of new varieties as well as to validate MAS selection. In this study, the Rpv mediated defense responses were investigated in 31 genotypes, encompassing susceptible and resistant varieties and 26 seedlings, following inoculation of leaf discs with P. viticola. The offspring differed in Rpv loci inherited (none, one or two): Rpv3-3 and Rpv10 from Solaris and Rpv3-1 and Rpv12 from Kozma 20-3. To improve the assessment of different resistance responses, pathogen reaction (sporulation) and host reaction (necrosis) were scored separately as independent features. They were differently expressed depending on Rpv locus: offspring carrying Rpv3-1 and Rpv12 loci showed the strongest resistance response (scarce sporulation and necrosis), those carrying Rpv3-3 locus showed the highest levels of necrosis while Rpv10 carrying genotypes showed intermediate levels of both sporulation and necrosis.