Early detection of pre-symptomatic downy mildew (Pseudoperonospora cubensis) infection in cucumbers by using deep learning tools based on hyperspectral imagery

Nitrogen is a critical nutrient that influences plant growth and resistance to pathogens; however, its impact on disease dynamics, particularly downy mildew infection, and the associated physiological responses in cucumber during early growth stages remains poorly understood. To evaluate the combined effects of downy mildew (caused by Pseudoperonospora cubensis) infection and nitrogen application on cucumber growth and physiological traits during the seedling and vine development stages, two downy mildew treatments— infected (B0) and non-infected(B1)—and three nitrogen levels—T1 (N-50%), T2 (N-100%), and T3 (N-150%)—were applied. Significant differences were observed between all treatments (p < 0.05). Among them, the B1T3 treatment had the most pronounced stimulatory effect, particularly on growth parameters (such as plant height, stem diameter, and leaf area). Without any disease infection (B1), the B1T2 treatment showed an increasing trend in photosynthetic rate and a more notable rise in stomatal conductance. In contrast, with downy mildew infection (B0), photosynthetic rates declined under B0T1 and B0T2. Moreover, with downy mildew infection (B0), the intracellular CO2 concentration, stomatal conductance, and transpiration rate of cucumber leaves decreased in the B0T1, B0T2, and B0T3 treatments. Plant height, stem diameter, and leaf area responded variably to nitrogen levels and downy mildew infection. The total root length, root surface area, average root diameter, total root volume, and total root tips of cucumber plants were significantly different under different experimental conditions (p < 0.05). Consequently, this study provides a theoretical basis for stress-resistant cucumber cultivation in greenhouses and has practical implications for advancing the sustainable development of the greenhouse cucumber industry.

BackgroundGrapevine (Vitis vinifera) productivity has been severely affected by various bacterial, viral and fungal diseases worldwide. When a plant is infected with the pathogen, various defense mechanisms are subsequently activated in plants at various molecular levels. Thus, for substantiating the disease control in an eco-friendly way, it is essential to understand the molecular mechanisms governing pathogen resistance in grapes.ResultsIn our study, we performed genome-wide identification of various defensive genes expressed during powdery mildew (PM) and downy mildew (DM) infections in grapevine. Consequently, we identified 6, 21, 2, 5, 3 and 48 genes of Enhanced Disease Susceptibility 1 (EDS1), Non-Race-specific Disease Resistance (NDR1), Phytoalexin deficient 4 (PAD4), Nonexpressor of PR Gene (NPR), Required for Mla-specified resistance (RAR) and Pathogenesis Related (PR), respectively, in the grapevine genome. The phylogenetic study revealed that V. vinifera defensive genes are evolutionarily related to Arabidopsis thaliana. Differential expression analysis resulted in identification of 2, 4, 7, 2, 4, 1 and 7 differentially expressed Nucleotide-binding leucine rich repeat receptor (NLR), EDS1, NDR1, PAD4, NPR, RAR1 and PR respectively against PM infections and 28, 2, 5, 4, 1 and 19 differentially expressed NLR, EDS1, NDR1, NPR, RAR1 and PR respectively against DM infections in V. vinifera. The co-expression study showed the occurrence of closely correlated defensive genes that were expressed during PM and DM stress conditions.ConclusionThe PM and DM responsive defensive genes found in this study can be characterized in future for impelling studies relaying fungal and oomycete resistance in plants, and the functionally validated genes would then be available for conducting in-planta transgenic gene expression studies for grapes.

The A2 mating type of Pseudoperonospora cubensis was first discovered in Israel in May 2010 on butternut gourd (Cucurbita moschata) (1). We monitored the occurrence of the A2 mating type of P. cubensis in isolates collected during May 2010 through September 2012 from downy mildew-infected cucurbit crops growing along the coastal plain of Israel. Mating type was determined by oospore production in melon leaf discs co-inoculated with sporangia of a test isolate mixed with sporangia of A1 or A2 tester isolates (2). The A1 and A2 tester isolates were maintained at 14°C (14 h light/day) by repeated inoculation of detached leaves of cucumber and pumpkin, respectively. The 29 isolates that were collected from cucumber (Cucumis sativum) were all A1. Of the 33 isolates collected from pumpkin (Cucurbita maxima), squash (C. pepo), or butternut gourd (C. moschata), 88% were A2 and 12% were A1. The host preference of mating type in P. cubensis was monitored at Bar-Ilan University farm during April to July 2012, among about 800 plants of eight cucurbit species (~100 plants per species) that were grown side-by-side in three adjacent net-houses (two 6 × 50 m and one 6 × 100 m) and exposed to natural infection. Downy mildew developed on cucumber, melon, pumpkin, squash, and butternut gourd, but not on watermelon, sponge gourd (Luffa cylindrica), or Momordica balsamina. Three-hundred and three isolates of P. cubensis were collected and tested for mating type: 123 from cucumber, 53 from melon, 30 from pumpkin, 48 from butternut gourd, and 41 from squash. The cucumber isolates expressed A1, A2, and A1A2 at a ratio of 94.3%, 3.3%, and 2.4%, respectively; the melon isolates 58.5%, 26.4%, and 15.1%; the pumpkin isolates 0%, 96.7%, and 3.3%; the butternut isolate 7.3%, 87.3%, and 5.5%; and the squash isolates 2.4%, 97.6%, and 0%, respectively. A1A2 isolates produce oospores when crossed with either A1 or A2 tester isolates. This is the first evidence suggesting a preference of A1 isolates to Cucumis spp. and of A2 isolates to Cucurbita spp. similar preference was recently observed among Chinese isolates of this pathogen (unpublished data). The mechanism(s) controlling this preference is not known. Classical genetics is currently employed to P. cubensis in order to understand if it derives from true linkage. The practical implication for downy mildew management is that growing cucumber/melon in close proximity to pumpkin/squash/butternut gourd should be avoided as it may enhance oospore production in nature. Oospores in soil were recently shown to serve as a primary source of downy mildew infection in cucumber (3).

Comparative proteomic profiles of resistant/susceptible cucumber leaves in response to downy mildew infection

Cucurbit production in Florida is impacted by downy mildew on a yearly basis. Cucurbit downy mildew (CDM), caused by Pseudoperonospora cubensis, is one of the most devastating cucurbit diseases and can lead to complete yield loss. Nearly continuous production of cucurbits occurs temporally throughout Florida, which puts extensive pressure on the pathogen population to select for individuals that are resistant to fungicides in use labeled for CDM. Loss of efficacy as a result of fungicide resistance developing is becoming a major concern for Florida cucurbit growers who rely on these products to manage CDM. This study was established to evaluate the field activity of 11 utilized fungicides by determining their duration of activity when applied at various intervals for the management of CDM in cucumber under Florida field conditions. By comparing levels of percent CDM control and area under the disease progress curve values, the fungicide's duration of field activity was established. Field activities were <1 week for dimethomorph and fluopicolide; 1 week for cymoxanil; 1 to 2 weeks for chlorothalonil and mancozeb; 2 weeks for ethaboxam; 1 to 3 weeks for propamocarb, cyazofamid, and ametoctradin + dimethomorph; and 2 to 4 weeks for oxathiapiprolin and fluazinam. Knowledge of duration of field activity can potentially improve the development of CDM management programs and slow the resistance selection.

Momordica balsamina (balsam apple) and M. charantia L. (bitter melon/bitter gourd/balsam pear) commonly grow in the wild in Africa and Asia; bitter melon is also cultivated for food and medicinal purposes in Asia (1). In the United States, these cucurbits grow as weeds or ornamentals. Both species are found in southern states and bitter melon is also found in Pennsylvania and Connecticut (3). Cucurbit downy mildew (CDM), caused by the oomycete Pseudoperonospora cubensis, was observed on bitter melon and balsam apple between August and October of 2013 in six North Carolina sentinel plots belonging to the CDM ipmPIPE program (2). Plots were located at research stations in Johnston, Sampson, Lenoir, Henderson, Rowan, and Haywood counties, and contained six different commercial cucurbit species including cucumbers, melons, and squashes in addition to the Momordica spp. Leaves with symptoms typical of CDM were collected from the Momordica spp. and symptoms varied from irregular chlorotic lesions to circular lesions with chlorotic halos on the adaxial leaf surface. Sporulation on the abaxial side of the leaves was observed and a compound microscope revealed sporangiophores (180 to 200 μm height) bearing lemon-shaped, dark sporangia (20 to 35 × 10 to 20 μm diameter) with papilla on one end. Genomic DNA was extracted from lesions and regions of the NADH dehydrogynase subunit 1 (Nad1), NADH dehydrogynase subunit 5 (Nad5), and internal transcribed spacer (ITS) ribosomal RNA genes were amplified and sequenced (4). BLAST analysis revealed 100% identity to P. cubensis Nad1 (HQ636552.1, HQ636551.1), Nad5 (HQ636556.1), and ITS (HQ636491.1) sequences in GenBank. Sequences from a downy mildew isolate from each Momordica spp. were deposited in GenBank as accession nos. KJ496339 through 44. To further confirm host susceptibility, vein junctions on the abaxial leaf surface of five detached leaves of lab-grown balsam apple and bitter melon were either inoculated with a sporangia suspension (10 μl, 104 sporangia/ml) of a P. cubensis isolate from Cucumis sativus ('Vlaspik' cucumber), or with water as a control. Inoculated leaves were placed in humidity chambers to promote infection and incubated using a 12-h light (21°C) and dark (18°C) cycle. Seven days post inoculation, CDM symptoms and sporulation were observed on inoculated balsam apple and bitter melon leaves. P. cubensis has been reported as a pathogen of both hosts in Iowa (5). To our knowledge, this is the first report of P. cubensis infecting these Momordica spp. in NC in the field. Identifying these Momordica spp. as hosts for P. cubensis is important since these cucurbits may serve as a source of CDM inoculum and potentially an overwintering mechanism for P. cubensis. Further research is needed to establish the role of non-commercial cucurbits in the yearly CDM epidemic, which will aid the efforts of the CDM ipmPIPE to predict disease outbreaks.

Downy mildew caused by Hyaloperonospora brassicae is a severe disease in Brassica oleracea that significantly reduces crop yield and marketability. This study aims to evaluate different vegetation indices to assess different downy mildew infection levels in the Brassica variety Mildis using hyperspectral data. Artificial inoculation using H. brassicae sporangia suspension was conducted to induce different levels of downy mildew disease. Spectral measurements, spanning 350 nm to 1050 nm, were conducted on the leaves using an environmentally controlled setup, and the reflectance data were acquired and processed. The Successive Projections Algorithm (SPA) and signal sensitivity calculation were used to extract the most informative wavelengths that could be used to develop downy mildew indices (DMI). A total of 37 existing vegetation indices and three proposed DMIs were evaluated to indicate downy mildew (DM) infection levels. The results showed that the classification using a support vector machine achieved accuracies of 71.3%, 80.7%, and 85.3% for distinguishing healthy leaves from DM1 (early infection), DM2 (progressed infection), and DM3 (severe infection) leaves using the proposed downy mildew index. The proposed new downy mildew index potentially enables the development of an automated DM monitoring system and resistance profiling in Brassica breeding lines.

A meta-analysis of the effect of fungicides on cucurbit downy mildew was conducted using data previously published in Fungicide & Nematicide Tests and Plant Disease Management Reports from 2000 to 2008. Standardized mean effect size (the difference in disease intensity expressed in standard deviation units between the fungicide treatment and its corresponding untreated control) was calculated for each of the 105 field studies evaluating the effects of fungicides on cucurbit downy mildew. Fixed- and random-effects meta-analyses were performed on the log-transformed standardized mean effect sizes to estimate the overall mean effect size (L+), and to determine the variability in the effect size among studies. Fungicides led to a significant (P<0.001) reduction in disease with an L+ value of 1.198, indicating that, overall, fungicides were highly effective against cucurbit downy mildew. Fixed- and random-effects meta-analyses were then conducted to determine the effects of moderator variables on L+. The fixed-effects model resulted in narrower 95% confidence intervals and generally lower estimates of moderator subgroup mean effect size (L) than the random-effects models. Fungicide efficacy was significantly (P<0.001) greater in cucumber, with L being 26.5% higher in cucumber than in other cucurbits. Year of study, number of sprays, and disease pressure in the control significantly (P<0.001) affected L. Fungicide efficacy was significantly lower during the 2004-05 season than prior to or after the 2004-05 season. Studies in which disease pressure was moderate had a significantly higher L than studies with either low or high disease pressure. Fungicide efficacy was ≥22% in studies that received 5 to 6 sprays than in studies that received 1 to 4 or >6 sprays. Fungicide products led to a significant (P<0.001) reduction in disease, although there was substantial between-study variability. The pyridinylmethyl-benzamide group of fungicides (fluopicolide) was the most effective, followed by the carbamate (propamocarb) and quinone inside inhibitors (cyazofamid) group of fungicides, while the carboxylic acid amide group (mandipropamid and dimethomorph) was the least effective. A combination of protectant and systemic fungicides resulted in a proportional increase in L compared with sole application of either protectant or systemic fungicides.

Foreword.- Biology and Evolution.- Phylogenetic investigations in the genus Pseudoperonospora reveal overlooked species and cryptic diversity in the P. cubensis species cluster.- Host matrix has major impact on the morphology of Pseudoperonospora cubensis.- Cucurbit downy mildew (Pseudoperonospora cubensis) - biology, ecology, epidemiology, host-pathogen interaction and control.- Spinach: better management of downy mildew and white rust through genomics.- Advances in sunflower downy mildew research.- Resistance mechanisms and host-parasite interactions.- Histological responses of host and non-host plants to Hyaloperonospora parasitica.- Distribution of race-specific resistance against Bremia lactucae in natural populations of Lactuca serriola.- Site-specific field resistance of grapevine to Plasmopara viticola correlates to altered gene expression and was not modulated by the application of organic amendments.- Influence of nitric oxide and reactive oxygen species on development of lettuce downy mildew in Lactuca spp..- Identification of genes expressed during the compatible interaction of grapevine with Plasmopara viticola through suppression subtractive hybridization (SSH).- Population ecology and control.- Quantitative studies on downy mildew (Peronospora destructor Berk. Casp.) affecting onion seed production in southern Uruguay.- Diversity and fitness of Plasmopara viticola isolates resistant to QoI fungicides.- Evolution of Qol resistance in Plasmopara viticola oospores.- EMS and UV irradiation induce unstable resistance against CAA fungicides in Bremia lactucae.- Influence of dosage, storage time and temperature on efficacy of metalaxyl-treated seed for the control of pearl millet downy mildew.

Anggraini et al, 2018. Host-Range Analysis of Downy Mildew (Pseudoperonospora cubensis) and Anthracnose (Colletotrichum Sp.) on some Cucurbitaceae Plants. JLSO 7(2): Inhibiting factors of cucumber cultivation is Downy mildew disease that is caused by Pseudoperonospora cubensis and Anthracnose by Colletotrichum sp. The objective of this research was to observe pathogenicity of activity caused by Pseudoperonospora cubensis and Colletotrichum sp. isolated from cucumber towards Cucurbitaceae plants. The research was conducted at greenhouse and Phytopathology Laboratory, Department of Plant Pests and Diseases Faculty of Agriculture, University of Sriwijaya in September to December 2016. This study used Randomized Block Design (RBD), with four types of plants, 2 treatments and 4 replications. The results showed that the incubation period of the plant inoculation Downy mildew by Pseudoperonospora cubensis range between 4.5-5.0 days. Result of broadest spot area was on melon leaf 2.05 cm2 with the chlorophyll content of 0.4928 mg/g and the lowest result was on cucumber leaf 0.90 cm with the chlorophyll content of 0.7628 mg/g. While the results showed that the incubation period of the applied plant pathogen Colletotrichum sp. ranging from 3.0-4.5 days. Result of broadest spot area was on melon leaf 4.00 cm2 with the chlorophyll content of 0.3932 mg/g and the lowest result was on cucumber leaf 2.42 cm2 with the chlorophyll content of 0.7588 mg/g.

To identify new bacterial antagonists for cucurbit downy mildew (CDM) caused by Pseudoperonospora cubensis, 163 bacterial isolates were recovered from different microenvironments of field-grown cucumber plants. In the greenhouse, 19 representative isolates were applied to cucumber plants as a foliar spray (FS); 7 isolates achieved the efficacy over 60% against CDM, with 5 (DS22, HS10, DP14, HP4, and DS57) identified as Bacillus pumilus, B. licheniformis, Enterobacter sp., Bacillus sp., and Stenotrophomonas maltophilia, respectively. Strains DP14, DS22, and HS10 were assessed for their biocontrol effect on naturally occurring CDM in 2-year field trials (2010 and 2011), in which their overall efficacy relative to that of propamocarb was 106.25 to 117.17% with foliar spray plus root drench (FS+RD) but only 70.98 to 84.03% with FS. Coincidently, DP14 and HS10 applied as root drench (RD) alone also significantly reduced CDM. Under field conditions, DP14, DS22, and HS10 all successfully colonized cucumber leaves and the rhizosphere, and also significantly increased fruit yield by 37.60 to 51.03%, as well as nutrient levels. Taken together, Enterobacter sp. DP14, B. licheniformis HS10, and B. pumilus DS22 are plant-growth-promoting rhizobacteria effective in controlling CDM in the field, whose efficacy increased with FS+RD compared with FS alone.

Partial resistance to downy mildew (Pseudoperonospora cubensis) and complete resistance to powdery mildew (Podosphaera xanthii races 1, 2, 3, and 5 and Golovinomyces cichoracearum race 1) were studied using a recombinant inbred line population between 'PI 124112' (resistant to both diseases) and 'Védrantais' (susceptible line). A genetic map of melon was constructed to tag these resistances with DNA markers. Natural and artificial inoculations of Pseudoperonospora cubensis were performed and replicated in several locations. One major quantitative trait loci (QTL), pcXII.1, was consistently detected among the locations and explained between 12 to 38% of the phenotypic variation for Pseudoperonospora cubensis resistance. Eight other Pseudoperonospora cubensis resistance QTL were identified. Artificial inoculations were performed with several strains of four races of Podosphaera xanthii and one race of G. cichoracearum. Two independent major genes, PmV.1 and PmXII.1, were identified and shown to be involved in the simple resistance to powdery mildew. Three digenic epistatic interactions involving four loci were detected for two races of Podosphaera xanthii and one race of G. cichoracearum. Co-localization between PmV.1, resistance genes, and resistance genes homologues was observed. Linkage between the major resistance QTL to Pseudoperonospora cubensis, pcXII.1, and one of the two resistance genes to powdery mildew, PmXII.1, was demonstrated.

Diseases constitute an important economic problem in oilseed rape (Brassica napus) cultivation. Although downy mildew has been counted so far as a minor disease, under intensive cultivation system and short rotation interval, the impact of diseases could increase in the future, especially under predicted more humid northern climatic conditions. This research study is the first report about the severity of downy mildew infection on cruciferous crops in Estonia. During two years (2010 − 2011), downy mildew infection severity was assessed in six different cruciferous crops (B. napus, B. juncea, B. nigra, Sinapis alba, Raphanus sativus, Eruca sativa) in field trials located in Eerika, Tartu County, Estonia. On both study years, four disease assessments were done. Downy mildew infection started a week earlier in 2010, but the overall disease pressure was lower compared to 2011. Based on our field trial results, S. alba, E. sativa and R. sativus plants were significantly more resistant to downy mildew compared to other tested crops. Furthermore, in 2010 downy mildew symptoms were not recorded on two cruciferous species E. sativa and R. sativus. Brassica nigra and B. napus plants were the most susceptible to downy mildew, with especially severe infection in 2011. Based on our two years data, downy mildew can damage foliage on spring oilseed rape (B. napus) and black mustard (B. nigra) in a notable extent. We conclude that downy mildew is an important pathogen, which should be monitored on different oilseed cruciferous crops in Estonia.

A study was conducted to determine the inheritance of resistance to downy mildew caused by Pseudoperonospora cubensis in the cucumber genotypes; cv. Beit Alpha, line 1902 M and line THG-1. The common cultivated cultivar in Egypt Beit Alpha was found to be highly susceptible to downy mildew infection. Two crosses were made as follows: 1-“line 1902 M“X”Beit Alpha“ 2-”line TMG-1“ דBeit Alpha”. The resulting F1, F2 and backcross generations of the first cross and F1 and F2 populations of the second cross were observed for downy mildew reactions in the field under natural infestiation conditions. Susceptibility of the F1 populations to downy mildew was completely dominant in both crosses. Segregation ratios of the F2 and backcrose populations of the cross “line 1902 M × Beit Alpha” indicated that two pairs of recessive genes controlled the tolerance reaction of Line 1902 to the fungus. Segregation ratio for reaction to downy mildew in the F2 population of cross “line TMG-1 × Beit Alpha” indicated that three pairs of recessive genes were controlled the resistance reaction to the fungus. The following tentative genotypes for downy mildew reactions are proposed: Cv. Beit Alpha: Dm1Dm1Dm2Dm2Dm3Dm3 (Suscceptible), Line 1902 M: dm1dm1dm2dm2Dm3Dm3 (Tolerant) and Line TMG-1 dm1dm1dm2dm2dm3dm3 (Resistant).

Agrochemicals suitable for downy mildew control in New Zealand boysenberry production