Surfactin and fengycin contribute to the protection of a Bacillus subtilis strain against grape downy mildew by both direct effect and defence stimulation

Bacillus subtilis GLB191 (hereafter GLB191) is an efficient biological control agent against the biotrophic oomycete Plasmopara viticola, the causal agent of grapevine downy mildew. In this study, we show that GLB191 supernatant is also highly active against downy mildew and that the activity results from both direct effect against the pathogen and stimulation of the plant defences (induction of defence gene expression and callose production). High-performance thin-layer chromatography analysis revealed the presence of the cyclic lipopeptides fengycin and surfactin in the supernatant. Mutants affected in the production of fengycin and/or surfactin were thus obtained and allowed us to show that both surfactin and fengycin contribute to the double activity of GLB191 supernatant against downy mildew. Altogether, this study suggests that GLB191 supernatant could be used as a new biocontrol product against grapevine downy mildew.     

Loop-mediated isothermal amplification assay for the detection of Plasmopara viticola infecting grapes

Grapes downy mildew caused by obligate oomycete plant pathogen Plasmopara viticola is a devastating disease worldwide, resulting in significant yield and quality losses. A field survey was conducted in two major grapes cultivated areas of Tamil Nadu for the incidence of grapevine downy mildew. The disease incidence was 43.42%–76.69%, and the highest disease incidence of 76.69% was observed in the Theni district. Totally eight P. viticola isolates were collected from different places in Coimbatore and Theni districts. These isolates were confirmed through microscopic observation and sequencing of COX 2 gene, and the phylogenetic tree was developed to study their phylogenetic relationship among the isolates which shows 97–100% sequence similarity with other P. viticola isolates and less sequence similarity with Plasmopara species. The loop-mediated isothermal amplification (LAMP) assay was developed based on the CesA4 gene sequence of P. viticola. The assay developed was more sensitive as it detected P. viticola genomic DNA up to 20 fmg. LAMP assay specificity was proved by carrying out the assay with genomic DNA extracted from other Oomycetes and fungal plant pathogens. Finally, LAMP assay was validated by testing seventy-eight grapevine leaf samples collected from seven different locations. LAMP assay showed a positive reaction in sixty-two samples tested out of seventy-eight samples tested. Therefore, the LAMP assay described should helpful for early and specific detection of downy mildew pathogen and help in mitigating disease incidence.     

Breakdown of resistance to grapevine downy mildew upon limited deployment of a resistant variety

Background  

Natural disease resistance is a cost-effective and environmentally friendly way of controlling plant disease. Breeding programmes need to make sure that the resistance deployed is effective and durable. Grapevine downy mildew, caused by the Oomycete Plasmopara viticola, affects viticulture and it is controlled with pesticides. Downy mildew resistant grapevine varieties are a promising strategy to control the disease, but their use is currently restricted to very limited acreages. The arising of resistance-breaking isolates under such restricted deployment of resistant varieties would provide valuable information to design breeding strategies for the deployment of resistance genes over large acreages whilst reducing the risks of the resistance being defeated. The observation of heavy downy mildew symptoms on a plant of the resistant variety Bianca, whose resistance is conferred by a major gene, provided us with a putative example of emergence of a resistance-breaking isolate in the interaction between grapevine and P. viticola.     

Agrobacterium rhizogenes—transformed plant roots as a source of grapevine viruses for purification

Agrobacterium rhizogenes-mediated transformation was applied toVitis spp. andNicotiana spp. infected by different grapevine phloem-limited viruses (grapevine fleck virus, grapevine virus A, grapevine virus B) to obtain root cultures for virus purification. All plant species were successfully transformed, and several clones were established in liquid culture. Transformed grapevine roots contained as much virus as non transformed roots and more than leaves, as assessed by ELISA and thin sectioning. Likewise, transformed roots ofNicotiana benthamiana Domin. contained in average more GVA than leaves, especially those at the base and the top of the plant, whereas withNicotiana occidentalis wheel., GVB was apparently less concentrated than in leaves.Nicotiana root grew faster than those ofVitis. All viruses multiplied and persisted in root cultures, which were successfully used for purification. Virus yields were the same (GFkV and GVB) or higher (GVB) than those reported in the literature. Grapevine roots may prove useful for culturing and purifying other non-mechanically transmissible grapevine viruses.     

不同品种葡萄抗霜霉病特性与叶片POD、PPO活性关系的研究

在霜霉病盛发期,对8804、梅尔诺、品丽珠3个葡萄品种(系)叶片中的PPO和POD活性变化进行了测定.结果显示,8804的PPO和POD活性较大,并保持相当长时间的高活性值,而梅尔诺、品丽珠叶片中PPO和POD活性较小;8804的PPO酶活性变化范围高于其它2个品种,但POD酶活性变化范围却低于后者.葡萄叶片中PPO和POD活性与葡萄霜霉病抗性之间存在一定的相关性,且不同抗感品种间PPO和POD酶活性存在极显著差异.研究结果表明,8804较梅尔诺、品丽珠对霜霉病具有较强的抗性.     

Relative Prevalence of Grapevine Leafroll-Associated Virus Species in Wine Grape-Growing Regions of California

Some diseases manifest as one characteristic set of symptoms to the host, but can be caused by multiple pathogens. Control treatments based on plant symptoms can make it difficult to effectively manage such diseases, as the biology of the underlying pathogens can vary. Grapevine leafroll disease affects grapes worldwide, and is associated with several viral species in the family Closteroviridae. Whereas some of the viruses associated with this disease are transmitted by insect vectors, others are only graft-transmissible. In three regions of California, we surveyed vineyards containing diseased vines and screened symptomatic plants for all known viral species associated with grapevine leafroll disease. Relative incidence of each virus species differed among the three regions regions, particularly in relation to species with known vectors compared with those only known to be graft-transmitted. In one region, the pathogen population was dominated by species not known to have an insect vector. In contrast, populations in the other surveyed regions were dominated by virus species that are vector-transmissible. Our survey did not detect viruses associated with grapevine leafroll disease at some sites with characteristic disease symptoms. This could be explained either by undescribed genetic diversity among these viruses that prevented detection with available molecular tools at the time the survey was performed, or a misidentification of visual symptoms that may have had other underlying causes. Based on the differences in relative prevalence of each virus species among regions and among vineyards within regions, we expect that region and site-specific management strategies are needed for effective disease control.     

Grapevine virome and production of healthy plants by somatic embryogenesis

Grapevine (Vitis spp.) is a widespread fruit tree hosting many viral entities that interact with the plant modifying its responses to the environment. The production of virus-free plants is becoming increasingly crucial for the use of grapevine as a model species in different studies. Using high-throughput RNA sequencing, the viromes of seven mother plants grown in a germplasm collection vineyard were sequenced. In addition to the viruses and viroids already detected in grapevine, we identified 13 putative new mycoviruses. The different spread among grapevine tissues collected in vineyard, greenhouse and in vitro conditions suggested a clear distinction between viruses/viroids and mycoviruses that can successfully be exploited for their identification. Mycoviruses were absent in in vitro cultures, while plant viruses and viroids were particularly accumulated in these plantlets. Somatic embryogenesis applied to the seven mother plants was effective in the elimination of the complete virome, including mycoviruses. However, different sanitization efficiencies for viroids and grapevine pinot gris virus were observed among genotypes. The absence of mycoviruses in in vitro plantlets, associated with the absence of all viral entities in somaclones, suggested that this regeneration technique is also effective to eradicate endophytic/epiphytic fungi, resulting in gnotobiotic or pseudo-gnotobiotic plants.     

A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death

Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.     

Detection of Grapevine Viruses in Poland

During a 3‐year study, grapevines from 23 vineyards in Poland were surveyed for virus diseases and tested to determine the prevalence of the most economically important viruses by RT‐PCR. The rate of positive samples was 2.2% for grapevine leafroll‐associated virus 1 (GLRaV‐1), 1.9% for grapevine leafroll‐associated virus 2 (GLRaV‐2), 1.5% grapevine leafroll‐associated virus 3 (GLRaV‐3), 1.9% for grapevine virus A (GVA), 0.2% for grapevine virus B (GVB), 0.2% for grapevine virus E (GVE), 0.65% for grapevine fanleaf virus (GFLV), 20.4% for grapevine fleck virus (GFkV) and 71.9% for grapevine rupestris stem pitting‐associated virus (GRSPaV). These viruses were found to occur as single or mixed infections of different combinations in individual grapevines. The overall viral infection rate in the surveyed grapevines was 82.6%. GRSPaV is the most widely distributed virus of all the viruses currently detected in the region. DNA sequencing confirmed the identification of the viruses in selected samples, and analysis indicated that the Polish isolates shared a close molecular identity with the corresponding isolates in GenBank. To our knowledge, this is the first detection of GLRaV‐1, ‐2, ‐3, GVA, GVB, GVE, GFLV, GFkV and GRSPaV in Poland.     

Cloning of molecular markers for disease resistance in sunflower, Helianthus annuus L.

 A candidate-gene approach to analyse the resistance of plants to phytopathogenic fungi is presented. The resistance of sunflower (Helianthus annuus L.) to downy mildew (Plasmopara halstedii) shows a gene-for-gene interaction (monogenic resistance), whereas resistance to white rot (Sclerotinia sclerotiorum) is quantitative, with different levels of resistance for different plant parts. By homology cloning, probes were obtained homologous to some plant resistance genes (nucleotide binding site-like, NBS, genes and serine-threonine protein kinase-like, PK, genes). These clones were used as probes for linkage mapping of the corresponding genes. It was demonstrated that at least three NBS-like loci are located on linkage-group 1, in the region where downy mildew resistance loci have been described. Quantitative trait loci for S. sclerotiorum resistance to penetration or extension of the mycelium in different tissues were studied in three crosses. Major QTLs for resistance were found on linkage group 1, with up to 50% of the phenotypic variability explained by peaks at the map position of the PK locus, 25 cM from the downy mildew loci. Received: 24 September 1997 / Accepted: 21 October 1997     

Cryptic diversity of <Emphasis Type="Italic">Plasmopara viticola</Emphasis> (Oomycota,Peronosporaceae) in North America

Plasmopara viticola is the causal agent of grapevine downy mildew and is among the most important diseases in viticulture. It originates from North America, where it coevolved with wild Vitis species. Beginning in the 1870s it turned into a global epidemic that has been causing severe yield losses. It is generally believed that a single species is causing downy mildew on a large variety of economically important cultivars. Here we report, based on one nuclear and two mitochondrial markers, that isolates from vineyards in the United States fall into three highly distinct phylogenetic lineages. One of these contains European strains and affects Vitis vinifera cultivars, while the other two lineages affect also other species of Vitis. The divergence between these lineages is high, and, judging from the genetic variation in other Plasmopara lineages, might reflect distinct species. Due to the potentially significant implications for quarantine regulations and resistance breeding, detailed studies will be necessary to clarify whether these genetically distinct lineages occur outside of North America or are still confined there.     

Yield and quality of grapes cultivated under plastic coverings with different downy mildew control strategies

Viticulture has been expanding in tropical regions. However, the climate in these areas is generally favourable to the incidence of plant diseases, especially downy mildew. Plastic covers and warning systems have shown very positive results in disease control, but they are tools that have never been used simultaneously in a tropical area. The Vitis vinifera cv. BRS Morena table grape was evaluated as regards yield and quality under different downy mildew control strategies as carried out on vineyards trained on an overhead trellis system, covered by a black shading screen (BSS) or a braided polypropylene film (BPF), over a 3‐year period. Different grapevine downy mildew management approaches defined the treatments: Co) Control (no spraying); Ca) Conventional control (calendar); Ba) “Rule 3–10” (Atti Istituto Botanico, 8, 1947, 45); Ma25) Low‐infection efficiency—i₀>25%; and Ma75) High‐infection efficiency—i₀>75% (Plant Disease, 84, 2000, 549). The occurrence of downy mildew and the amount of damage inflicted on vine yield and grape quality are directly related to the period of the crop cycle when there is rainfall. The use of the Ma75 warning system (Plant Disease, 84, 2000, 549) under braided polypropylene film resulted, for the most part, in similar vineyard productivity compared to Ca, but did not influence the number of branches and its fertility. The other warning systems decreased productivity by 31.9% compared to Ca. It was not possible to establish a relationship between the occurrence of downy mildew and its influence on grape sweetness and acidity. The use of warning systems led to a substantial reduction in fungicide sprays, approximately 66.7 to 71.3%, compared to the calendar system commonly used by the vine growers, with the Ba (Atti Istituto Botanico, 8, 1947, 45) and Ma75 controls (Plant Disease, 84, 2000, 549) leading to the highest fungicide saving.     

Genetic dissection of a TIR‐NB‐LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR‐NB‐LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated r esistance to P lasmopara v iticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south‐eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1‐mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR‐NB‐LRR genes at this locus share a common ancestor.     

Development of PCR markers for the<Emphasis Type="Italic"> Pl5/Pl8</Emphasis> locus for resistance to<Emphasis Type="Italic"> Plasmopara halstedii</Emphasis> in sunflower,<Emphasis Type="Italic"> Helianthus annuus</Emphasis> L. from complete CC-NBS-LRR sequences

Sunflower downy mildew, caused by Plasmopara halstedii, is one of the major diseases of this crop. Development of elite sunflower lines resistant to different races of this oomycete seems to be the most efficient method to limit downy mildew damage. At least two different gene clusters conferring resistance to different races of P. halstedii have been described. In this work we report the cloning and mapping of two full-length resistance gene analogs (RGA) belonging to the CC-NBC-LRR class of plant resistance genes. The two sequences were then used to develop 14 sequence tagged sites (STS) within the Pl5/Pl8 locus conferring resistance to a wide range of P. halstedii races. These STSs will be useful in marker-assisted selection programs.Communicated by C. Möllers     

Effect of climate change on infection of grapevine by downy and powdery mildew under controlled environment

Plant responses to elevated CO2 and temperature have been much studied in recent years, but effects of climate change on pathological responses are largerly unknown. The pathosystems grapevine (Vitis vinifera) - downy mildew (Plasmopara viticola) and powdery mildew (Erysiphe necatrix) were chosen as models to assess the potential impact of increased CO2 and temperature on disease incidence and severity under controlled environment. Grapevine potted plants were grown in phytotrons under 4 different simulated climatic conditions: (1) standard temperature (ranging from 18 degrees to 22 degrees C) and standard CO2 concentration (450 ppm); (2) standard temperature and elevated CO2 concentration (800 ppm); (3) elevated temperature (ranging from 22 degrees to 26 degrees C, 4 degrees C higher than standard) and standard CO2 concentration; (4) elevated temperature and CO2 concentration. Each plant was inoculated with a spore suspension containing 5x10(5) cfu/ml. Disease index and physiological parameters (chlorophyll content, fluorescence, assimilation rate) were assessed. Results showed an increase of the chlorophyll content with higher temperatures and CO2 concentration, to which consequently corresponded an higher fluorescence index. Disease incidence of downy mildew increased when both CO2 and temperatures were higher, while an increase in CO2 did not influenced powdery mildew incidence, probably due to the increased photosynthetic activity of plants under such conditions. Considering that the rising concentrations of CO2 and other greenhouse gases will lead to an increase in global temperature and longer seasons, we can assume that this will allow more time for pathogens evolution and could increase pathogen survival, indirectly affecting downy and powdery mildews of grapevine.     

Exploiting medicinal plants and phylloplane microflora for the management of grapevine downy mildew

Abstract

Grapevine downy mildew is the most devastating disease throughout the world causing huge monetary losses. Twenty medicinal plant extracts and six phylloplane microfloras were evaluated for their efficacy against sporangial germination of grapevine downy mildew pathogen Plasmopara viticola in vitro. The results revealed that the Neem Seed Kernel Extract (NSKE) at 5% significantly inhibited the sporangial germination (75.36%) of P. viticola. Among the phylloplane microflora Pseudomonas fluorescens was highly effective in reducing the sporangial germination (64.26%). Post inoculation spraying of NSKE (5%) and P. fluorescens (0.2%) effectively inhibited the disease development in the greenhouse. Three sprays with NSKE (5%) and phylloplane P. fluorescens (0.2%): first spray after initial appearance of disease and the second and third at 10 day intervals were found to be promising in reducing disease incidence in the field.