Causal Agent Of Downy Mildew Research Articles

Rpv10.2: A Haplotype Variant of Locus Rpv10 Enables New Combinations for Pyramiding Downy Mildew Resistance Traits in Grapevine.

In viticulture, pathogens like the oomycete Plasmopara viticola, the causal agent of downy mildew, can cause severe yield loss and require extensive application of plant protection chemicals. Breeders are generating pathogen-resistant varieties exploiting American and Asian wild Vitis germplasm as sources of resistance. Several loci mediating resistance to P. viticola have been identified in the past but may be overcome by specifically adapted strains of the pathogen. Aiming to find and characterize novel loci, a cross population with Vitis amurensis ancestry was investigated searching for resistance-correlated quantitative trait loci (QTL). As a prerequisite, a genetic map was generated by analyzing the 244 F1 individuals derived from a cross of the downy mildew susceptible Vitis vinifera cultivar 'Tigvoasa' and the resistant V. amurensis pBC1 breeding line We 90-06-12. This genetic map is based on the information from 627 molecular markers including 56 simple sequence repeats and 571 rhAmpSeq markers. A phenotypic characterization of the progeny showed a clear segregation of the resistance traits in the F1 population after an experimental inoculation of leaf discs with downy mildew. Combining genetic and phenotypic data, an analysis for QTL revealed a major locus on linkage Group 9 that correlates strongly with the resistance to downy mildew. The locus was mapped to a region of about 80 kb on the PN40024 (12x.V2) grapevine reference genome. This genomic region co-localizes with the formerly identified locus Rpv10 from the grapevine cultivar 'Solaris'. As we found different allele sizes of the locus-linked SSR markers than those characterizing the known Rpv10 locus and differences in the sequence of a candidate gene, it was regarded as a haplotype variant and named Rpv10.2.

Traditional and Emerging Approaches for Disease Management of Plasmopara viticola, Causal Agent of Downy Mildew of Grape

The oomycete Plasmopara viticola, which causes downy mildew, is currently one of the most destructive pathogens affecting grape production. Although native to the eastern United States, P. viticola was introduced into Europe in the mid-to-late 1800s and is now found in virtually every grape-growing region of the world. Since its discovery, much effort has been made to understand the life cycle and infection process of the pathogen to develop more effective management practices. Widespread application of fungicides, especially those which have only one mode of action, has led to an increased occurrence of resistance to these treatments. Thus, with increased fungicide resistance and rising environmental concerns surrounding their use, traditional chemical management practices have begun to fall out of favor. Newer approaches, from targeted breeding utilizing quantitative trait loci to biological control agents, are continually being investigated and adapted to limit the damage caused by downy mildew. This review summarizes the current knowledge of the pathogen and methods of its control and explores potential avenues for future research focused on hypovirulence and biological control agents.

Sulfur — a potential additive to increase the efficacy of copper-based fungicides against grapevine downy mildew

Control of downy mildew on grapevines in organic viticulture in Europe is mainly based on using copper-based fungicides. However, the effect of these agents is often insufficient to prevent losses when infestation pressure is high. The addition of sulfur, which is primarily used to control powdery mildew, improves the effect of copper on downy mildew. In three out of five trial years, sulfur had a clear positive effect when it was added during spray treatments against Plasmopara viticola, the causal agent of downy mildew on grapevines. This was evident mainly in the fruits; no further positive effect on the leaves was evident. In addition, the present study shows that copper-based fungicides are effective on leaves but have a rather poor effect on grapevine fruit. Furthermore, the study demonstrates that the effect of copper is significantly worse than that of the chemical synthetic contact fungicide folpet.

Remote detection of fungal pathogens in viticulture using laser-induced fluorescence: an experimental study on infected potted vines

IntroductionPathogenic fungi, such as Plasmopara viticola and Erysiphe necator, severely threaten the annual yield of grapes in both quantity and quality. In contrast to other crop production systems, fungicides are intensively applied in viticulture as a countermeasure. The goal of precision viticulture is to optimize vineyard performance as well as the environmental impact by reducing fungicides and applying different techniques and combined strategies. Therefore, new emerging technologies are required, including non-invasive detection, as well as monitoring and tools for the early and in-field detection of fungal development. MethodsIn this study, we investigated leaves of potted vines (Vitis vinifera cv. ‘Riesling’) and traced the development of the inoculated leaves using our new remote detection system vinoLAS®, which is based on laser-induced fluorescence spectroscopy. We ran a measurement campaign over a period of 17 days.ResultsWe were able to detect a leaf infection with P. viticola, the causal agent of downy mildew, between 5 and 7 days after inoculation. Our results provide evidence for a successful application of laser-based standoff detection in vineyard management in the future. Thus, the vinoLAS system can serve as a model technology for the detection of pathogenic disease symptoms and thus monitoring complete vineyard sites. This allows for early countermeasures with suitable crop protection approaches and selected hot-spot treatments.DiscussionAs P. viticola is considered one of the most damaging fungi in European viticulture, disease mapping via this monitoring tool will help to reduce fungicide applications, and will, therefore, support the implementation of the European Green Deal claims.

Combining Trichoderma koningiopsis and chitosan as a synergistic biocontrol and biostimulating complex to reduce copper rates for downy mildew control on grapevine

Plasmopara viticola, causal agent of downy mildew and one of the most severe grapevine diseases, is commonly controlled by multiple fungicide applications, primarily based on copper compounds in organic viticulture. Biostimulants and biocontrol agents could make a major contribution to pesticide reduction. A synergistic biocontrol and biostimulating complex consisting of Trichoderma sp. and chitosan was investigated to achieve copper fungicide reduction in the control of P. viticola. To ensure complex efficacy, the interactions and effects of all agents need to be investigated at the tri-trophic level of grapevine, downy mildew, and biocontrol and biostimulating complex. To guarantee the compatibility of Trichoderma sp. and chitosan, the chitosan tolerance of different copper-tolerant Trichoderma isolates was investigated in mycelial growth and germination tests. A T. koningiopsis isolate proved to be highly chitosan-tolerant was identified for further studies. The timing of spray application with T. koningiopsis and various chitosans was evaluated for efficacy against P. viticola. In the period 0 to 1 day before inoculation with the pathogen, the formation of the sporangia number was significantly reduced by the tested agents. To further describe the mode of action and efficacy of T. koningiopsis and chitosan against P. viticola, the application of single agents on upper and lower grapevine leaves inoculated with the pathogen in comparison to resistance inducers and copper fungicide were tested. Moreover, the combination of T. koningiopsis with different chitosans and a reduced copper rate was investigated on the respective leaf sides of greenhouse plants. Both, a direct and indirect mode of action against downy mildew was demonstrated. Regardless of the treated leaf side, sporulation of downy mildew was clearly inhibited compared to the untreated control. Moreover, microscopic images showed successful establishment of T. koningiopsis and uniform distribution of chitosan on leaves. The tested biocontrol and biostimulating complex might show a promising combatting tool against P. viticola while simultaneously reducing copper rate.

Chitosan application reduces downy mildew severity on grapevine leaves by positively affecting gene expression pattern

Grapevine (Vitis vinifera) is one of the most economically relevant crops in the world, producing more than sixty million tonnes a year. Unfortunately, almost all cultivars currently used for grape or wine production are sensitive to various pathogens, such as the oomycete causal agent of downy mildew, Plasmopara viticola, so that, generally, up to 15 fungicide treatments are needed to prevent diseases on leaves and berries. Nevertheless, fungicides have many different problems in terms of their impact on the environment and human health. To this end, some new chemicals have been used in the recent years. These are bio-stimulants and defence inducers, which are mainly used in the organic agriculture. Previous research has led to different results regarding their effectiveness, especially in the field. In 2020, an experimental product (chitosan - (C6H11O4N)n), capable of inducing defence mechanisms against downy mildew in grapevine, was tested in vineyard. Our study clearly showed that disease severity on canopy was reduced by 30%, on average, in the chitosan-treated grapevines in field and that several genes, encoding for proteins and enzymes of key defence-related metabolic pathways are up-regulated upon treatment.

Toxicity and Preventive Activity of Chitosan, Equisetum arvense, Lecithin and Salix Cortex against Plasmopara viticola, the Causal Agent of Downy Mildew in Grapevine

Grapevine, a crop of global economic importance, is annually affected by diseases that can compromise the quality and quantity of the harvest, producing large economic losses. Downy mildew caused by Plasmopara viticola (Berk. & M.A. Curtis) Berl. & de Toni is one of the most important diseases in the vineyard. To fight this pathogen, winegrowers often rely on conventional chemical fungicides or copper-based formulations, whose use is determined to be reduced by the European Commission due to their environmental consequences. Hence, alternative plant protection products (PPP) in grapevine must be considered and studied. In this context, we selected several alternative commercial products, based on basic substances (BS) or low-risk active substances (LRAS), to evaluate their suitability to deal with P. viticola. We measured the preventive activity of the products, both in vitro and in planta, as well as their toxicity against the sporangia and zoospores of the pathogen. Results showed that four commercial products were effective against the pathogen directly and preventively, being composed of approved basic substances, more concretely, chitosan, Equisetum arvense, lecithins, and Salix cortex. Among those, the products composed of lecithins and Salix cortex were the most toxic and active preventively. Therefore, these basic substances should be promoted in the vineyard as an alternative to conventional treatments in order to transition to a more sustainable viticulture.

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.

A grapevine by-product extract enriched in oligomerised stilbenes to control downy mildews: focus on its modes of action towards <i>Plasmopara viticola</i>

Natural extracts, and particularly plant by-product extracts, can represent an eco-friendly alternative to synthetic pesticides to control crop diseases. We produced an extract from a mix of grapevine trunk and roots that contain a high amount of complex stilbenes (grapevine phytoalexins), especially ε- and r-viniferins. This extract conferred protection to grapevines against Plasmopara viticola (the causal agent of downy mildew) by displaying antimicrobial activities towards zoospore mobility and sporulation and by stimulating plant defences. From the perspective of its application in the field, a formulated preparation of the extract was proposed using polysorbate and sophorolipids. The formulated extract reduced the development of various oomycetes impacting grapevine, potato, tomato and melon in semi-controlled conditions. Thus, such grapevine extract constitutes a promising strategy to protect major cultivated plants against downy mildews.

Pathogenesis of Plasmopara viticola Depending on Resistance Mediated by Rpv3_1, and Rpv10 and Rpv3_3, and by the Vitality of Leaf Tissue.

Grapevine cultivars vary in their resistance to Plasmopara viticola, causal agent of downy mildew. Genes from various Vitis species confer pathogen resistance (Rpv), resulting in reduced compatibility of the host-pathogen interaction and partial disease resistance that may become apparent at different stages of pathogenesis. This study describes the pathogenesis of P. viticola on the partially resistant cultivars Regent (Rpv3-1) and Solaris (Rpv3-3, Rpv10) as compared with the susceptible cultivar Mueller-Thurgau using various microscopic techniques, visual disease rating as well as qPCR. Host plant resistance had no effect on the initial steps of pathogenesis outside the host plant cells (zoospore attachment, formation of substomatal vesicle) and became detectable only after the formation of primary haustoria. The restricted compatibility resulted in reductions in haustorium size and in the number of secondary haustoria and was associated with callose depositions around haustoria and stomatal guard cells, collapsed mesophyll cells (hypersensitive reaction), and additional production of an amorphous substance in the intercellular space of cultivar Solaris. Resistance mechanisms reduced the efficiency of P. viticola haustoria and largely restricted tissue colonization to the spongy parenchyma; resistance of cultivar Solaris having thicker leaves was more effective than that of cultivar Regent. Despite of the effects of resistance genes, P. viticola was able to complete its life cycle by forming sporangiophores with sporangia through the stomata on both resistant cultivars indicating partial resistance. Differences in the pathogenesis on detached and attached grapevine leaves highlighted the impact of host tissue vitality on both resistance and susceptibility to the biotrophic pathogen.

Plasmopara viticola the Causal Agent of Downy Mildew of Grapevine: From Its Taxonomy to Disease Management.

Plasmopara viticola (P. viticola, Berk. & M. A. Curtis; Berl. & De Toni) causing grapevine downy mildew is one of the most damaging pathogens to viticulture worldwide. Since its recognition in the middle of nineteenth century, this disease has spread from America to Europe and then to all grapevine-growing countries, leading to significant economic losses due to the lack of efficient disease control. In 1885 copper was found to suppress many pathogens, and is still the most effective way to control downy mildews. During the twentieth century, contact and penetrating single-site fungicides have been developed for use against plant pathogens including downy mildews, but wide application has led to the appearance of pathogenic strains resistant to these treatments. Additionally, due to the negative environmental impact of chemical pesticides, the European Union restricted their use, triggering a rush to develop alternative tools such as resistant cultivars breeding, creation of new active ingredients, search for natural products and biocontrol agents that can be applied alone or in combination to kill the pathogen or mitigate its effect. This review summarizes data about the history, distribution, epidemiology, taxonomy, morphology, reproduction and infection mechanisms, symptoms, host-pathogen interactions, host resistance and control of the P. viticola, with a focus on sustainable methods, especially the use of biocontrol agents.

Effectiveness of COPPION 5.2 WV and Rhapynal to control downy mildew of lettuce

The most important lettuce pathogen under Greek agro-ecological conditions is Bremia lactucae, the causal agent of downy mildew. Chemical control methods are widely used to manage this pathogen, with copper fungicides of tensed as the first line of defence, especially for growers who prefer environmentally friendly methods. The main aim of this work was to investigate the effectiveness of the biosurfactant Rhapynal (rhamnolipids) and the commercial copper product COPPION 5.2 WV (an ionic form copper) against downy mildew of lettuce. The experiments were conducted under both greenhouse and field conditions. COPPION 5.2 WV was applied at a rate of 2 mLL−1, while Rhapynal was applied on its own at a rate of 1.5 gL−1 or in a mixture with COPPION 5.2 WV at rates of 0.5, 1.0 and 1.5 gL−1. In greenhouse experiments, COPPION 5.2 WV alone or in a mixture with 0.5, 1.0, or 1.5 gL−1 Rhapynal were the most effective treatments to control downy mildew of lettuce. In field experiments, the percentage of infection in plants treated with COPPION 5.2 WV in a mixture with 0.5, 1, or 1.5 gL−1 Rhapynal was statistically similar. No significant differences were found among treatments with COPPION 5.2 WV, COPPION 5.2 WV in a mixture with 0.5 or 1 gL−1 Rhapynal, or Rhapynal on its own at 1.5 gL−1. Based on the results of this study, a mixture of COPPION 5.2 WV with Rhapynal seems to be a promising alternative method to control downy mildew on lettuce plants.

Crossing Plasmopara viticola strains in controlled conditions to uncover the genomic bases of downy mildew resistance breakdown in grapevine

The biotrophic oomycete Plasmopara viticola is the causal agent of downy mildew, one of the major grapevine diseases. We report here a successful strategy to cross compatible strains of this pathogen and obtain a large viable progeny. We used this method to study the offspring between two P. viticola strains able to overcome two major grapevine resistance factors (Rpv3, Rpv10). Thanks to the genomic resources now available, we will genotype this progeny to build an unprecedented linkage map and uncover the genomic bases of grapevine resistance breakdown displayed by P. viticola virulent strains.

Phytopathological Threats Associated with Quinoa (Chenopodium quinoa Willd.) Cultivation and Seed Production in an Area of Central Italy.

In 2017, in a new Chenopodium quinoa cultivation area (Central Italy), emergence failures of the Titicaca, Rio Bamba, and Real varieties, whose seeds were obtained the previous year (2016) in the same location, were observed. Moreover, leaf disease symptoms on the Regalona variety, whose seeds came from Chile, were detected. Visual and microscopic analyses showed the presence of browning/necrotic symptoms on the seeds of the three varieties whose emergence in the field had failed. In addition, their in vitro germination rates were strongly compromised. Fusarium spp. was isolated with high incidence from Titicaca, Rio Bamba, and Real seeds. Among the detected Fusarium species, in the phylogenetic analysis, the dominant one clustered in the sub-clade Equiseti of the Fusarium incarnatum-equiseti (FIESC) species complex. Instead, the pathogen associated with Regalona leaf symptoms was identified, by morphological and molecular features, as Peronospora variabilis, the causal agents of downy mildew. This is the first report of both P. variabilis and F. equiseti on C. quinoa in Italy. Species-specific primers also detected P. variabilis in Regalona seeds. These results underline the importance of pathogen monitoring in new quinoa distribution areas, as well as of healthy seed production and import for successful cultivation.

Responses to Drought Stress Modulate the Susceptibility to Plasmopara viticola in Vitis vinifera Self-Rooted Cuttings.

Climate change will increase the occurrence of plants being simultaneously subjected to drought and pathogen stress. Drought can alter the way in which plants respond to pathogens. This research addresses how grapevine responds to the concurrent challenge of drought stress and Plasmopara viticola, the causal agent of downy mildew, and how one stress affects the other. Self-rooted cuttings of the drought-tolerant grapevine cultivar Xynisteri and the drought-sensitive cultivar Chardonnay were exposed to full or deficit irrigation (40% of full irrigation) and artificially inoculated with P. viticola in vitro or in planta. Leaves were sampled at an early infection stage to determine the influence of the single and combined stresses on oxidative parameters, chlorophyll, and phytohormones. Under full irrigation, Xynisteri was more susceptible to P. viticola than the drought-sensitive cultivar Chardonnay. Drought stress increased the susceptibility of grapevine leaves inoculated in vitro, but both cultivars showed resistance against P. viticola when inoculated in planta. Abscisic acid, rather than jasmonic acid and salicylic acid, seemed to play a prominent role in this resistance. The irrigation-dependent susceptibility observed in this study indicates that the practices used to mitigate the effects of climate change may have a profound impact on plant pathogens.

A method for scoring the risk of fungicide resistance in vineyards

Because the risk of resistance towards single-site fungicides in populations of Plasmopara viticola and Erysiphe necator, the causal agents of downy mildew (DM) and powdery mildew (PM), respectively, is substantial, fungicide resistance management is important in vineyards. Available methods for assessing resistance risk mainly focus on single fungicides and do not consider the risk of resistance of a complete fungicide schedule, which typically includes multiple fungicides, used solo or in mixture and at different doses, and other actions (behaviors) of the farmer. A simple scoring method (fungicide resistance risk score, FRRS) was developed to account for both (i) the intrinsic risk due to the fungicides used, the target pathogens, and agronomic factors, and (ii) the behavioral risk, which includes how the fungicides are used during the season. FRRSs were then calculated for 10 representative vineyards (10 cases) and ranked from 1 (highest score) to 10 (lowest score). For comparison, 20 assessors (four winegrowers and 16 experts including public advisors, field technicians, and consultants) ranked the same cases based on their individual “perception of risk” from 1 (highest risk) to 10 (lowest risk).When the FRRS-based rankings were compared with the modes of the rankings provided by the assessors, the correlation coefficients were significant for all experts (P < 0.001) and for each category of experts (P < 0.05); i.e., public advisors, field technicians from agro-chemical companies, and consultants; for non-experts, i.e., winegrowers, the FFRS values were significantly correlated with the modes of rankings for PM (P < 0.001) but not for DM (P = 0.293).The results indicated that the use of the new scoring method, i.e., the use of FRRSs, would reduce the variability in assessments of resistance risks and would provide a common method for evaluating resistance risk by both experts and winegrowers.

Detection of Airborne Sporangia of Pseudoperonospora cubensis and P. humuli in Michigan Using Burkard Spore Traps Coupled to Quantitative PCR.

Cucurbit downy mildew (CDM), caused by the oomycete pathogen Pseudoperonospora cubensis, is a devastating foliar disease on cucumber resulting in reduced yields. In 2004, the pathogen re-emerged in the United States, infecting historically resistant cucumber cultivars and requiring the adoption of an intensive fungicide program. The pathogen cannot overwinter in Michigan fields but because of an influx of airborne sporangia CDM occurs annually. In Michigan, spore traps are used to monitor the presence of airborne P. cubensis sporangia in cucumber growing regions to guide the initiation of a fungicide program. However, Pseudoperonospora humuli sporangia, the causal agent of downy mildew on hop, are morphologically indistinguishable from P. cubensis sporangia. This morphological similarity reduces the ability to accurately detect P. cubensis from spore trap samples when examined with the aid of light microscopy. To improve P. cubensis detection, we adapted a qPCR-based assay to allow the differentiation between P. cubensis and P. humuli on Burkard spore trap samples collected in the field. Specifically, we evaluated the specificity and sensitivity of P. cubensis detection on Burkard spore trap tapes using a morphological-based and quantitative-PCR (qPCR)-based identification assay and determined whether sporangia of P. cubensis and P. humuli on Burkard samples could be distinguished using qPCR. We found that the qPCR assay was able to detect a single sporangium of each species on spore trap samples collected in the field with Cq values <35.5. The qPCR assay also allowed the detection of P. cubensis and P. humuli in samples containing sporangia from both species. However, the number of sporangia quantified using light microscopy explained only 54 and 10% of the variation in the Cq values of P. cubensis and P. humuli, respectively, suggesting a limited capacity of the qPCR assay for the absolute quantification of sporangia in field samples. After 2 years of monitoring using Burkard spore traps coupled with the qPCR in cucumber fields, P. humuli sporangia were detected more frequently than P. cubensis early in the growing season (May and June). P. cubensis sporangia were detected ∼5 to 10 days before CDM symptoms were first observed in cucumber fields during both years. This research describes an improved sporangial detection system that is key for the monitoring and management of P. cubensis in Michigan.

Analysis of the virome associated to grapevine downy mildew lesions reveals new mycovirus lineages.

The obligatory biotrophic oomycetes Plasmopara viticola is the causal agent of downy mildew, a destructive disease of grapevine worldwide. So far, chemical fungicides are widely employed to limit this pathogen, but their adverse effects are stimulating the quest for environmentally friendly alternative approaches. Here, we report on the search for mycoviruses that might be later developed as biocontrol agents for this pathogen. Symptomatic leaves were collected from various regions in Spain and Italy and mycelia associated to leaf lesions was harvested. Total RNA extractions were depleted of rRNA and metatranscriptomes were generated using a high-throughput sequencing approach. The virome associated to leaf lesions was then characterized through a bioinformatic pipeline relying on blast searches against current viral databases. Here, we present an inventory of 283 new RNA viruses: 222 positive strand RNA viruses, 29 negative strand RNA viruses, 27 double-stranded RNA viruses and 5 ORFan virus RdRP segments, which could not be reliably assigned to any existing group in the Riboviria. In addition to ORFan viruses, we found other surprising new evolutionary trajectories in this wide inventory of viruses. The most represented viruses in our collection are those in phylum Lenarviricota, and, among them, a group of mycovirus segments distantly related to narnaviruses, but characterized by a polymerase palm domain lacking subdomain C, with the putative GDD catalytic triad. We also provided evidence of a strict association between two RNA segments that form a new mycovirus clade of positive strand RNA in the phylum Kitrinoviricota, order Martellivirales. In the phylum Negarnaviricota, we report for the first time in the order Mononegavirales a clade of viruses that is ambisense, a feature that so far was present only in the order Bunyavirales. Furthermore, in the same phylum we detected the widespread occurrence and abundant accumulation in our libraries of a distinct mycovirus clade distantly related to the Muvirales and Goujanvirales orders, which so far include only viruses infecting invertebrates. Possible new oomycetes-specific virus clades are also described in the phylum Duplornaviricota. These data greatly expand the evolutionary history of mycoviruses adding new layers of diversity to the realm Riboviria.

Preliminary Investigation of Effect of Neem-Derived Pesticides on Plasmopara halstedii Pathotype 704 in Sunflower under In Vitro and In Vivo Conditions.

Two neem-derived pesticides were examined under in vitro and in vivo conditions to test their efficacy in controlling Plasmopara halstedii pathotype 704, a causal agent of downy mildew in sunflower. All the tested concentrations of neem leaf extract and the highest concentration of commercial neem product significantly reduced the sporangial germination under in vitro conditions. In in vivo experiment, 3-days old pre-treated seedlings with both concentrations of neem leaf extract and the highest concentration of commercial product showed a significant reduction in the infection indicating possible systemic effect of neem. When the seedlings were treated following the infection with P. halstedii (i.e., post-treatment), only the highest concentrations of neem leaf extract and the commercial product showed a significant reduction in the infection indicating curative effect of neem. Possibilities for the control of P. halstedii with neem-derived pesticides are discussed.

Tracking host infection and reproduction of Peronospora salviae‐officinalis using an improved method for confocal laser scanning microscopy

AbstractPeronospora salviae‐officinalis, the causal agent of downy mildew on common sage, is an obligate biotrophic pathogen. It grows in the intercellular spaces of the leaf tissue of sage and forms intracellular haustoria to interface with host cells. Although P. salviae‐officinalis was described as a species of its own 10 years ago, the infection process remains obscure. To address this, a histological study of various infection events, from the adhesion of conidia on the leaf surface to de novo sporulation is presented here. As histological studies of oomycetes are challenging due to the lack of chitin in their cell wall, we also present an improved method for staining downy mildews for confocal laser scanning microscopy as well as evaluating the potential of autofluorescence of fixed nonstained samples. For staining, a 1:1 mixture of aniline blue and trypan blue was found most suitable and was used for staining of oomycete and plant structures, allowing discrimination between them as well as the visualization of plant immune responses. The method was also used to examine samples of Peronospora lamii on Lamium purpureum and Peronospora belbahrii on Ocimum basilicum, demonstrating the potential of the presented histological method for studying the infection processes of downy mildews in general.