Kaolin Particle Film Protects Grapevine cv. Cabernet Sauvignon Against Downy Mildew by Forming Particle Film at the Leaf Surface, Directly Acting on Sporangia and Inducing the Defense of the Plant

Downy mildew is a major threat to viticulture, leading to severe yield loss. The use of traditional copper-based fungicides is effective, but has adverse effects on the environment and human health, making it urgent to develop an environmentally friendly disease management program. Multi-functional kaolin particle film (KPF) is promising as an effective and safer treatment strategy, since this material lacks chemically active ingredients. In this study, ability of Kaolin particle film (KPF) pretreatment to protect grapevine leaves from Plasmopara viticola was tested and the mode of action of KPF was analyzed. KPF application reduced the disease severity and the development of intercellular hyphae. Additionally, there was reduced accumulation of H2O2 and malondialdehyde (MDA) with pretreatment. The observation of ultrastructure on the leaf surface showed KPF deposition and stomatal obstruction, indicating that KPF protected plants against disease by preventing the adhesion of pathogens to the leaf surface and blocking invasion through the stomata. KPF pretreatment also activated host defense responses, as evidenced by increased activities of anti-oxidative enzymes [superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)] and defense-related enzymes [phenylalanine ammonia-lyase (PAL), chitinases, and β-1,3-glucanases], increased phytohormone signals [abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA)] and the up-regulation of defense genes related to plant defense. Overall, these results demonstrate that KPF treatment counters grapevine downy mildew by protecting leaves and enhancing plant defense responses.

Revealing the secrets beneath grapevine and Plasmopara viticola early communication: a picture of host and pathogen proteomes

Plant apoplast is the first hub of plant-pathogen communication where pathogen effectors are recognized by plant defensive proteins and cell receptors and several signal transduction pathways are activated. As a result of this first contact, the host triggers a defence response that involves the modulation of several extra and intracellular proteins. In grapevine-pathogen interactions, little is known about the communication between cells and apoplast. Also, the role of apoplastic proteins in response to pathogens still remains a blackbox. In this study we focused on the first 6 hours after Plasmopara viticola inoculation to evaluate grapevine proteome modulation in the apoplastic fluid (APF) and whole leaf tissue. Plasmopara viticola proteome was also assessed enabling a deeper understanding of plant and pathogen communication. Our results showed that oomycete recognition, plant cell wall modifications, ROS signalling and disruption of oomycete structures are triggered in Regent after P. viticola inoculation. Our results highlight a strict relation between the apoplastic pathways modulated and the proteins identified in the whole leaf proteome. On the other hand, P. viticola proteins related to growth/morphogenesis and virulence mechanisms were the most predominant. This pioneer study highlights the early dynamics of extra and intracellular communication in grapevine defence activation that leads to the successful establishment of an incompatible interaction.

Eucalyptus staigeriana essential oil can control downy mildew in grapevine

Downy mildew (Plasmopara viticola) is the primary disease in viticulture worldwide, and your control requires synthetic fungicides applications to avoid quality and yield loss in the grapevines. However, alternatives to reduce synthetic fungicides are needed to ensure the consumer’s health and the environment. Essential oils (EOs) are amongst the most promising natural plant protection alternatives because of their antifungal properties on several crop diseases. The present study objective was to determine the effect of Eucalyptus staigeriana EO in vitro and in vivo against P. viticola. The EO exhibited the highest activity in vitro, inhibiting 90% of the incidence and severity of disease caused by P. viticola in leaves of grapevines in the greenhouse. In the field (in vivo), treatment with EO could not control the disease; however, treatment with EO in consortium with conventional treatment reduced approximately 50% of the incidence and more than 90% of the severity of downy mildew disease in leaves, decreasing the application of synthetic fungicides by 50%.

Warburgia ugandensis Leaf and Bark Extracts: An Alternative to Copper as Fungicide against Downy Mildew in Organic Viticulture?

In organic viticulture, copper-based fungicides are commonly used to suppress Downy Mildew infection, caused by the oomycete Plasmopara viticola. However, the frequent and intensive use of such fungicides leads to accumulation of the heavy metal in soil and nearby waters with adverse effects on the ecosystem. Therefore, alternative, organic fungicides against Downy Mildew are urgently needed to reduce the copper load in vineyards. In this study, the use of Warburgia ugandensis Sprague (Family Canellacea) leaf and bark extracts as potential fungicides against Downy Mildew were evaluated. In vitro (microtiter) and in vivo (leaf discs, seedlings) tests were conducted, as well as field trials to determine the efficacy of the extracts against Downy Mildew. The results revealed an MIC100 of 500 µg/mL for the leaf extract and 5 µg/mL for the bark extract. Furthermore, experiments with leaf discs and seedlings demonstrated a strong protective effect of the extracts for up to 48 h under (semi-) controlled conditions. However, in field trials the efficacy of the extracts distinctly declined, regardless of the extracts’ origin and concentration.

“The PLCP gene family of grapevine (Vitis vinifera L.): characterization and differential expression in response to Plasmopara Viticola”

Background Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases, are structurally related to papain. The members belonging to PLCPs family contribute to plant immunity, senescence, and defense responses in plants. The PLCP gene family has been identified in Arabidopsis, rice, soybean, and cotton. However, no systematic analysis of PLCP genes has been undertaken in grapevine. Since Plasmopara viticola as a destructive pathogen could affect immunity of grapes in the field, we considered that the members belonged to PLCPs family could play a crucial role in defensive mechanisms or programmed cell death. We aimed to evaluate the role of PLCPs in 2 different varieties of grapevines and compared the changes of their expressions with the transcriptional data in response to P. viticola. Results In this study, 23 grapevine PLCP (VvPLCP) genes were identified by comprehensive bioinformatics analysis. Subsequently, the chromosomal localizations, gene structure, conserved domains, phylogenetic relationship, gene duplication, and cis-acting elements were analyzed. Numerous cis-acting elements related to plant development, hormone, and stress responses were identified in the promoter of the VvPLCP genes. Phylogenetic analysis grouped the VvPLCP genes into nine subgroups. The transcription of VvPLCP in different inoculation time points and varieties indicated that VvPLCP may have vital functions in grapevine defense against Plasmopara viticola. According to transcriptome data and qPCR analysis, we observed the increasing expression levels of VvRD21–1 at 72 h after inoculation in resistant variety, inferring that it was related to grape downy mildew resistance. Meanwhile, 3 genes including VvXBCP1, VvSAG12–1, and VvALP1 showed higher expression at 24 h after pathogen inoculation in the susceptible variety and might be related to the downy mildew phenotype. We nominated these four genes to function during hypersensitive response (HR) process, inferring that these genes could be associated with downy mildew resistance in grapes. Conclusions Our results provide the reference for functional studies of PLCP gene family, and highlight its functions in grapevine defense against P. viticola. The results help us to better understand the complexity of the PLCP gene family in plant immunity and provide valuable information for future functional characterization of specific genes in grapevine.

Rapid Detection of Airborne Inocula of Grapevine Mildews Using PCR and LAMP Assay

Grapes powdery mildew and downy mildew caused by Erysiphe necator and Plasmopara viticola respectively are the most devastating diseases worldwide resulting in significant loss of yield and quality. Epidemics of grapevine mildews are caused by airborne inocula such as conidia and sporangia. Rapid detection of airborne inocula will help to face up timely management strategies under field conditions. The aim of the current study was to design a suction spore trap to trap the airborne mildew inocula and their early detection by molecular methods of PCR and LAMP assay. A total of twelve airborne inocula samples were collected the weekly intervals from 3 to 14 standard weeks of 2021 during the cropping season. The presence of airborne inocula of E. necator was detected on standard weeks 3,6,10 and 13 through PCR assay which yielded an amplicon of 470 bp. Similarly, airborne inocula of P.viticola were detected on standard week 6 only through PCR which yielded an amplicon of 520 bp. A rapid, highly specific , sensitive Loop mediated isothermal amplification (LAMP) assay was performed to detect the E. necator and P. viticola using six sets of LAMP primers constructed by targeting rDNA region of ITS and the 5S rRNA and CesA4 a gene, respectively. LAMP assay efficiently detected the presence of airborne inocula of E.necator in most of the samples collected from standard week 3 – 14 except 7, 8, and 9. However, the presence of airborne inocula of P.viticola from standard week 3 – 14 was confirmed by LAMP assay. The LAMP assay is absolutely the best in identifying airborne inocula of grapevine mildews compared to PCR and phenotypic microscopic observation.

Grapevine Rpv3-, Rpv10- and Rpv12-mediated defense responses against Plasmopara viticola and the impact of their deployment on fungicide use in viticulture

Background The high susceptibility of European grapevine cultivars (Vitis vinifera) to downy mildew (Plasmopara viticola) leads to the intensive use of fungicides in viticulture. To reduce this input, breeding programs have introgressed resistance loci from wild Vitis species into V. vinifera, resulting in new fungus-resistant grapevine cultivars (FRC). However, little is known about how these different resistance loci confer resistance and what the potential reduction in fungicide applications are likely to be if these FRCs are deployed. To ensure a durable and sustainable resistance management and breeding, detailed knowledge about the different defense mechanisms mediated by the respective Rpv (Resistance to P. viticola) resistance loci is essential. Results A comparison of the resistance mechanisms mediated by the Rpv3–1, Rpv10 and/or Rpv12-loci revealed an early onset of programmed cell death (PCD) at 8 hours post infection (hpi) in Rpv12-cultivars and 12 hpi in Rpv10-cultivars, whereas cell death was delayed in Rpv3-cultivars and was not observed until 28 hpi. These temporal differences correlated with an increase in the trans-resveratrol level and the formation of hydrogen peroxide shortly before onset of PCD. The differences in timing of onset of Rpv-loci specific defense reactions following downy mildew infection could be responsible for the observed differences in hyphal growth, sporulation and cultivar-specific susceptibility to this pathogen in the vineyard. Hereby, Rpv3- and Rpv12/Rpv3-cultivars showed a potential for a significant reduction of fungicide applications, depending on the annual P. viticola infection pressure and the Rpv-loci. Furthermore, we report on the discovery of a new P. viticola isolate that is able to overcome both Rpv3- and Rpv12-mediated resistance. Conclusion This study reveals that differences in the timing of the defense reaction mediated by the Rpv3-, Rpv10- and Rpv12-loci, result in different degrees of natural resistance to downy mildew in field. Vineyard trials demonstrate that Rpv12/Rpv3- and Rpv3-cultivars are a powerful tool to reduce the dependence of grape production on fungicide applications. Furthermore, this study indicates the importance of sustainable breeding and plant protection strategies based on resistant grapevine cultivars to reduce the risk of new P. viticola isolates that are able to overcome the respective resistance mechanism.

Hyperparasitism on Plasmopara viticola by Simplicillium lanosoniveum

A previously unknown association between Plasmopara viticola, the causal pathogen of grape downy mildew, and the mycoparasite strain F2 was described in this paper. F2 could be consistently isolated from the mildew of diseased spots on grape leaves of Kyoho (Vitis labrusca × V. vinifera cv. Kyoho) infected with P. viticola, but neither from the surfaces of healthy grape leaves nor within lesions of other diseases. It could be observed that strain F2 was capable of coiling around sporangiophores and sporangia of P. viticola under microscope, causing hyphae deformation, decreasing the pathogen growth, and reducing the number of sporangia. This fungal strain was identified as Simplicillium lanosoniveum on the basis of morphological characterizations and 28S rDNA sequencing. To our knowledge, this is the first report of S. lanosoniveum with hyperparasitic characteristics on P. viticola, and the hyperparasite has potential application in the control of grape downy mildew.

Evidence for a Novel Partitivirus Isolated From Entomopathogenic Nematode Steinernema Ceratophorum

Nematodes are abundant, yet little is known about their viruses. In this study, we report a novel partitivirus isolated from Entomopathogenic nematode specie “Steinernema ceratophorum, named as Steinernema ceratophorum Partiti-like Virus 1 (ScPV1). The complete genome of ScPV1 is comprised of two dsRNA segments, dsRNA1 (2352 bp) and dsRNA2 (2196 bp) in length. Both dsRNAs contained a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. The sequences of the RdRp and CP showed the highest similarity (47% and 33% identity, respectively) to Plasmopara viticola associated Partitivirus 7. Multiple sequence alignments and phylogenetic analysis of RdRp of ScPV1 with other selected viruses indicated that ScPV1 is the new member of genus Betapartitivirus in the family Partitiviridae.