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The genus Peronospora is the largest genus of the oomycetes, fungus-like members of the kingdom Straminipila that also contains amoeboid (e.g., Leukarachnion) and plant-like (e.g., Laminaria) life forms. Peronospora species are obligate biotrophic plant pathogens, causing high economic losses in various crops and ornamentals, including Plantago species. Several species of Plantago are used as specialty crops and medicinal plants. In this study, Peronospora species parasitic on Plantago were investigated based on morphology and phylogenetic analyses using two nuclear (ITS, nrLSU) loci and one mitochondrial (cox2) locus. As a result of these investigations, 10 new species are added to the already known Peronospora species on Plantago. Interestingly, it was found that four independent species are parasitic to Plantago major, highlighting that the reliance on the host plant for pathogen determination can be misleading in Peronospora. Taking this into account, morphological and phylogenetic analyses should be conducted as a prerequisite for effective quarantine regulations and phytosanitary measures.

Biotic stress leads to enormous yield, quality, and economic losses in grapevine (Vitis vinifera L.). However, the crucial genes and underlying mechanisms in grapevine response to biotic stress are still not fully understood. Therefore, a meta-analysis was done using microarray expression data from 114 samples from five biotic stress investigations to find differentially expressed genes (DEGs). Subsequently, 1005 DEGs yielded from the meta-analysis were subjected to further analysis using a combination of systems biology and machine learning algorithms. A total of 75 transcription factors (TFs) and 80 pathogen recognition genes (PRGs) were identified among the 1005 DEGs. The majority of upregulated PRGs belonged to a class of kinases (KIN), followed by the RLK, RLP, TRAN, N, and CK classes. Furthermore, the MYB and bHLH families had the highest number of up-regulated TFs in response to biotic stress. Their co-expression with biotic stress-related pathways highlights the crucial role of these families in the response to biotic stress in grapevine. Additionally, weighted gene co-expression network analysis identified six modules, three of which were upregulated and relevant to defense responses, photosynthesis process, and cell wall reinforcement. Using eight attribute weighting algorithms on the identified key genes, we prioritized eight genes with the highest relevance to both control and disease conditions. Our findings indicate that the transcriptome responses of grapevine plants to various biotic stimuli, including downy mildew, powdery mildew, grapevine leaf-roll-associated virus-3, and Bois noir phytoplasma, exhibit commonalities. These shared responses could serve as breeding targets for enhancing grapevine resistance to diverse biotic stresses.

Downy mildew of cucumber caused by the oomycete Pseudoperonospora cubensis (P. cubensis) is currently the most destructive disease of this crop, causing high yield losses. Visual evaluation by experts is the most established method for the detection of P. cubensis infection, but depends on visual disease symptoms. Detection of fungal infection before visual symptoms appear is highly desirable, allowing to deploy disease control measures before extensive crop damage occurs. The capacity of remote sensing approaches, such as hyperspectral imaging or high-resolution spectrometry, have proved to be powerful tools for detecting plant fungal diseases at pre-symptomatic stages over the last years. However, these approaches are expensive and processing the massive amount of multidimensional data, in case of spectroscopy and hyperspectral imaging, remains complex. Affordable, easy to handle, devices enabling the identification of P. cubensis infected cucumber plants at the pre-symptomatic stage in-situ may represent a high-value alternative for farmers. This study compared the performance of a high-cost high-resolution spectroradiometer (FieldSpec4) and a lower-cost leaf-clip sensor (DUALEX) for the early detection of P. cubensis infection in cucumber. Screening of cucumber plants, grown in a growth chamber, 24 h and 48 h post inoculation, allowed to identify a subset of spectral bands and leaf pigments for differentiating between infected and healthy cucumber plants at the pre-symptomatic stage. In the case of the FieldSpec 4, models based on spectral bands at 402, 576, 690, 708, and 723 nm differentiated between cucumber plants infected with P. cubensis and healthy plants with a F1 value of 0.67. The use of the lower-cost leaf-clip DUALEX sensor differentiated between infected and healthy cucumber plants with a F1 value of 0.60. Furthermore, the epidermal flavonol content obtained with the + leaf-clip DUALEX sensor was shown to be decreased in cucumber plants infected with

The genetic erosion of the European grapevine diversity in the last century has promoted the conservation of varieties in germplasm banks to prevent their disappearance. The study of these varieties is necessary as it would allow the diversification of the wine market, as well as provide a source of genes to face new pathogens or climate constraints. In this work, the grapevine varieties preserved in the "Estación de Viticultura e Enoloxía de Galicia" (EVEGA) Germplasm Bank (Ourense, Spain) were widely characterized, combining ampelography, ampelometry, agronomy, and phytopathology. Moreover, genetic characterization was carried out through the analysis of 48 single-nucleotide polymorphisms (SNPs). A Bayesian analysis based on the SNP data was carried out to define the genetic structure of the EVEGA Germplasm Bank, which allowed the differentiation of two main reconstructed panmictic populations (RPPs), confirming previous results obtained based on microsatellite markers (SSRs). A great diversity between varieties was found for almost every parameter evaluated for ampelography, ampelometry, phytopatology, phenology, and berry quality. A principal component analysis (PCA) performed with these phenotypical data allowed discrimination among some groups of varieties included in different genetic populations. This study allowed us to evaluate the grapevine diversity maintained in the EVEGA Germplasm Bank and characterize varieties of potential value for breeding programs of interest for the Galician viticulture.

In this thesis, I have investigated microbiomes of plants that are under attack by obligate biotrophic pathogens that cause downy mildew disease. In particular, I have studied the phyllosphere bacterial communities of laboratory cultures of the downy mildews Hyaloperonospora arabidopsidis (Hpa) and Peronospora effusa (Pe) on their respective hosts Arabidopsis thaliana and Spinacia oleracea (henceforth Arabidopsis and spinach). Within these two pathosystems, we observed consistent enrichment of specific bacteria in distinct cultures (Chapters 2 and 4), and for the Arabidopsis system we demonstrated that the genomes of the specific bacteria enriched in distinct laboratory cultures across Europe were isogenic (Chapter 2). These bacteria were further shown to reach higher abundances in the phyllosphere upon Hpa infection (Chapter 2). Also in the rhizosphere, we observed increased colonization by downy mildew-associated bacteria on plants that were grown on soil that was conditioned by downy mildew-infected plants. This suggests that the downy mildew-associated microbes are part of a soil-borne legacy of disease that can be inherited by future generations of plants grown on the same soil (Chapter 3). Moreover, the microbes that are enriched in downy mildew-associated communities appear to be geared towards plant protection (Chapter 3), suggesting that their assembly is indeed directed by the host plant. Lastly, similarities were observed between Pe-associated microbiomes in laboratory cultures and naturally Pe-infected field-grown plants (Chapter 4), highlighting that these microbiomes are not only a laboratory phenomenon. Together, these findings suggest that phyllosphere bacterial communities of plants that are under downy mildew attack are modulated to benefit the plant, meaning that a plant’s cry for help towards the microbiome upon pathogen attack may be a contributing factor to phyllosphere microbiome assembly. Finally, these findings are discussed in a broader per

Downy mildew is one of the most destructive diseases affecting grapevines (Vitis vinifera L.). Caused by the oomycete Plasmopara viticola (Berk. and Curt.) Berl. and de Toni, it can appear anywhere where vines are cultivated. It is habitually controlled by the application of phytosanitary agents (copper-based or systemic) at different stages of the vine growth cycle. This, however, is costly, can lead to reduced yields, has a considerable environmental impact, and its overuse close to harvest can cause fermentation problems. All grapevines are susceptible to this disease, although the degree of susceptibility differs between varieties. Market demands and European legislation on viticulture and the use of phytosanitary agents (art. 14 of Directive 128/2009/EC) now make it important to know the sensitivity of all available varieties, including minority varieties. Such knowledge allows for a more appropriate use of phytosanitary agents, fosters the commercial use of these varieties and thus increases the offer of wines associated with different terroirs, and helps identify material for use in crop improvement programmes via crossing or genetic transformation, etc. Over 2020–2021, the susceptibility to P. viticola of 63 minority vine varieties from different regions of Spain was examined in the laboratory using the leaf disc technique. Some 87% of these varieties were highly susceptible and 11% moderately susceptible; just 2% showed low susceptibility. The least susceptible of all was the variety Morate (Madrid, IMIDRA). Those showing intermediate susceptibility included the varieties Sanguina (Castilla la Mancha, IVICAM), Planta Mula (Comunidad Valenciana, ITVE), Rayada Melonera (Madrid, IMIDRA), Zamarrica (Galicia, EVEGA), Cariñena Roja (Cataluña, INCAVI), Mandrègue (Aragón, DGA) and Bastardo Blanco (Extremadura, CICYTEX). The highly susceptible varieties could be differentiated into three subgroups depending on sporulation severity and density.

Downy mildew, caused by Peronospora variabilis, is the most important quinoa disease worldwide. However, little is known about the resistance mechanisms acting against this disease. The study goals were to identify quinoa accessions showing resistance to P. variabilis under Spanish field conditions and to characterize the resistance mechanism involved. Towards these objectives, a total amount of 229 accessions of Chenopodium quinoa and one accession of each of the species Chenopodiun berlandieri subs. nutillae, Chenopodium ugandae, and Chenopodium opulifolium were screened for resistance to P. variabilis under field conditions in Córdoba, Spain, during two seasons. The response to P. variabilis in the accessions showed a continuous distribution ranging from complete resistance to high susceptibility. Fifteen resistant and one susceptible accessions were selected for further histological studies. Histological results showed that resistance to downy mildew in quinoa acts mainly at the stage of colony establishment. In resistant accessions, no colonies were formed or success in colony establishment was significantly reduced compared with the susceptible control. Hypersensitive response was associated with colony abortion in a number of the resistant accessions. This work is the first proof of hypersensitive reaction occurrence in quinoa as a response to P. variabilis.

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.

Downy mildew pathogens of graminicolous hosts (Poaceae) are members of eight morphologically and phylogenetically distinct genera in the Peronosporaceae (Oomycota, Peronosporales). Graminicolous downy mildews (GDMs) cause severe losses in crops such as maize, millets, sorghum, and sugarcane in many parts of the world, especially in tropical climates. In countries where the most destructive GDMs are not endemic, these organisms are often designated as high-risk foreign pathogens and subject to oversight and quarantine by regulatory officials. Thus, there is a need to reliably and accurately identify the causal organisms. This paper provides an overview of the Peronosporaceae species causing graminicolous downy mildew diseases, with a description of their impact on agriculture and the environment, along with brief summaries of the nomenclatural and taxonomic issues surrounding these taxa. Key diagnostic characters are summarized, including DNA sequence data for types and/or voucher specimens, morphological features, and new illustrations. New sequence data for cox2 and 28S rDNA markers are provided from the type specimens of three species, Peronosclerospora philippinensis, Sclerospora iseilematis, and Sclerospora northii. Thirty-nine species of graminicolous downy mildews are accepted, and seven previously invalidly published taxa are validated. Fifty-five specimens are formally designated as types, including lectotypification of 10 species, neotypification of three species, and holotype designation for Sclerophthora cryophila. Citation: Crouch JA, Davis WJ, Shishkoff N, Castroagudín VL, Martin F, Michelmore R, Thines M (2022). Peronosporaceae species causing downy mildew diseases of Poaceae, including nomenclature revisions and diagnostic resources. Fungal Systematics and Evolution 9: 43-86. doi: 10.3114/fuse.2022.09.05.

In this study, we carried out experiments to evaluate the effect of resistance inducers (RIs) on downy mildew (DM). In a first experiment, we focused on the use of the RIs in a protection strategy based on the combination with copper-based products applied following the indications of the decision support system (DSS) vite.net® provided by Horta. In a second experiment, we studied the efficacy of resistance inducers in controlling DM following artificial inoculation of Plasmopara viticola at different timings after treatment.

Grapevine downy mildew is an important disease in vineyards. It requires continuous work on plant protection products to supply alternative solutions for pest management, avoid fungicides extensive use, and comply with the EU regulations. Fungicides used to control Plasmopara viticula, the causal agent of downy mildew in grapevine are well known in their mode of action. However, a further understanding of the fungicide characteristics, namely, the physical mode of action (PMoA) is important to consider also. The PMoA refers to aspects other than the biochemical mode of action (Moa), that impact the molecule activity within the plant, pathogen, and environment relationships, namely the protection temporal efficacy, the application time, and the product retention and movement. The integration of this information in a plant protection strategy allows for timely and precise management of grapevine pathogens. This work had the purpose to study the Folpet, a well-known contact fungicide, and describing its protection temporal dynamic on grapevine plant material.

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.

Melon (Cucumis melo L.) production is often restricted by a plethora of pests and diseases, including powdery mildew and downy mildew caused respectively by the fungal species Podosphaera xanthii/Golovinomyces orontii and oomycete species Pseudoperonospora cubensis. Many efforts have been directed on identification of resistant sources by screening (wild) melon germplasm. In the current review, we summarized such efforts from various publications of the last 50 plus years. Resistance to powdery mildew has been identified in 239 melon accessions and downy mildew resistance in 452 accessions of both C. melo and the wild relative species C. figarei. Among the resistance sources, C. melo var. cantalupensis accessions PMR 45, PMR 5, PMR 6, and WMR 29 as well as C. melo var. momordica accessions PI 124111, PI 124112, and PI 414723 have been considered as the most valuable germplasm because multiple resistance genes have been identified from these accessions and are widely used in melon resistance breeding. Further genetic mapping in a number of resistant sources has enabled identification of 25 dominant genes, two recessive genes and seven QTLs conferring powdery mildew resistance, as well as eight dominant genes and 11 QTLs for downy mildew resistances. Based on the reported sequences of associated markers, we anchored physically (many of) these genes and QTLs to chromosomes of the melon cv. DHL92 genome. In addition to presenting a comprehensive overview on powdery mildew and downy mildew resistance in (wild) melon germplasm, we suggest strategies aiming at breeding melon with durable and broad-spectrum resistance to pathogens and pests.

Downy mildew is common disease of sunflower. If not controlled, damage inflicted on sunflower can be severe. Widely used method of disease control is growing resistant genotypes. Pathogen is adapting to changes in host by gradual spread and in the end dominance of physiological races that overcome host resistance genes. To minimize major damages from such event, determination of pathogen virulence is needed. The aim of this work was determination of P. halstedii races from samples collected in Serbia. Samples of plants with symptoms of downy mildew were collected and analyzed. For virulence determination standardized set of differential lines was used with additional inbred lines. Inoculation of sunflower seedling was done following the WSI method. The most important finding in this research was P. halstedii race 735.

When growing grapevines there are common risks for disease outbreaks when the pathogen has remained from earlier growing cycles and the weather and soil conditions work in the diseases' favour. The diseases may spread a lot before being highly noticeable. This means that the harvests of grapes for wine production are in jeopardy. This thesis concerns a branch of a, from the Swedish Board of Agriculture funded, project "IoT and AI to increase the competiveness in Swedish fruit and wine production" with the special focus for the thesis to use machine learning tools such as convolutional neural networks to detect the most common grapevine diseases downy mildew and powdery mildew. By using an image inventory from the project staff the purpose is to give decision support regarding the mentioned diseases of an input image using neural networks and image analysis. As being able to notice different types and stages of the grapevine diseases is desired, the network was sought to have 5 class outputs but also testing to have fewer (3 and 2). The resulting model, after training to reach a good validation accuracy, reached an accuracy of ∼65 % when predicting to 5 labels, but with lower recall on the infected classes with fewer image samples. Training for predicting 3 and 2 different outputs gave higher results of ∼70 % and ∼75 % respectively and better tendencies of finding non-healthy leaves. It is believed that a larger amount of training images could improve the performance of the network.

When growing grapevines there are common risks for disease outbreaks when the pathogen has remained from earlier growing cycles and the weather and soil conditions work in the diseases' favour. The diseases may spread a lot before being highly noticeable. This means that the harvests of grapes for wine production are in jeopardy. This thesis concerns a branch of a, from the Swedish Board of Agriculture funded, project "IoT and AI to increase the competiveness in Swedish fruit and wine production" with the special focus for the thesis to use machine learning tools such as convolutional neural networks to detect the most common grapevine diseases downy mildew and powdery mildew. By using an image inventory from the project staff the purpose is to give decision support regarding the mentioned diseases of an input image using neural networks and image analysis. As being able to notice different types and stages of the grapevine diseases is desired, the network was sought to have 5 class outputs but also testing to have fewer (3 and 2). The resulting model, after training to reach a good validation accuracy, reached an accuracy of ∼65 % when predicting to 5 labels, but with lower recall on the infected classes with fewer image samples. Training for predicting 3 and 2 different outputs gave higher results of ∼70 % and ∼75 % respectively and better tendencies of finding non-healthy leaves. It is believed that a larger amount of training images could improve the performance of the network.

The management of downy mildew (Plasmopara halstedii) in sunflower, is heavily dependent on genetic resistance, whilst entomopathogenic fungi (EF) can reduce other sunflower diseases. In this work, we characterized P. halstedii from Spain and other countries collected in the past few years. Twenty-three races were identified (the most frequent in Spain being 310, 304, 705 and 715), with an increasing proportion of highly virulent races. Five isolates from countries other than Spain overcame the resistance in RHA-340. In addition, we assessed the efficacy of five EF against downy mildew and their effects on sunflower growth in axenic conditions. None of the entomopathogens reduced disease severity, nor did they have any effect on plant growth when applied together with P. halstedii. In contrast, three EF reduced some of the plant growth variables in the absence of the pathogen. Microbiological and molecular diagnostics suggest that the axenic system and the short experimental time used in this study did not favor the successful establishment of EF in the plants or their potential biocontrol effect. Our results show a shift in P. halstedii racial patterns and suggest that soil as a growth substrate and long infection times are needed for EF effectiveness against downy mildew.

A mechanistic model was developed to predict secondary infections of Plasmopara viticola and their severity as influenced by environmental conditions; the model incorporates the processes of sporangia production and survival on downy mildew (DM) lesions, dispersal and deposition, and infection. The model was evaluated against observed data (collected in a 3-year vineyard) for its accuracy to predict periods with no sporangia (i.e., for negative prognosis) or with peaks of sporangia, so that growers can identify periods with no/low risk or high risk. The model increased the probability to correctly predict no sporangia [P(P−O−) = 0.67] by two times compared to the prior probability, with fewer than 3% of the total sporangia found in the vineyard being sampled when not predicted by the model. The model also correctly predicted peaks of sporangia, with only 1 of 40 peaks unpredicted. When evaluated for the negative prognosis of infection periods, the model showed a posterior probability for infection not to occur when not predicted P(P−O−) = 0.87 with only 9 of 108 real infections not predicted; these unpredicted infections were mild, accounting for only 4.4% of the total DM lesions observed in the vineyard. In conclusion, the model was able to identify periods in which the DM risk was nil or very low. It may, therefore, help growers avoid fungicide sprays when not needed and lengthen the interval between two sprays, i.e., it will help growers move from calendar-based to risk-based fungicide schedules for the control of P. viticola in vineyards.

Breeding technologies exploiting marker-assisted selection have accelerated the selection of new cross-bred pathogen-resistant grapevine varieties. Several genotypes have been patented and admitted to cultivation; however, while their tolerance to fungal diseases has been the object of several in vitro and field studies, their productive and fruit composition traits during ripening are still poorly explored, especially in warm sites. In this study, five white pathogen-resistant varieties (PRVs) listed as UD 80–100, Soreli, UD 30–080, Sauvignon Rytos, Sauvignon Kretos were tested over two consecutive seasons in a site with a seasonal heat accumulation of about 2000 growing degree days (GDDs), and their performances were compared to two Vitis vinifera L. traditional varieties, Ortrugo and Sauvignon Blanc. Berries were weekly sampled from pre-veraison until harvest to determine total soluble solids (TSS) and titratable acidity (TA) dynamics. All tested PRV exhibited an earlier onset of veraison and a faster sugar accumulation, as compared to Ortrugo and Sauvignon Blanc, especially in 2019. At harvest, Sauvignon Blanc was the cultivar showing the highest titratable acidity (8.8 g/L). Ortrugo and PRV varieties showed very low TA (about 4.7 g/L), with the exception of Sauvignon Rytos (6.5 g/L). However, data disclose that Sauvignon Rytos higher acidity at harvest relies on higher tartrate (+1.1 to +2.2 g/L, as compared to other PRV), whereas in Sauvignon Blanc, high TA at harvest is due to either tartaric (+1 g/L, compared to PRV) and malic (+2.5 g/L, compared to PRV) acid retention. Overall, Sauvignon Rytos is the most suited PRV to be grown in a warm climate, where retaining adequate acidity at harvest is crucial to produce high-quality white wines. Nevertheless, canopy and ripening management strategies must be significantly adjusted, as compared to the standard practice employed for the parental Sauvignon Blanc.

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.

In India, Brassica is attacked by many bacterial, fungal and viral pathogens causing various diseases among which, downy mildew caused by Hyaloperonospora brassicae, an oomycete is the most severe one. The present investigation was conducted to reduce fungicide load on Brassica by testing an alternate method of disease control. Different Systemic Acquired Resistance (SAR) compounds were tested as foliar sprays like Salicylic acid, Jasmonic acid and Bion (Benzothiadiazole-BTH) for inducing resistance in different genotypes of Raya (Brassica juncea) against downy mildew pathogen. Protein content in plants ranged between 43.5 to 57.7 mg/g fresh weight compared to 37.2 mg/g fresh weight in control. Induction of proteins and defense related enzymes was systemic in nature. The SAR compounds also surged the levels of defense related proteins, i.e. Polyphenol oxidase (PPO), Phenylalanine ammonia lyase (PAL) and pathogenesis related Pr- proteins i.e. ?-1,3 glucanase, Peroxidase (POD), from 21 to 130 per cent indicating induction of resistance. Protein profiling of treated Brassica plants was also done electrophoreticaly, which further confirmed the induction of pathogenesis-related proteins ranging from 15- 75 kDa along with some other proteins. Salicylic acid @ 500µM showed best results with 71.27 per cent disease control followed by Jasmonic acid with 69.6 per cent; whereas both, Bion and ?eta amino butyric acid gave almost 63 per cent disease control as compared to control plants. Integration of disease tolerance in Brassica varieties/genotypes combined with prophylactic spray of salicylic acid proved to be very economical for managing downy mildew disease.

Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here, we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was around 35.4 Mbp in length, with an N50 scaffold length of around 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of around 40.1 kbp. From the repeat-masked genome, 9,049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16% of the genome consists of repetitive sequences, and, based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of P. belbahrii. P. belbahrii has undergone a high degree of convergent evolution with other obligate parasitic pathogen groups, reflecting its obligate biotrophic lifestyle. Features of its secretome, signaling networks, and promoters are presented, and some patterns are hypothesized to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.

Disease development in plants requires a susceptible host, a virulent pathogen, and a favourable environment. Oomycete pathogens cause many important diseases and have evolved sophisticated molecular mechanisms to manipulate their hosts. Day length has been shown to impact plant-oomycete interactions but a need exists for a tractable reference system to understand the mechanistic interplay between light regulation, oomycete pathogen virulence, and plant host immunity. Here we present data demonstrating that light is a critical factor in the interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We investigated the role of light on spore germination, mycelium development, sporulation, and oospore formation of Hpa, along with defence responses in the host. We observed abundant Hpa sporulation on compatible Arabidopsis under day lengths ranging from 10 to 14 hr. In contrast, exposure to constant light or constant dark suppressed sporulation. Exposure to constant dark suppressed spore germination, mycelial development, and oospore formation, whereas exposure to constant light stimulated these three stages of development. A biomarker of plant immune system activation was induced under both constant light and constant dark. Altogether, these findings demonstrate that Hpa has the molecular mechanisms to perceive and respond to light and that both the host and pathogen responses are influenced by the light regime. Therefore, this pathosystem can be used for investigations to understand the molecular mechanisms through which oomycete pathogens like Hpa perceive and integrate light signals, and how light influences pathogen virulence and host immunity during their interactions.

Research has linked chlorothalonil exposure to declines in pollinator health due to an increased likelihood of Nosema ceranae infection, altered gut microbial community, and a reduction in colony fitness and survival of honey bees (Apis mellifera). Therefore, a reduction in use of chlorothalonil, a large component of cucurbit disease management, may be needed. Without chlorothalonil, a widely used, broad-spectrum fungicide, the fungal and oomycete pathogens in cucurbit cropping systems can more quickly evolve resistance to targeted fungicides due to a limited number of efficacious modes of action and frequent sprays. Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew, for example, has a short life cycle, experiences repeated applications of fungicides, and has a wide host range making it a high risk for fungicide resistance development. Our research focused on the development of an alternative fungicide spray program in melons to reduce the use of chlorothalonil, identifying the fungicide insensitivities of local P. cubensis populations and determining the efficacy of fungicides used to manage cucurbit downy mildew, and investigating the clade-host relationship and formation of oospores in regional P. cubensis samples. Efficacy on two important diseases in melon, powdery mildew and gummy stem blight, can be largely maintained without chlorothalonil but anthracnose control was not adequate without the inclusion of chlorothalonil. Currently, there are a number of highly effective targeted fungicides available to growers for management of cucurbit downy mildew including oxathiapiprolin, zoxamide + chlorothalonil, chlorothalonil, and cyazofamid. Our research shows evidence of P. cubensis clade-host associations, with clade 1 preferentially infecting acorn and summer squash (Cucurbita pepo), butternut squash (Cucurbita moschata), and watermelon (Citrullus lanatus), while clade 2 preferentially infects cucumber (Cucumis sativus). Melons (Cucumis melo) a

Disease development in plants requires a susceptible host, a virulent pathogen, and a favourable environment. Oomycete pathogens cause many important diseases and have evolved sophisticated molecular mechanisms to manipulate their hosts. Day length has been shown to impact plant-oomycete interactions but a need exists for a tractable reference system to understand the mechanistic interplay between light regulation, oomycete pathogen virulence, and plant host immunity. Here we present data demonstrating that light is a critical factor in the interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We investigated the role of light on spore germination, mycelium development, sporulation, and oospore formation of Hpa, along with defence responses in the host. We observed abundant Hpa sporulation on compatible Arabidopsis under day lengths ranging from 10 to 14 hr. In contrast, exposure to constant light or constant dark suppressed sporulation. Exposure to constant dark suppressed spore germination, mycelial development, and oospore formation, whereas exposure to constant light stimulated these three stages of development. A biomarker of plant immune system activation was induced under both constant light and constant dark. Altogether, these findings demonstrate that Hpa has the molecular mechanisms to perceive and respond to light and that both the host and pathogen responses are influenced by the light regime. Therefore, this pathosystem can be used for investigations to understand the molecular mechanisms through which oomycete pathogens like Hpa perceive and integrate light signals, and how light influences pathogen virulence and host immunity during their interactions.

Notre époque est indéniablement marquée par les changements climatiques et la baisse drastique de la biodiversité, questionnant la durabilité de l'ensemble de nos systèmes productifs. En agriculture, la remise en cause des pratiques dites conventionnelles est de plus en plus prégnante. L'usage des pesticides est l'une des pratiques les plus controversées. Bien qu'ayant contribués à l'accroissement sans précédent des rendements agricoles dans les années 1970, ces produits inquiètent désormais par leur dangerosité, autant pour la santé humaine que pour celle de l'environnement. Ils impactent notamment de façon directe la santé des populations d'insectes, dont les pollinisateurs, et du microbiote des sols. Sur le long terme, si l'usage actuel persiste, un déséquilibre des écosystèmes est à craindre. Il y a donc une urgence à sortir du système actuel de gestion de problèmes phytosanitaires. Cette évolution ne va pas être du même niveau de simplicité pour toutes les cultures. La viticulture utilise des quantités importantes de pesticides. En France, en 2006, la vigne représentait 3,3 % de la surface agricole mais 14,4 % des pesticides utilisés. Réaliser des traitements adaptés à la situation phytosanitaire réelle de la parcelle contribuerait à réduire cette consommation. Cependant, connaître l'état de santé précis d'une parcelle donnée n'est pas une information facile à obtenir. Prospecter tout un vignoble prend beaucoup de temps, ce qui rend sa réalisation régulière difficile. Dans ce projet, nous souhaitons faciliter la prospection par son automatisation. Celle-ci pourrait se matérialiser par une caméra montée sur un robot, un tracteur ou un drone, dont les images seraient ensuite analysées automatiquement, permettant à l'agriculteur d'obtenir une carte de l'état de santé de ses parcelles. Toutefois, développer un tel outil est loin d'être simple du fait de la complexité des cultures. Celui-ci doit être capable de mener une analyse malgré la diversité des variétés, des, Climate change and the severe decline in biodiversity undeniably mark our era, questioning the sustainability of all our production systems. In agriculture, the reconsideration of so-called conventional practices is more and more prevalent. Use of pesticides is one of the most controversial practices. Despite their contribution to the unprecedented increase in agricultural yields in the 1970s, these products are now a matter of concern because of their danger to both human health and the environment. In particular, they directly impact the health of insect populations, including pollinators, as well as soil microbiota. In the long term, if current use persists, imbalance in the ecosystems is to be expected. Therefore, urgent action is needed to move away from the current phytosanitary management system. This change will not be as simple for all crops. Viticulture uses significant quantities of pesticides. In France, in 2006, vineyards covered 3.3 % of the agricultural surface but accounted for 14.4 % of the pesticides used. Applying treatments according to the real health conditions of the field would reduce this consumption. However, it is not simple to know the precise health status of a given plot. Scouting an entire vineyard requires a significant amount of time, which makes it difficult to do so on a regular basis. In this project, we intend to facilitate scouting by its automation. It could be achieved by a camera mounted on a robot, a tractor or a drone, whose images would then be automatically analyzed, providing the farmer with a health map of his fields. However, developing such a tool is not easy given the crops complexity. It must be able to analyze images despite the diversity of varieties, phenological stages, as well as the diversity of pests, diseases and all their symptoms. The variety of acquisition conditions and the complexity of the foliage and background also constitute challenges for the analysis, hindering the development of a reli

Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here, we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was around 35.4 Mbp in length, with an N50 scaffold length of around 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of around 40.1 kbp. From the repeat-masked genome, 9,049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16% of the genome consists of repetitive sequences, and, based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of P. belbahrii. P. belbahrii has undergone a high degree of convergent evolution with other obligate parasitic pathogen groups, reflecting its obligate biotrophic lifestyle. Features of its secretome, signaling networks, and promoters are presented, and some patterns are hypothesized to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.

Grapevine downy mildew (DM) is caused by the dimorphic oomycete Plasmopara viticola, which incites epidemics through primary and secondary infection cycles that occur throughout the season. The secondary infection cycles are caused by the sporangia produced on DM lesions. The current research examined the relationship between numbers of airborne sporangia and DM development on grape leaves to determine whether spore sampler data can be useful to predict the potential for secondary infections of P. viticola. Three years (2015–2017) of spore sampler data confirmed that sporangia are a common component of the airborne microflora in a DM-infested vineyard and that their numbers depend on weather conditions. For a total of 108 days, leaf samples were collected from the vineyard at 2- to 3-day intervals and incubated under optimal conditions for P. viticola infection. The numbers of airborne sporangia sampled on 1 to 7 days before leaf sampling were significantly correlated with the numbers of DM lesions on the leaves. The best correlation (r=0.59), however, was found for the numbers of viable airborne sporangia (SPV), which were assessed by using equations driven by the vapour pressure deficit. In Bayesian and ROC curve analyses, SPV was found to be a good predictor of P. viticola infection of grape leaves, with AUROC=0.821 and false positive predictions mainly occurring at low SPV. A binary logistic regression showed that a threshold of 2.52 viable sporangia m-3 air day-1 enables a prediction of no infection with a posterior probability of 0.870, which was higher than the prior probability of 0.574. Numbers of viable sporangia in the vineyard air is therefore a useful predictor of infection and especially of no infection. The predictor missed some observed infections, but these infections were not severe (they accounted for only 31 of 374 DM lesions).

Plant-pathogenic oomycetes secrete effector proteins to suppress host immune responses. Resistance proteins may recognize effectors and activate immunity, which is often associated with a hypersensitive response (HR). Transient expression of effectors in plant germplasm and screening for HR has proven to be a powerful tool in the identification of new resistance genes. In this study, 14 effectors from the lettuce downy mildew Bremia lactucae race Bl:24 were screened for HR induction in over 150 lettuce accessions. Three effectors-BLN06, BLR38 and BLR40-were recognized in specific lettuce lines. The recognition of effector BLR38 in Lactuca serriola LS102 did not co-segregate with resistance against race Bl:24, but was linked to resistance against multiple other B. lactucae races. Two unlinked loci are both required for effector recognition and are located near known major resistance clusters. Gene dosage affects the intensity of the BLR38-triggered HR, but is of minor importance for disease resistance.

The control of Plasmopara viticola and Botrytis cinerea, two of the most dangerous pathogens on grapevine, requires frequent treatments with chemicals based on weather conditions. Numerous applications of fungicides have resulted in developing fungicide resistance. Active ingredients based on copper have been used very successfully for a long time to protect grapevines against these pathogens. Application of a copper citrate formulation with high degree dissociation at a very low concentration (1%) was evaluated in field trials. The efficacy of two concentrations of copper citrate, 0.5 and 1.0%, were tested against P. viticola on grapevine in three locations, and against B. cinerea in two locations during 2018. Our results demonstrated that the concentration of 1.0% copper citrate was highly effective against P. viticola (87.4%) and B. cinerea (63.7%), compared to standard treatment (90.6 and 53.1%), under a high level of infection., Zaštita vinove loze od prouzrokovača plamenjače vinove loze - Plasmopara viticola i sive truleži - Botrytis cinerea, je vrlo kompleksna i zahteva primenu većeg broja hemijskih tretmana, u skladu sa vremenskim uslovima. Učestala primena fungicida uslovljavala je pojavu rezistentnih izolata patogena na fungicide. Različite forme bakarnih jedinjenja primenjuju se u zaštiti vinove loze dugi niz godina prilično uspešno. Primena bakar-citrata - formulacije sa visokim stepenom disocijacije u niskoj koncentraciji (1,0%) ispitivana je u poljskim uslovima. Efikasnost dve koncentracije bakar-citrata - 0.5 i 1.0% je testirana u suzbijanju P. viticola i B. cinerea na vinovoj lozi na tri (dva) lokaliteta, tokom 2018 godine. Naši rezultati pokazuju da je ispitivana koncentracija od 1,0% bakar-citrata ispoljila zadovoljavajući efekat na P. viticola (87,4%) i B. cinerea (63,7%) u odnosu na primenjene standarde (90,6% i 53,1%) u uslovima visokih zaraza.

The control of Plasmopara viticola and Botrytis cinerea, two of the most dangerous pathogens on grapevine, requires frequent treatments with chemicals based on weather conditions. Numerous applications of fungicides have resulted in developing fungicide resistance. Active ingredients based on copper have been used very successfully for a long time to protect grapevines against these pathogens. Application of a copper citrate formulation with high degree dissociation at a very low concentration (1%) was evaluated in field trials. The efficacy of two concentrations of copper citrate, 0.5 and 1.0%, were tested against P. viticola on grapevine in three locations, and against B. cinerea in two locations during 2018. Our results demonstrated that the concentration of 1.0% copper citrate was highly effective against P. viticola (87.4%) and B. cinerea (63.7%), compared to standard treatment (90.6 and 53.1%), under a high level of infection., Zaštita vinove loze od prouzrokovača plamenjače vinove loze - Plasmopara viticola i sive truleži - Botrytis cinerea, je vrlo kompleksna i zahteva primenu većeg broja hemijskih tretmana, u skladu sa vremenskim uslovima. Učestala primena fungicida uslovljavala je pojavu rezistentnih izolata patogena na fungicide. Različite forme bakarnih jedinjenja primenjuju se u zaštiti vinove loze dugi niz godina prilično uspešno. Primena bakar-citrata - formulacije sa visokim stepenom disocijacije u niskoj koncentraciji (1,0%) ispitivana je u poljskim uslovima. Efikasnost dve koncentracije bakar-citrata - 0.5 i 1.0% je testirana u suzbijanju P. viticola i B. cinerea na vinovoj lozi na tri (dva) lokaliteta, tokom 2018 godine. Naši rezultati pokazuju da je ispitivana koncentracija od 1,0% bakar-citrata ispoljila zadovoljavajući efekat na P. viticola (87,4%) i B. cinerea (63,7%) u odnosu na primenjene standarde (90,6% i 53,1%) u uslovima visokih zaraza.

Production of fresh herbs is a trendy market that continues to grow in popularity in the United States. Basil (Ocimum baasilicum) is such an herb that is experiencing growth in popularity, especially sweet basil. In Virginia, sweet basil production is increasing and can occur both within the field and greenhouses. However, basil production faces certain challenges, with the most serious one being disease losses caused by downy mildew. With few effective control measures available it is important to find management practices that can assist in controlling this disease, particularly in organic systems. Thus, studies were performed examining eight different sweet basil cultivars during the summer growing season of 2019. The study took place at Adam’s Apples and Herbs, located in Shenandoah County in the Shenandoah Valley of Virginia and at Virginia Tech’s Eastern Shore Agricultural Research and Extension (ESAREC) in Painter. At harvest, basil was weighed, by leaf and by stem, and then an average of the weight was calculated as was the height of each cultivar. Disease incidence and severity for each basil cultivar was recorded throughout the course of the study. Little disease occurred during the course of the study in the Shenandoah Valley. However, significant differences in basil cultivar growth and appearance were noted. At the ESAREC, downy mildew did develop and resistant cultivars proved to be effective at suppressing disease.

Directive 128/2009/EC on the Sustainable Use of Pesticides makes mandatory the adoption of integrated pest management (IPM) across Europe. IPM promotes low pesticide control of harmful organisms based on crop monitoring and decision support tools. Vite.net® is a Decision Support System (DSS) developed by Horta (www.horta-srl.com), a spin-off company of the Università Cattolica del Sacro Cuore, in Italy. The DSS is a web-based tool able to: i) collect multiple information/data in real-time about different vineyard components (air, soil, plants, pests, and diseases) by using IoT technologies; ii) analyze these data by advanced modelling and bigdata solutions; and iii) make up-to-date information, alerts and decision supports for vineyard management. The DSS considers grapevine growth and development, risk for diseases (downy and powdery mildews, black-rot and Botrytis bunch rot) and pests (berry moth, American leafhopper and mealybugs), protection dynamics of fungicide applications, and abiotic stresses (drought, low and high temperatures). The DSS is available for Italian growers since 2013 and it is used by hundreds of farmers. Since 2015, the DSS is available also in Spain and Portugal. In this work, we show the results of two pilot runs developed in Spain and Portugal to evaluate the use of vite.net, primarily for the management of downy mildew in vineyards. In Spain, the DSS was evaluated in the Penedès region (Nort-East Spain) by the regional service of plant health (SISTEMIO project). The evaluation was performed in 18 vineyards in 2016 and 2017, by comparing the DSS output concerning primary and secondary infections of Plasmopara viticola, fungicide treatments performed by the growers, and the disease development. In Portugal, the DSS was evaluated in the Alto Douro region (North Portugal) by the Association for the Development of Viticulture in the Douro Region (ADVID). In this case, the evaluation was performed in two vineyards from 2016 to 2018, by compar

Downy mildew caused by Sclerospora graminicola is the most devastating disease in pearl millet which leads to decrease in fodder and grain yield. Application of nanoparticles has become a major thrust for disease management in agriculture. In this study, green synthesized zinc oxide nanoparticles were evaluated for their efficiency to suppress downy mildew. Saponin rich fraction from aqueous extract of Eclipta alba was used to synthesise zinc oxide nanoparticles. The synthesized nanoparticles were characterized using UV spectroscopy, FTIR, SEM, XRD, DLS and EDAX analysis. Under laboratory and greenhouse conditions, seed treatment with synthesized nanoparticles (NP) significantly enhanced pearl millet seed germination, vigor, plant height, fresh and dry weight of seedlings. In inductively coupled plasma mass spectrometry analysis, the zinc content in nanoparticle treated seedlings was found to be higher compared to control seedlings. Sporangicidal assay of ZnO NP showed that 50 ppm of nanoparticle treatment led to plasmolysis and inhibition of spore germination of S. graminicola zoospore. ZnO NP was applied as seed treatment and foliar spray which resulted in 35% reduction in downy mildew incidence in comparison to untreated control. ZnO NP treated seedlings showed high lignification and callose deposition upon downy mildew infection. Analysis of defense enzymes showed that nanoparticle treatment significantly enhanced the activities of peroxidase, phenylalanine ammonia-lyase, lipoxygenase and polyphenol oxidase in comparison to untreated control. Semi-quantitative RT-PCR analysis revealed differentially expressed transcripts of the defense enzymes where the genes were over expressed in treated seedlings compared to low expression in control. These results indicate that synthesized ZnO NP could promote growth and induce systemic resistance in pearl millet against S. graminicola and can be effectively used to manage downy mildew.

The control of Plasmopara viticola and Botrytis cinerea, two of the most dangerous pathogens on grapevine, requires frequent treatments with chemicals based on weather conditions. Numerous applications of fungicides have resulted in developing fungicide resistance. Active ingredients based on copper have been used very successfully for a long time to protect grapevines against these pathogens. Application of a copper citrate formulation with high degree dissociation at a very low concentration (1%) was evaluated in field trials. The efficacy of two concentrations of copper citrate, 0.5 and 1.0%, were tested against P. viticola on grapevine in three locations, and against B. cinerea in two locations during 2018. Our results demonstrated that the concentration of 1.0% copper citrate was highly effective against P. viticola (87.4%) and B. cinerea (63.7%), compared to standard treatment (90.6 and 53.1%), under a high level of infection., Zaštita vinove loze od prouzrokovača plamenjače vinove loze - Plasmopara viticola i sive truleži - Botrytis cinerea, je vrlo kompleksna i zahteva primenu većeg broja hemijskih tretmana, u skladu sa vremenskim uslovima. Učestala primena fungicida uslovljavala je pojavu rezistentnih izolata patogena na fungicide. Različite forme bakarnih jedinjenja primenjuju se u zaštiti vinove loze dugi niz godina prilično uspešno. Primena bakar-citrata - formulacije sa visokim stepenom disocijacije u niskoj koncentraciji (1,0%) ispitivana je u poljskim uslovima. Efikasnost dve koncentracije bakar-citrata - 0.5 i 1.0% je testirana u suzbijanju P. viticola i B. cinerea na vinovoj lozi na tri (dva) lokaliteta, tokom 2018 godine. Naši rezultati pokazuju da je ispitivana koncentracija od 1,0% bakar-citrata ispoljila zadovoljavajući efekat na P. viticola (87,4%) i B. cinerea (63,7%) u odnosu na primenjene standarde (90,6% i 53,1%) u uslovima visokih zaraza.

During the evolution plants have developed a large set af defense mechanisms aerinetpashogens, Defense responses have been sudlein dealin model lant speces such as Arabidopsis. However, to complete our itis i to study defense in del plants, Suhoe In our experiment we aimed to study expression of defense-related genes in the sunflower host plant during combined Infection with downy mildew and broomrape and elucidate thelr role in sunflower reaction to attack of different pathogens. Downey-mildew-resistant genotype was inoculated with the pathogen and planted Into the broomrape inoculated soll. Far gene expression analysis, leaf samples were taken at different stages from planis with combined broomrape-downy mildew infection, plants only infected with broomrape, and control plants, Expression of defense related genes is analyzed using RT-PCR, with special focus on defensin.

The aims of this thesis were to identify genes involved in cucumber-mildew interactions, in order to better understand these pathosystems, thus providing new leads for the breeding of mildew resistant cucumbers. As resistances against both PM and DM were previously shown to be usually recessive, special attention is given to the concept of susceptibility genes (S genes), loss-of-function alleles of which can contribute to effective and durable resistance.

One of the most dangerous fungal diseases in viticulture is downy mildew. Copper-based active ingredients have been used for a long time to protect vines against Plasmopara viticola and they have always maintained an important role in the defense of vines especially for organic production. On the other hand, copper accumulates in the soil causing toxicity with negative consequences on plants and soil fauna. For this reason, new formulations with lower copper content have been considered. In recent years, foliar fertilizers have been studied, in particular those based on phosphites that, along with the nutritional function, stimulate the production of phytoalexins. Those metabolites cause the induced systemic resistance and act directly in the break of cell membranes of pathogens. This study aims to investigate the efficacy against downy mildew of a commercial product based on phosphites and of a new copper- based foliar fertilizer, where copper is chelated with gluconic acid and aminoacids of plant origin. Based on present results, phosphites significantly reduced the infection, without anyway achieving the defense effectiveness of the copper-based treatment however they ensure a significant reduction of the doses of copper necessary to achieve adequate levels of vineyard protection.

In modern agriculture, cultivated lettuce (Lactuca sativa) is typically grown in large monocultures with multiple crops per year and is susceptible to disease outbreaks. In particular the plant pathogenic oomycete Bremia lactucae that causes downy mildew disease on lettuce, has devastating effects and is considered the most important disease affecting the production of cultivated lettuce worldwide. Consequently, the introgression of resistance (R) genes that provide high levels of resistance, is a major aspect of breeding programs. Deployment of R genes exerts strong selection pressure on the pathogen resulting in the rapid breakdown of resistance. Achieving durable resistance against B. lactucae is therefore still a major challenge. Activation of R proteins is dependent on the recognition of effectors. Effector proteins are produced by plant pathogens to facilitate infection. Following the recognition of effectors, an immune response ensues, which is frequently associated with localized cell death (necrosis). In this thesis, we exploited this phenomenon to discover novel R genes. Expression of effector BLR38 resulted in necrosis in the prickly lettuce (Lactuca serriola) accession LS102. Plants that recognized BLR38 were resistant to multiple B. lactucae isolates but not isolate Bl:24 from which BLR38 was cloned. Possibly, isolate Bl:24 contains one or more effectors that are absent in other B. lactucae isolates and effectively interfere with BLR38-triggered immune responses in lettuce. Interestingly, recognition of BLR38 required two loci on distinct chromosomes and was gene dosage dependent. An improved understanding of effector targets in lettuce can help to elucidate the underlying molecular mechanisms leading to disease susceptibility and, potentially, lead to alternative sources of durable resistance. To identify effector targets in lettuce, we performed a protein-protein interaction study using the yeast-two-hybrid (Y2H) system. We identified 61 interacti

The transition from conventional to sustainable viticulture requires increased knowledge of the farming system and makes decision making for crop management complex (Rossi et al., 2012). In Europe, the framework for sustainable viticulture is the Directive 128/2009/EC on the Sustainable Use of Pesticides that makes integrated pest management (IPM) mandatory across Europe. IPM promotes low pesticide control of harmful organisms based on crop monitoring and tools for decision-making. A decision support system (DSS), called vite.net, was developed for vineyards, powdered by Horta, a spin-off of UNICATT. The DSS is a web-based tool able to: i) collect multiple information/data in real-time about different vineyard components (air, soil, plants, pests, and diseases) by using IoT technologies; ii) analyze these data by advanced modelling and bigdata solutions; and iii) make up-to-date information, alerts and decision supports for vineyard management. The DSS considers grapevine growth and development, risk for diseases (downy and powdery mildews, black-rot and Botrytis bunch rot) and pests (berry moth, American leafhopper and mealybugs), protection dynamics of fungicide applications, and abiotic stresses (drought, low and high temperatures). In season 2017, the DSS was used by 300 farmers on approximately 12.000 ha across Italy and in 36 pilot vineyards in Spain. The use of the DSSs significantly increase the farmer’s profit and decrease the environmental impact of crop production.

La vite (Vitis vinifera) è una delle più importanti colture a livello mondiale, soprattutto in termini economici. La maggior parte delle varietà usate per la produzione di uva da tavola e da vino sono però suscettibili a numerose malattie, tra le quali la peronospora (causata da Plasmopara viticola) ed l’oidio (causato da Erysiphe necator). Queste malattie sono controllate dall’uso frequente di fungicidi chimici che, oltre ad avere un elevato costo economico per il produttore, hanno effetti nocivi sull’uomo e sull’ambiente e possono causare l’insorgenza di resistenza del patogeno. Per questo motivo vi è un enorme interesse nello sviluppo di alternative eco-compatibili per il controllo di queste due malattie. Ad esempio, l'ottenimento di varietà di vite resistenti ai patogeni rappresenta un'opzione promettente per limitare l'applicazione dei pesticidi chimici in viticoltura. La comprensione approfondita dei meccanismi di difesa della vite a peronospora ed oidio e l'identificazione dei geni chiave della resistenza sono quindi fondamentali per aiutare i programmi di miglioramento genetico finalizzati all’ottenimento di varietà resistenti. Oltre ai geni di resistenza, i geni di suscettibilità sono regolatori chiave dei processi di difesa nelle piante, come i geni Mildew Locus O (MLO) noti per essere responsabili della suscettibilità all’oidio in piante erbacee. Le analisi di espressione genica sono state ampiamente impiegate per comprendere i processi di regolazione dell'interazione della vite con la peronospora e l'oidio e per individuare i geni marcatori della resistenza, ma tutti gli studi condotti finora hanno preso in considerazione la foglia intera. Tuttavia, solo poche cellule sono realmente in contatto con il patogeno ai primi stadi di infezione e i cambiamenti trascrizionali altamente localizzati nelle cellule infette possono essere mascherati dalle restanti cellule non infette nelle analisi di organi interi. L'obiettivo finale di questo progetto di dottorato e, Grapevine (Vitis vinifera) is one of the most economically important fruit crops worldwide, but many cultivars conventionally used for table grape and wine production are susceptible to two several diseases, such as downy mildew (caused by Plasmopara viticola) and powdery mildew (caused by Erysiphe necator). These pathogens are controlled by frequent applications of chemical fungicides to avoid significant losses in yield and berry quality. However, the ecological drawbacks of pesticides and the rapid appearance of resistant pathogen strains have sparked crescent interest in sustainable control alternatives. The development of resistant grapevine hybrids represents a promising option to limit the application of chemical pesticides in viticulture. Thus, a better understanding of the mechanisms responsible for the grapevine defense against downy and powdery mildew and the identification of key resistance genes will help breeding programs to develop resistant varieties. In addition to the resistance genes, the susceptibility genes are key regulators of defense processes in plants, such as the Mildew Locus O (MLO) genes that are known to be responsible for powdery mildew susceptibility in herbaceous plants. Gene expression profiling has been used extensively to investigate regulation processes of the grapevine interaction with mildews and to identify gene markers of the grapevine resistance. However, all studies to date have involved the use of whole leaves, while only a small fraction of host cells is in contact with the pathogen at the early infection stages. Therefore, highly localized transcriptional changes of infected cells may be masked by the large portion of non-infected cells when analyzing the whole leaf. The final goal of this PhD project was to develop innovative methods for disease control, such as resistant varieties and efficient resistance inducers, based on a better understanding of the key genes and cellular processes involved in the defense and susce, Dottorato di ricerca in Genetica e biologia cellulare

Downy mildew (DM) of opium poppy (Papaver somniferum) caused by Peronospora somniferi is one of the most destructive diseases of this crop due to the systemic nature of infection as compared with local infections caused by Peronospora meconopsidis, the other downy mildew pathogen of this crop. We developed an inoculation method using Peronospora somniferi sporangia as inoculum and demonstrated for the first time that local infection of leaves by sporangia give rise to systemic infections in the plant as well as of seeds. Our results also showed that this inoculation protocol was very effective in reproducing disease symptoms and assessing the resistance response to DM in opium poppy genotypes under field conditions. More interestingly, results indicate that up to 100% of seed samples from some genotypes showing a complete (symptomless) resistant phenotype were infected by the pathogen when seeds were analyzed by a P. somniferi-specific nested-PCR protocol. This latter aspect deserves further attention while breeding opium poppy for resistance to P. somniferi.

Downy mildew (DM) of opium poppy (Papaver somniferum) caused by Peronospora somniferi is one of the most destructive diseases of this crop due to the systemic nature of infection as compared with local infections caused by Peronospora meconopsidis, the other downy mildew pathogen of this crop. We developed an inoculation method using Peronospora somniferi sporangia as inoculum and demonstrated for the first time that local infection of leaves by sporangia give rise to systemic infections in the plant as well as of seeds. Our results also showed that this inoculation protocol was very effective in reproducing disease symptoms and assessing the resistance response to DM in opium poppy genotypes under field conditions. More interestingly, results indicate that up to 100% of seed samples from some genotypes showing a complete (symptomless) resistant phenotype were infected by the pathogen when seeds were analyzed by a P. somniferi-specific nested-PCR protocol. This latter aspect deserves further attention while breeding opium poppy for resistance to P. somniferi.

Downy mildew (DM) of opium poppy (Papaver somniferum) caused by Peronospora somniferi is one of the most destructive diseases of this crop due to the systemic nature of infection as compared with local infections caused by Peronospora meconopsidis, the other downy mildew pathogen of this crop. We developed an inoculation method using Peronospora somniferi sporangia as inoculum and demonstrated for the first time that local infection of leaves by sporangia give rise to systemic infections in the plant as well as of seeds. Our results also showed that this inoculation protocol was very effective in reproducing disease symptoms and assessing the resistance response to DM in opium poppy genotypes under field conditions. More interestingly, results indicate that up to 100% of seed samples from some genotypes showing a complete (symptomless) resistant phenotype were infected by the pathogen when seeds were analyzed by a P. somniferi-specific nested-PCR protocol. This latter aspect deserves further attention while breeding opium poppy for resistance to P. somniferi.

During the 2016 growing season, biological efficacy of fungicides mixture propamocarb hydrochloride + chlorothalonil, metalaxyl-M + mancozeb and metalksil-M + chlorothalonil in the control of downy mildew in pea crop (Peronospora viciae), was investigated. The experiments were conducted in the localities of Sakule and Glogonj. The experiments were carried out as randomized block system in four replications EPPO (EPPO, 1997). Two fugicide applications were conducted , one at the beginning of flowering (BBCH 61) (1st treatment), and second at the stage when 10% of pods reached typical size (BBCH 71) (2nd treatment). Favorable conditions resulted in significant disease intensity on untreated plots, which amounted from 19.9 to 24.8% in the first, and from 32.3 to 44.5% in the second assessment. Fungicide mixture propamocarb-hydrochloride + chlorothalonil exhibited efficacy ranging from 92.7-93.9% at the first and from 85.5 to 91.9% at the second assessment. Fungicide metalaxyl-M + mancozeb, reached the efficacy of 93.1-95.4% at the first, and 86.3-93.2% at the second assessment, while the highest efficacy was achieved by a combination metalaxyl-M + chlorothalonil, which ranged from 86.9 to 100% at the first, and from 86.9 to 95.3% at the second assessment., U toku 2016. godine, vršena su ispitivanja biološke efikasnosti fungicida na bazi kombinacije propamokarb-hidrohlorid + hlorotalonil, metalaksil-M + mankozeb i metalksil-M + hlorotalonil u suzbijanju prouzrokovača plamenjače (Peronospora viciae) u usevu konzumnog graška. Ogledi su izvedeni na lokalitetima Sakule i Glogonj, po tipu slučajnog blok sistema u četiri ponavljanja (EPPO, 2004, 2012). Fungicidi su primenjeni dva puta, i to kada su biljke bile u fenofazi početka cvetanja (BBCH 61) (prvo tretiranje) i kada je 10% mahuna dostiglo tipičnu veličinu (BBCH 71) (drugo tretiranje). Povoljni agrometeorološki uslovi, rezultirali su značajnom pojavom oboljenja u kontroli koja je iznosila 19,9- 24,8% u vreme prve, odnosno, 32,3-44,5% u vreme druge ocene efekata. Primenom kombinacije fungicida propamokarb-hidrohlorid + hlorotalonil, ostvarena je efikasnost u iznosu 92,7-93,9% u prvoj, odnosno, 85,5-91,9%, u drugoj oceni. Kombinacijom fungicida metalaksil-M + mankozeb, postignuta je efikasnost od 93,1-95,4% u prvoj i 86,3-93,2% u drugoj oceni, dok je najbolja efikasnost postignuta primenom fungicida na bazi kombinacije metalaksil-M + hlorotalonil, koja je iznosila 86,9-100% u prvoj, odnosno, 86,9-95,3% u drugoj oceni.

The use of resistant varieties against downy mildew (DM, Plasmopara viticola) is increasingly gaining ground in Europe to reduce fungicide applications. A research was conducted to characterize the resistance components to DM in 16 grapevine genotypes bringing one or more of the following resistance genes from Muscadinia and American Vitis spp.: Rpv3, Rpv4, Rpv10, Rpv11, and Rpv12. The following resistance components were assessed in monocyclic experiments with artificial inoculation on leaf discs: i) infection efficiency of sporangia (percentage of the inoculation sites showing DM symptoms); ii) AUDPC (area under the disease progress curve); iii) incubation length; iv) latency period (in degree-days); v) sporangia produced per unit of DM lesion (number of sporangia per mm2 of lesion area); v) infectivity of the produced sporangia (by re-inoculation on susceptible variety); and vi) infectiousness (time a DM lesion produces sporangia, evaluated through repeated washings). Leaf discs were excised from fully developed young leaves collected in 2014 and 2015 at shoot growing, flowering and fruit set. Resistance components were expressed at different degrees in the different genotypes and at the different growth stages, with significant differences in comparison to the susceptible Vitis vinifera variety used as reference (Merlot). Data from monocyclic experiments were used for calibrating a DM epidemiological model able to predict disease progress in resistant genotypes. The research was funded by the FP7 European collaborative project “Innovine” (grant agreement n° 311775). Federica Bove carried out this work within the Doctoral School Agrisystem of the Università Cattolica del Sacro Cuore (Italy).

During the 2016 growing season, biological efficacy of fungicides mixture propamocarb hydrochloride + chlorothalonil, metalaxyl-M + mancozeb and metalksil-M + chlorothalonil in the control of downy mildew in pea crop (Peronospora viciae), was investigated. The experiments were conducted in the localities of Sakule and Glogonj. The experiments were carried out as randomized block system in four replications EPPO (EPPO, 1997). Two fugicide applications were conducted , one at the beginning of flowering (BBCH 61) (1st treatment), and second at the stage when 10% of pods reached typical size (BBCH 71) (2nd treatment). Favorable conditions resulted in significant disease intensity on untreated plots, which amounted from 19.9 to 24.8% in the first, and from 32.3 to 44.5% in the second assessment. Fungicide mixture propamocarb-hydrochloride + chlorothalonil exhibited efficacy ranging from 92.7-93.9% at the first and from 85.5 to 91.9% at the second assessment. Fungicide metalaxyl-M + mancozeb, reached the efficacy of 93.1-95.4% at the first, and 86.3-93.2% at the second assessment, while the highest efficacy was achieved by a combination metalaxyl-M + chlorothalonil, which ranged from 86.9 to 100% at the first, and from 86.9 to 95.3% at the second assessment., U toku 2016. godine, vršena su ispitivanja biološke efikasnosti fungicida na bazi kombinacije propamokarb-hidrohlorid + hlorotalonil, metalaksil-M + mankozeb i metalksil-M + hlorotalonil u suzbijanju prouzrokovača plamenjače (Peronospora viciae) u usevu konzumnog graška. Ogledi su izvedeni na lokalitetima Sakule i Glogonj, po tipu slučajnog blok sistema u četiri ponavljanja (EPPO, 2004, 2012). Fungicidi su primenjeni dva puta, i to kada su biljke bile u fenofazi početka cvetanja (BBCH 61) (prvo tretiranje) i kada je 10% mahuna dostiglo tipičnu veličinu (BBCH 71) (drugo tretiranje). Povoljni agrometeorološki uslovi, rezultirali su značajnom pojavom oboljenja u kontroli koja je iznosila 19,9- 24,8% u vreme prve, odnosno, 32,3-44,5% u vreme druge ocene efekata. Primenom kombinacije fungicida propamokarb-hidrohlorid + hlorotalonil, ostvarena je efikasnost u iznosu 92,7-93,9% u prvoj, odnosno, 85,5-91,9%, u drugoj oceni. Kombinacijom fungicida metalaksil-M + mankozeb, postignuta je efikasnost od 93,1-95,4% u prvoj i 86,3-93,2% u drugoj oceni, dok je najbolja efikasnost postignuta primenom fungicida na bazi kombinacije metalaksil-M + hlorotalonil, koja je iznosila 86,9-100% u prvoj, odnosno, 86,9-95,3% u drugoj oceni.

During the 2016 growing season, biological efficacy of fungicides mixture propamocarb hydrochloride + chlorothalonil, metalaxyl-M + mancozeb and metalksil-M + chlorothalonil in the control of downy mildew in pea crop (Peronospora viciae), was investigated. The experiments were conducted in the localities of Sakule and Glogonj. The experiments were carried out as randomized block system in four replications EPPO (EPPO, 1997). Two fugicide applications were conducted , one at the beginning of flowering (BBCH 61) (1st treatment), and second at the stage when 10% of pods reached typical size (BBCH 71) (2nd treatment). Favorable conditions resulted in significant disease intensity on untreated plots, which amounted from 19.9 to 24.8% in the first, and from 32.3 to 44.5% in the second assessment. Fungicide mixture propamocarb-hydrochloride + chlorothalonil exhibited efficacy ranging from 92.7-93.9% at the first and from 85.5 to 91.9% at the second assessment. Fungicide metalaxyl-M + mancozeb, reached the efficacy of 93.1-95.4% at the first, and 86.3-93.2% at the second assessment, while the highest efficacy was achieved by a combination metalaxyl-M + chlorothalonil, which ranged from 86.9 to 100% at the first, and from 86.9 to 95.3% at the second assessment., U toku 2016. godine, vršena su ispitivanja biološke efikasnosti fungicida na bazi kombinacije propamokarb-hidrohlorid + hlorotalonil, metalaksil-M + mankozeb i metalksil-M + hlorotalonil u suzbijanju prouzrokovača plamenjače (Peronospora viciae) u usevu konzumnog graška. Ogledi su izvedeni na lokalitetima Sakule i Glogonj, po tipu slučajnog blok sistema u četiri ponavljanja (EPPO, 2004, 2012). Fungicidi su primenjeni dva puta, i to kada su biljke bile u fenofazi početka cvetanja (BBCH 61) (prvo tretiranje) i kada je 10% mahuna dostiglo tipičnu veličinu (BBCH 71) (drugo tretiranje). Povoljni agrometeorološki uslovi, rezultirali su značajnom pojavom oboljenja u kontroli koja je iznosila 19,9- 24,8% u vreme prve, odnosno, 32,3-44,5% u vreme druge ocene efekata. Primenom kombinacije fungicida propamokarb-hidrohlorid + hlorotalonil, ostvarena je efikasnost u iznosu 92,7-93,9% u prvoj, odnosno, 85,5-91,9%, u drugoj oceni. Kombinacijom fungicida metalaksil-M + mankozeb, postignuta je efikasnost od 93,1-95,4% u prvoj i 86,3-93,2% u drugoj oceni, dok je najbolja efikasnost postignuta primenom fungicida na bazi kombinacije metalaksil-M + hlorotalonil, koja je iznosila 86,9-100% u prvoj, odnosno, 86,9-95,3% u drugoj oceni.

Downy mildew (DM) caused by the oomycete Plasmopara viticola is one of the most important grapevine diseases. The use of DM resistant grapevine varieties is getting increasingly consideration and importance in Europe, as a possible mitigation measure to reduce the use of fungicides. The goal of the present work is to characterize the resistance components to DM in grapevine genotypes (specifically Regent, Rkatsiteli, Bronner, Johanniter, Solaris, 32-078, 34-111, Villaris, Calandro, Felicia, Reberger, Palava, Royal Merlot, Gf.93-22-6, Early Merlot) bringing different resistance genes (Rpv 3, 4, 10, 11 and 12) from Muscadinia and Vitis American species. The following resistance components were assessed in monocyclic experiments carried out under environment controlled conditions: (i) infection efficiency of sporangia (as a percentage of the inoculation sites showing DM symptoms); (ii) AUDPC (area under the disease progress curve); (iii) incubation length, (iv) latency period, (v) sporangia produced per unit of infected area (as number of sporangia per mm2 of lesion area); and (v) infectiousness of the produced sporangia. Resistance components were assessed after artificial inoculation of leaf discs with a natural population of P. viticola. Leaf discs were excised from fully developed, young leaves collected in 2014 and 2015 at shoot growing and at flowering/fruit set. Resistance components were expressed at different degrees in the different genotypes and at the different sampling times, with significant differences in comparison to the susceptible Vitis vinifera variety used as reference (Merlot). For instance, infection efficiency ranged between 60 and 99%, the latency period lasted between 104 and 132 degree-days, and the production of sporangia varied between 80 and 1.1×103 sporangia per mm2 of DM lesion. The research was partially funded by the European collaborative project “Innovine”, that has received funding from the European Union’s Seventh Framework Program