PLASMO: a computer program for grapevine downy mildew development forecasting

Effect of Y-trellis and vertical shoot positioning training systems on downy mildew and botrytis bunch rot of grape in highlands of southern Brazil

Summary The practical application of pyramiding resistance genes by the use of molecular markers was investigated in a F1 progeny derived from the cross of VHR 3082-1-42 x ‘Regent’. VHR 3082-1-42 is a cross between Muscadinia rotundifolia x Vitis vinifera, backcrossed another four times with V. vinifera (PAUQUET et al. 2001). It carries the Run1-gene which causes resistance to powdery mildew and the Rpv1-gene which is related to resistance against downy mildew. Both genes were introduced from Muscadinia rotundifolia (BOUQUET et al. 2000; WIEDEMANN-MERDINOGLU et al. 2006). ‘Regent’ is a new cultivar with quantitative resistance against downy and powdery mildew (EIBACH and TOPFER 2003) released in Germany in 1996 for commercial use. 119 individuals of the F1 progeny were screened with a molecular marker for the Run1-gene (DONALD et al. 2002), with two SSR-markers for the Rpv1-gene (WIEDEMANN-MERDINOGLU et al. 2006) and with several markers from ‘Regent’ that showed good correlation to powdery and downy mildew resistance (ZYPRIAN et al. 2002, SALAKHUTDINOV et al. 2003, AKKURT 2004, FISCHER et al. 2004, AKKURT et al. 2007). Phenotypic evaluation for downy mildew resistance was done by artificial inoculation of leaf discs, and for powdery mildew by natural infection in a greenhouse. Comparison of the phenotypic data with the results of the molecular marker analyses showed a clear correlation between the degree of resistance and the presence of the resistance related alleles. According to the phenotypic data, 20 genotypes of the offspring were free of powdery and downy mildew infections. Based on a marker-assisted evaluation, out of these 20 genotypes a subset of four carried all the resistance related alleles for powdery and downy mildew indicating that resistance genes from both parents were effectively combined.

Grapevine (Vitis vinifera) is susceptible to a variety of pathogenic fungi that affect yield and wine quality. Their control is generally achieved by widespread application of fungicides in the vineyards. The economic costs and negative environmental impact associated with these applications has led to a quest for alternative strategies, focusing on manipulation of host defence mechanisms. Here, we evaluated the ability of riboflavin (i.e. Vitamin B2) to induce resistance against downy mildew in grapevine. Our results showed that 2 mM riboflavin applied 1–3 days before pathogen inoculation provided a disease reduction efficiency of 86 %. A microscopic analysis of the time course of P. viticola colonization in riboflavin-treated leaf discs suggests early inhibition of hyphae spreading in the intercellular space. This resistance does not result from a direct fungitoxic effect of riboflavin. However, this vitamin activates host-defence responses including H2O2 generation, upregulation of an array of defence-related genes and synthesis of callose in stomata cells, while stilbene synthesis is not affected. Using a pharmacological approach, we deduced that both jasmonic acid and callose biosynthesis pathways are significantly involved in riboflavin-induced resistance against downy mildew in grapevine.

The oomycete Plasmopara viticola (Berk. & Curt.) Berl. & de Toni causes downy mildew, one of the most devastating diseases of grapevine (Vitis vinifera L.). Traditional European grapevine cultivars are highly susceptible to this obligate biotrophic pathogen. Large amounts of fungicides are necessary to protect the grapevine plants and secure harvest. This strong requirement for protective chemicals conflicts with the modern demand for sustainability in agriculture. A significant reduction of chemical protection is possible by generating novel robust grapevine cultivars through resistance breeding. Current grapevine breeding utilizes marker-assisted genetic selection. The aim is to combine diverse resistance loci for durable resistance. Markers tagging various resistance loci were elaborated during the last 10 years. However, knowledge about the conveyed defense mechanisms is still sparse but would be essential to optimize the combination of resistance loci. Asian Vitis amurensis accessions carry resistance against downy mildew e.g. in the Rpv10 locus. This locus has been introgressed into the resistant grapevine cultivar ‘Solaris’ and was genetically mapped to chromosome nine. To understand its mode of action in early defense reactions we performed a comparative RNA sequencing analysis after pathogen challenge of Rpv10-carriers, Rpv10-carriers containing additionally the resistance locus Rpv3 from American Vitis sp. origin and non-Rpv carriers. This study indicated comprehensive transcriptional re-programming and a large number of differentially expressed genes. The data indicates that the difference between resistant and susceptible grapevines relies in the increased amount of responsive genes and the efficiency of early signal transduction. This results in the fast activation of large gene clusters encoding phenylalanine ammonium lyase and stilbene synthase on chromosome 16.

14-3-3 proteins are highly conserved regulatory molecules in plants. In grapevine (Vitis vinifera L.), 14-3-3 proteins are studied under abiotic stress. However, the role of 14-3-3 proteins in the interaction between grapevine and downy mildew is yet to be studied. In this study, we identified a highly conserved 14-3-3 protein in grapevine and performed a phylogenetic analysis, revealing a close relationship between one of its homologs, 14-3-3ω proteins from Arabidopsis thaliana and Nicotiana benthamiana. We designated this homolog as Vv14-3-3ω. Subcellular localization studies showed that Vv14-3-3ω resides in the plasma membrane and cytoplasm. Expression analysis revealed a strong induction of Vv14-3-3ω at early time points following Plasmopara viticola infection, correlating with enhanced pathogen sporulation in grapevine. Furthermore, transient overexpression of Vv14-3-3ω in N. benthamiana increased susceptibility to the Phytophthora capsici pathogen and suppressed Flg22-induced pattern-triggered immunity (PTI) responses. Overexpression of Vv14-3-3ω in Nb14-3-3-silenced N. benthamiana plants resulted in increased susceptibility to P. capsici, suggesting functional conservation of this isoform. These findings indicate that Vv14-3-3ω functions as a susceptibility factor, facilitating pathogen infection and disease progression in grapevine, and highlight its potential role for improving resistance against downy mildew.

Members of the plant-specific GASA (gibberellic acid-stimulated Arabidopsis) gene family have multiple potential roles in plant growth and development, particularly in flower induction and seed development. However, limited information is available about the functions of these genes in fruit plants, particularly in grapes. We identified 14 GASA genes in grapevine (Vitis vinifera L.) and performed comprehensive bioinformatics and expression analyses. In the bioinformatics analysis, the locations of genes on chromosomes, physiochemical properties of proteins, protein structure, and subcellular positions were described. We evaluated GASA proteins in terms of domain structure, exon-intron distribution, motif arrangements, promoter analysis, phylogenetic, and evolutionary history. According to the results, the GASA domain is conserved in all proteins and the proteins are divided into three well-conserved subgroups. Synteny analysis proposed that segmental and tandem duplication have played a role in the expansion of the GASA gene family in grapes, and duplicated gene pairs have negative selection pressure. Most of the proteins were predicted to be in the extracellular region, chloroplasts, and the vacuole. In silico promoter analysis suggested that the GASA genes may influence different hormone signaling pathways and stress-related mechanisms. Additionally, we performed a comparison of the expression between seedless (Thompson seedless) and seeded (Red globe) cultivars in different plant parts, including the ovule during different stages of development. Furthermore, some genes were differentially expressed in different tissues, signifying their role in grapevine growth and development. Several genes (VvGASA2 and 7) showed different expression levels in later phases of seed development in Red globe and Thompson seedless, suggesting their involvement in seed development. Our study presents the first genome-wide identification and expression profiling of grapevine GASA genes and provides the basis for functional characterization of GASA genes in grapes. We surmise that this information may provide new potential resources for the molecular breeding of grapes.

Comparison of grape seedlings population against downy mildew by using different provocation methods.

Grapevine (Vitis vinifera) is a valuable crop in Europe for both economical and cultural reasons, but highly susceptible to Downy mildew (DM). The generation of resistant vines is of critical importance for a sustainable viticulture and can be achieved either by introgression of resistance genes in susceptible varieties or by mutation of Susceptibility (S) genes, e.g., by gene editing. This second approach offers several advantages: it maintains the genetic identity of cultivars otherwise disrupted by crossing and generally results in a broad-spectrum and durable resistance, but it is hindered by the poor knowledge about S genes in grapevines. Candidate S genes are Downy mildew Resistance 6 (DMR6) and DMR6-Like Oxygenases (DLOs), whose mutations confer resistance to DM in Arabidopsis. In this work, we show that grapevine VviDMR6-1 complements the Arabidopsis dmr6-1 resistant mutant. We studied the expression of grapevine VviDMR6 and VviDLO genes in different organs and in response to the DM causative agent Plasmopara viticola. Through an automated evaluation of causal relationships among genes, we show that VviDMR6-1, VviDMR6-2, and VviDLO1 group into different co-regulatory networks, suggesting distinct functions, and that mostly VviDMR6-1 is connected with pathogenesis-responsive genes. Therefore, VviDMR6-1 represents a good candidate to produce resistant cultivars with a gene-editing approach.

Grape downy mildew, caused by Plasmopara viticola, is a relatively new disease in Western Australia (WA). The effect of temperature and light intensity on zoospore germination and penetration of stomatal apertures by a WA isolate of P. viticola in the presence of free water on leaf discs of grapevine (Vitis vinifera) is described. Germination and penetration commenced within the first hour following inoculation. Zoospores germinated at temperatures ranging from 5 to 30°C, but not at 35°C. Germ tubes penetrated stomatal apertures at temperatures ranging from 10 and 25°C, but not at 30 or 35°C and rarely at 5°C. A 20°C dark environment favoured zoospore germination and host penetration, whereas both infection events occurred at a lower frequency in the light. Light exerted an inhibitory effect on the development of zoospores soon after their release from sporangia. This in turn influenced the infectivity of the pathogen.

As grapevine (Vitis vinifera L.) is rarely produced in Bangladesh because of unavailability of improved varieties, so this study was designed to solve this problem through evaluating the effects of hormonal combination for the duration of in vitro micro propagation of grapevine (Vitis vinifera L.) from shoot tips and nodal segments. Firstly, surface sterilization process was carried out by using HgCl2 (mercuric chlorite) at 0.1% for 3 min and best result was found. During establishment stage, explants were cultured on MS (Murashige and Skoog) basal medium supplemented with BAP (6-benzylamino purine) 0.5, 1.0 and 2.0 mg/l and NAA (β-naphthalene acetic acid). 0.1mg/l where MS+ BAP 1.0 mg/l + NAA 0.1mg/l displayed best potential result. During shoot multiplication stage, BAP 2.0 and 3.0 mg/l and NAA 0.1, 0.2 and 0.3 mg/l and their combination were used and highest number of proliferated shoots was obtained from MS+ BAP 3.0 mg/l + NAA 0.2 mg/l. For rooting stage, NAA 0.5 and 1.0 mg/l and IBA (Indol-3-butyric acid) 0.5, 1.0 and 1.5 mg/l were used and tested. The highest rooting percentage, number of roots per shoot and root length found in MS+ 0.5 mg/l NAA + IBA 1.0 mg/l. Finally, neo-formed plantlets were transferred into pots containing peat moss and sand (1:1 v/v) and potential growth of these plantlets in environment indicates that through using the adequate amount of hormonal combination could give a better solution for the improvement and availability of grapevine (Vitis vinifera L.) for Bangladeshi farmers.

Microtubules play important roles during growth and morphogenesis of plant cells. Multiple isoforms of alpha- and beta-tubulin accumulate in higher plant cells and originate either by transcription of different genes or by post-translational modifications. The use of different tubulin isoforms involves the binding of microtubules to different associated proteins and therefore generates microtubules with different organizations and functions. Tubulin isoforms are differentially expressed in vegetative and reproductive structures according to the developmental program of plants. In grapevine (Vitis vinifera L.), vegetative and reproductive structures appear on the same stem, making this plant species an excellent model to study the accumulation of tubulin isoforms. Proteins were extracted from grapevine samples (buds, leaves, flowers and tendrils) using an optimized extraction protocol, separated by two-dimensional electrophoresis and analyzed by immunoblot with anti-tubulin antibodies. We identified eight alpha-tubulin and seven beta-tubulin isoforms with pI around 4.8-5 that group into separate clusters. More acidic alpha-tubulin isoforms were detected in buds, while more basic alpha-isoforms were prevalently found in tendrils and flowers. Similarly, more acidic beta-tubulin isoforms were used in the bud stage while a basic beta-tubulin isoform was essentially used in leaves and two central beta-tubulin isoforms were characteristically used in tendrils and flowers. Acetylated alpha-tubulin was not detected in any sample while tyrosinated alpha-tubulin was essentially found in large latent buds and in bursting buds in association with a distinct subset of tubulin isoforms. The implication of these data on the use of different tubulin isoforms during grapevine development is discussed.

Sweet basil ( Ocimum basilicum ) is the most economically important culinary herb in the United States. In 2007, a new disease, basil downy mildew (BDM), caused by the oomycete pathogen Peronospora belbahrii , was introduced into the United States and has since caused significant losses in commercial basil production. Although no commercial sweet basils available are resistant to P. belbahrii , other species of Ocimum have exhibited potential tolerance, resistance, or both. The objectives of this work were to determine if leaf morphological characteristics including stomata density and leaf curvature correlated with infection of plants by P. belbahrii , and thus could be used as selected characters in plant breeding. In 2011, 20 Ocimum cultivars including sweet ( O. basilicum ), cinnamon ( O. basilicum ), clove ( O. basilicum ), citrus ( Ocimum × africanum syn. Ocimum citriodorum ), spice ( Ocimum americanum syn. Ocimum canum ), and holy basils ( Ocimum tenuiflorum syn. Ocimum sanctum ) were evaluated for susceptibility to downy mildew. Sweet basils were determined to be the most susceptible; cinnamon, clove, and Thai types were moderately susceptible; and citrus, spice, and holy types were least susceptible to downy mildew. Using those same 20 Ocimum species and cultivars, stomata length and density and leaf curvature were measured and correlated with downy mildew incidence and severity. In general, basil species with higher stomatal densities had higher downy mildew incidence and severity. High stomatal densities were mainly found in the sweet, cinnamon, and clove basils. Citrus and spice species with longer stomatal lengths generally exhibited lower downy mildew incidence. Holy basil, the least susceptible of all Ocimum sp. to P. belbahrii evaluated in this study, had the greatest stomatal density and shortest stomatal length. Some sweet basil cultivars with the highest downy mildew incidence also had the greatest downward leaf curvature, whereas other sweet basil cultivars with moderate downy mildew incidence had leaves that were nearly flat or curved upward. Holy, citrus, and spice basils with low downy mildew incidence had leaves that were nearly flat or curved upward. This study suggests that leaf curvature and stomatal density and length affect downy mildew development and sporulation. Considerations of these leaf morphological characteristics may be useful phenotypic traits in breeding for downy mildew resistance in Ocimum .

Analysis of histone acetyltransferase and deacetylase families of Vitis vinifera

Methods of inoculum delivery and timing of test‐row planting were assessed for efficacy in promoting development of downy mildew (Peronosclerospora sorghi) in susceptible maize, and for cost of implementation in terms of personnel and labour. Direct inoculation of pre‐germinated seed of spreader rows resulted in consistent and high incidence of downy mildew infected plants and required substantially less labour and inoculum than the spray inoculation of spreader rows. The optimum time to plant test rows was around 15 days after inoculation of spreader rows. Eight breeding lines were screened for resistance to downy mildew using the revised inoculation method. After one cycle of screening with the new method, resistance levels improved by a range of 0–44% over the previous cycle.

Soil management in semi-arid vineyards: Combined effects of organic mulching and no-tillage under different water regimes