The Novel Oomycide Oxathiapiprolin Inhibits All Stages in the Asexual Life Cycle of Pseudoperonospora cubensis - Causal Agent of Cucurbit Downy Mildew

Oxathiapiprolin is a new oomycide (piperidinyl thiazole isoxazoline class) discovered by DuPont which controls diseases caused by oomycete plant pathogens. It binds in the oxysterol-binding protein domain of Oomycetes. Growth chambers studies with detached leaves and potted plants showed remarkable activity of oxathiapiprolin against Pseudoperonospora cubensis in cucurbits. The compound affected all stages in the asexual life cycle of the pathogen. It inhibited zoospore release, cystospore germination, lesion formation, lesion expansion, sporangiophore development and sporangial production. When applied to the foliage as a preventive spray no lesions developed due to inhibition of zoospore release and cystospore germination, and when applied curatively, at one or two days after inoculation, small restricted lesions developed but no sporulation occurred. When applied later to mature lesions, sporulation was strongly inhibited. Oxathiapiprolin suppressed sporulation of P. cubensis in naturally-infected leaves. It exhibited trans-laminar activity, translocated acropetaly from older to younger leaves, and moved from the root system to the foliage. Seed coating was highly effective in protecting the developed cucumber plants against downy mildew. UV microscopy observations made with cucumber leaves infected with P. cubensis revealed that inhibition of mycelium growth and sporulation induced by oxathiapiprolin was associated with callose encasement of the haustoria.


Introduction
Downy mildew caused by Pseudoperonospora cubensis is a major disease of cucurbits. It attacks cucumber, melon, squash, pumpkin and various gourds [1,2]. The pathogen has undergone major changes during the last decade. Disease severity and epidemics became far more destructive and new genotypes made disease control through host plant resistance ineffective. Consequently, disease control relies mainly on oomycides applications [3,4]. Sixteen chemical classes with different modes of action are available for the control oomycete plant pathogens (www.

Pathogen
Most experiments were performed with cucumber inoculated with isolate 83c of P. cubensis (pathotype 3, mating type A1, resistant to mefenoxam, dimethomorph, mandipropamid and azoxysrrobin). The effect of oxathiapiprolin on infection and disease development of cucumber was also tested with additional 25 isolates of the pathogen of various origins, pathotypes, mating types and oomycide-resistance profiles (Table 1). In some experiments, other cucurbit hosts (see below) were used for experiments in the manner described for cucumber. Isolates were maintained by repeated inoculations of detached leaves of cucumber in growth chambers at 15°C (14h light/day). Sporangia were removed from freshly sporulating leaves into distilled water kept on ice, adjusted to 2000 sporangia/ml, and used for inoculation. In some experiments sporangial suspensions were used for in-vitro testing of zoospore release, zoospore motility, zoospore encystment and cystospore germination.

Plants
Cucumber plants (cv. Nadiojni or SMR-18) were used in most experiments. In some experiments, melon (cv. AY), squash (cv. Beruti), pumpkin (cv. Tripoli), butternut gourd (cv. Dalorit), watermelon (cv. Malali), bottle gourd (local cultivar) and ridge gourd (cv. SV) were used. Plants were grown in the greenhouse and used when reached the 2-3 leaf stage (3-4 weeks after sowing). Most assays were conducted with detached leaves, but some were done with whole potted plants or plants grown in net-houses.

Preventive treatment and inoculation
Detached leaves were placed on moistened filter paper in 14 cm Petri dishes lower surface uppermost (unless stated otherwise). They were sprayed with oxathiapiprolin of various concentrations with the aid of a fine glass atomizer. Leaves sprayed with water served as controls. At 2-3h after spray, leaves were drop inoculated with sporangia of P. cubensis (400 sporangia per 20 μl, 20 droplets per leaf, unless stated otherwise). Inoculated leaves were incubated in a growth chamber at 20°C (12h light/day, 60 μmole. m 2 .s -1 ) and evaluated for disease development at 7 dpi (days post inoculation), unless stated otherwise.

Inoculation and curative treatment
Detached leaves were placed on moistened filter paper inside 14 cm Petri dishes lower surface upper-most and drop inoculated with sporangia of P. cubensis in the manner described above. The inoculated leaves were incubated in a growth chamber at 20°C as above and sprayed at 1-6 (dpi) with water (control) or oxathiapiprolin of various concentrations.

Sporulation in naturally-infected leaves
Cucumber leaves, naturally-infected with P. cubensis, were collected at 8 am from Net-house 1 on campus on April 27 and May 1, 2015. Leaves were brought to the laboratory and the sporangia they carried were blown away in a laminar hood with the aid of a strong stream of compressed air. Leaves (n = 4) were laid upper surface uppermost on wet filter in 14 cm Petri dishes and sprayed with water (control) or oxathiapiprolin of various concentrations. Leaves were incubated at 20°C under light (100 μmole.m 2 .s -1 ) until 17 pm, the number of lesions counted (ranged from 120-180 per leaf) and then placed at 18°C in the dark for 16h to allow for sporulation. The sporangia produced were collected with a fine water jet into a beaker, their concentration was determined with the aid of a cytometer and the number of sporangia produced per lesion was calculated.
Similar experiments were conducted with naturally-infected leaves collected on June 9 and June 14, 2015 from Net-house 3 located at Bar-Ilan Farm. In these leaves, downy mildew lesions occupied the whole leaf area. Therefore, sporangial number was determined in 12 mm leaf discs removed from each leaf (n = 3). Leaf discs were placed in 3 ml water containing 0.01% Tween-20, agitated on Vortex (high speed for 1 minute), the number of sporangia was determined with the aid of a cytometer, and the number of sporangia produced per 1cm 2 leaf tissue was calculated.
The effect of oxathiapiprolin on sporulation of P. cubensis in naturally-infected cucumber plants was tested directly in Net-house 3. Downy mildew-infected plants (n = 10) were sprayed at 3 weeks after inoculation with oxathiapiroplin (3 mg/l ai) at 8 am 11.6.2015 while control plants (n = 10) were left untreated. Leaves (one per plant) were collected at 8 am 12.6.2015 (night was dewy). Leaves were brought to the laboratory, photographed, and the intensity of sporulation was visually estimated (0-3 scale, with 0 = no visible sporulation; 1 = weak sporulation; 2 = moderate sporulation; and 3 = heavy sporulation).

Translaminar movement
The following experiments were performed to learn if oxathiapiprolin can translocate from one surface of the leaf to the opposite (thereafter inoculated) surface of that leaf. Detached leaves (n = 3) of cucumber were laid in 14 cm Petri dishes on wet filter paper lower or upper surface uppermost. Leaves were sprayed with oxathiapiprolin of various concentrations and two hours later were drop-inoculated with sporangia (15 droplets, 25 μl each) on the treated surface or on the opposite, untreated surface. Leaves were incubated at 18°C in the dark for 20h to allow for infection and then at 20°C (12h light/day) to enable lesion development. Number and size of lesions was determined at 7 and 14dpi.

Translocation form leaves or hypocotyl
The following experiments were performed to learn if oxathiapiprolin translocates acropetally from a treated leaf 1 to the newly developed leaf 2. Cucumber plants having one true leaf (n = 3) were either sprayed on that leaf or treated with 10 droplets (25 μl each) of oxathiapiprolin of various concentrations. Control plants were left untreated. Plants were then incubated at 20°C (12h light/day) for 4 days to allow the second true leaf to expand. Plants were then sprayinoculated with the pathogen and disease development on the treated leaf 1 and the untreated leaf 2 was recorded at 7dpi.
In other experiments, oxathiapiprolin was applied as a fine spray to the hypocotyl of horizontally-laid 2-leaf plants (n = 6). To avoid contact with other organs, leaves were covered with a plastic bag and the soil surface covered with aluminum foil. Control plants remained untreated. Plants were inoculated at 6h after treatment and were evaluated for disease development at 7dpi.

Translocation from the root
Plants (n = 6) at the 2-leaf stage growing in 200 ml pots were irrigated to soil capacity and thereafter drenched around the stem base with 1 ml oxathiapiprolin of 100 mg/l. Plants were thereafter irrigated twice, at 4h intervals, with 5 ml tap water to facilitate the distribution of the oxathiapiprolin through the root system. Plants were incubated at 20°C (12h light/day) and inoculated one day after treatment. Disease records were taken at 7dpi.

Seed coating
One hundred cucumber seeds (2g) were mixed with 100 mg oxathiapirolin 10%. The mixture was rotated at 200 rpm for 1h and the seeds were allowed to rest on the bench for 10 days. The coated seeds weighed 2.04 g, indicating that each seed was coated with approximately 40 μg active ingredient of oxathiapiprolin. Plants were raised from control and coated seeds and were spray-inoculated P. cubensis at the 1-leaf 2-leaf or 3-leaf stage. A week later the number of lesions produced was counted on each plant (n = 10).

In-vitro assays
One ml aliquots of sporangial suspensions in 20 ml vials were supplemented with oxathiapiprolin of various concentrations (n = 3), kept on ice for 30 min and then on the bench to allow for zoospore release. Zoospore release and motility were examined in 10 μl droplets at a magnification of x63.
Temporary exposure of sporangia to oxathiapiprolin was tested as follows: sporangia were suspended in water or oxathiapiprolin of various concentrations. After 15 minutes, they were washed with excessive water over 8μ Millipore membrane and re-suspended in fresh water. Zoospore release was examined microscopically at x63 after 2 hours.
Motile zoospores in depression glass slides were amended with oxathiapiprolin of various concentrations (or water) and sucrose to final concentration of 0.1M. Slides were incubated inside moisten Petri dishes at 18°C in the dark. Cystospore germination was examined after 15 h at x63.

Microscopy
The procedures described by Cohen et al. [34,35] were employed. One cm diameter leaf discs were removed from the test leaves at various time intervals after inoculation or fungicidal treatment. Leaf discs were placed in 5 ml ethanol and boiled for 5 minutes to clarify. The ethanol was discarded and 5 ml of 0.01% aniline blue solution (in 0.1M dibasic-potassium phosphate pH 8.9 buffer) were added. The vials containing the leaf discs were placed at 4°C overnight. The leaf discs were then removed, placed on glass slides, lower surface upper-most, treated with 10μl of 0.01% calcofluor solution (Sigma), covered with a cover slip and examined with aid of Olympus A70 epifluorescent microscope. Electron micrographs were produced with the aid of Phillips SEM after standard processing of the infected tissue.

Data analysis
Experiments with detached leaves and potted plants were repeated twice. In vitro experiments were repeated three times. Results from the repeated experiments showed little variation. Data from repeated experiments were averaged and the mean and standard deviation values were calculated. Analysis of variance was done using JMP 1 software (SAS Institute). Significant difference between means were calculated for α = 0.05 using Tukey's-Kramer HSD (honest significant difference) test.
Temporary exposure of sporangia to oxathiapiprolin. Control sporangia in water released a mean of 2000±450 motile zoospores in 10μl suspension. Sporangia continuously exposed to 0.00001 mg/l oxathiapiprolin released 20±30 motile zoospores and sporangia temporarily exposed to 0.00001 mg/l oxathiapiprolin released 200±50 motile zoospores. Exposure to 0.001 mg/l oxathiapiprolin for 15 minutes was totally inhibitory to zoospore release, suggesting that oxathiapiprolin may bind quickly and irreversibly to its target in the pathogen.
Effect of oxathiapiprolin on zoospore motility. The oomycide had a weak effect on zoospore motility. At 30 min after adding it to motile zoospore suspension the proportion of halted zoospores was reduced by 25, 50 and 75%, at oxathiapiprolin of 0.0005, 0.05 and 5 mg/l, respectively.
Cystospore germination. Oxathiapiprolin had a strong inhibitory effect on cystospore germination in vitro. In various experiments, percent germinating cystospores in water amended with 0.1M sucrose ranged from 20 to 30%, as against 0% in oxathiapiprolin of 0.0001 mg/l (Fig 1A-1D).

Efficacy in vivo
Zoospore release and cystospore germination. Control-untreated and oxathiapiprolintreated cucumber leaves were drop-inoculated with sporangia, incubated at 20°C for one or two days and thereafter examined microscopically. Cystospores germinated nicely at 1 dpi on the surface of control untreated inoculated leaves and germ-tubes grew over the stomatal opening (Fig 1A, 1C and 1E). In oxathiapiprolin-treated (0.0001 mg/l) leaves no germinating cystospores were seen (Fig 1B, 1D and 1F). Extensive mycelium growth inside the control leaves was observed at 2 dpi (Fig 1G) while no visible mycelium growth was seen in the treated leaves ( Fig  1H) due to the lack of zoospores release and/or cystospore germination.

Infection and sporulation in detached leaves and potted plants
The effect of a preventive application in detached leaf assays is demonstrated in Fig 2A  (cucumber) and Fig 2B (melon). Treatment with oxathiapiprolin of 0.0001-0.001 mg/l totally inhibited disease development, conforming that no penetration has taken place due to the fact that oxathiapiprolin avoided zoospore release and/or cystospore germination. Preventive spray with oxathiapiprolin was highly effective against downy mildew in melon leaf discs. Percent area infected in leaf discs (4 cm diameter) treated with oxathiapiprolin of 0, 0.0001, 0.001, 0.01, 0.1, and 1 mg/l was 97.8±4.4, 4.4±6.8, 2.2±5.1, 0, 0, and 0%, respectively ( Fig 2C).
A series of experiment were performed to explore whether isolates of P. cubensis of various origins and characteristics differ in their sensitivity to oxathiapiprolin. All 26 isolates listed in Table 1, regardless of their origin, pathotype, mating type or sensitivity to other oomycides, produced no lesions when drop-inoculated onto detached cucumber leaves preventivelytreated with oxathiapiprolin of 0.0001 mg/l. A1 and A2 isolates were similarly controlled by 0.0001 mg/l of the compound (Fig 2D and 2E). Similarly, the isolates belonging to pathotypes 5, 6, and 10 produced no lesions when drop-inoculated onto detached leaves of their respective hosts watermelon, squash or ridge gourd, respectively, when treated with oxathiapiprolin of 0.0001 mg/l before inoculation.
In potted plants, the detrimental effect of oxathiapiprolin on infection was significantly expressed at 0.001 mg/l. Two leaf plants were sprayed with oxathiapiprolin on the upper leaf surface and 3h later were spray-inoculated on the upper leaf surface with sporangia of P. cubensis. Disease records (taken at 7dpi) showed a mean of 383±76 downy mildew lesions per plant in the controls; this number decreased by 96 and 100%, respectively relative to control, at oxathiapiprolin of 0.001 and 1 mg/l, respectively (Fig 3A and 3B).
The differential effects of oxathiapiprolin in preventive vs curative application is demonstrated in Fig 4. The compound, applied preventively (time zero) at a low dose of 0.0001 mg/l, totally prevented symptom production (Fig 4A upper panel), whereas when applied at 1 or 2dpi, symptoms were produced even at a dose of up to 0.01 mg/l ( Fig 4A middle and lower  panels). Very slight effect on lesion appearance was seen in leaves treated with oxathiapiprolin at 3dpi, even with a high of 10 mg/l (Fig 4B).
While oxathiapiprolin applied before inoculation suppressed zoospore release and/or cyst germination and therefore inhibited lesion formation, oxathiapiprolin applied after inoculation suppressed lesion expansion, sporangiophore formation and sporangial yield, depending on the time of its application.
Data in Fig 5A present lesion size in leaves treated with oxathiapiprolin at 0, 1, 2 or 3dpi. No lesions appeared at 0 dpi at 0.0001 mg/l oxathiapiprolin. At 1-3 dpi, suppression of lesion expansion was significantly stronger as the dose of the oomycide was higher. Higher doses were required at 3dpi compared to 2 or 1dpi (note the change in the dose values). Data in Fig 5B show that sporangiophore formation in such lesions was strongly suppressed. Thus, no sporangiophores were produced in leaves treated with the compound at 0 or 1dpi. Their number decreased significantly in a dose-dependent manner at 2 or 3dpi. The fluorescent micrographs presented in Fig 6A and 6B show that at increasing doses, oxathiapiprolin prevented sporangial formation, sporangiophore ramification or sporangiophore emergence from stomata.
In the experiments shown in Fig 7, detached leaves were treated with oxathiapiprolin at 2 or 5 dpi and the numbers of sporangia (Fig 7A) or sporangiophores (Fig 7B) produced were counted at 7 dpi. Sporangial yield was reduced by 88, 99 and 99.8% in leaves treated at 2 dpi with oxathiapiprolin of 0.01, 0.1 and 1 mg/l, respectively (Fig 7A). The number of sporangiophores in leaves treated at 5dpi was reduced by 84, 95.4 and 100% with oxathiapiprolin of 0.1,  1 and 10 mg/l, respectively (Fig 7B). A visual illustration of such inhibition is given in the fluorescent micrographs presented in Fig 7C and 7D. While abundant sporangiophores and sporangia were produced in the control leaves (Fig 7C) no such organs were produced in leaves treated at 5 dpi with oxathiapiprolin of 1 mg/l (Fig 7D). Oxathiapiprolin induced heavy callose Detached leaves were inoculated on lower leaf surface with sporangial suspension (25 droplets/leaf) and oxathiapiprolin was applied as a spray at 0, 1, 2 or 3dpi. A-No lesions developed when oxathiapiprolin was applied at 0 dpi. Lesions appeared when oxathiapiprolin was applied at 1 or 2dpi, but became smaller as its dose increased. B-Oxathiapiprolin was applied at 3dpi. Note no effect on lesion size. Photos were taken at 6 dpi.   Fig 8B). A visual representation of such inhibition is provided in Fig 8C (control) and 8D (oxathiapirolin of 0.0001 mg/l applied June 9).
Translaminar activity of oxathiapiprolin. Oxathiapiprolin exhibited translaminar properties: detached leaves sprayed with 0.0001-1 oxathiapiprolin mg/l and thereafter drop-inoculated with P. cubensis on the treated leaf surface (upper/upper or lower/lower) showed complete control of the disease at all doses at both 7 and 14dpi. Control inoculated leaves produced 15 lesions per leaf whose size ranged from 21-26 mm at 7 dpi and from 25-35 mm at 14dpi. Complete control of the disease was also seen in leaves treated with 0.001-1 mg/l, regardless of the surface treated/ inoculated (upper/lower, or lower/upper). However, at the  (Fig 10), indicating on partial translaminar movement of the compound when applied at a low concentration.
Systemic translocation of oxathiapiprolin. Oxathiapiprolin exhibited acropetal systemic mobility in cucumber plants. When sprayed at 50 or 100 mg/l to leaf 1, both leaf 1 and the newly-developed leaf 2 were fully protected against downy mildew whereas the control untreated inoculated plants developed 260±50 lesions per plant (Fig 11A). When droplets containing 10 mg/l were applied to leaf 1, the treated leaf 1 was fully protected but leaf 2 showed no protection (Fig 11B). However, application of droplets containing 100 or 1000 mg/l oxathiapiprolin were fully protective to both leaf 1 and leaf 2 (Fig 11B). A spray of 1 or 10 mg/l applied The effect of oxathiapiprolin on sporulation of P. cubensis in cucumber. A-Detached leaves were treated with oxathiapiprolin at 2dpi and examined for sporulation at 7 dpi. B-Detached leaves were treated with oxathiapiprolin at 5dpi and examined for sporulation at 7 dpi. Different letters on bars indicate on a significant difference at α = 0.05 (Tukey's-Kramer HDS test). C-Sporulation of P. cubensis on an untreated control leaf of cucumber at 7 dpi. D-Encasement with callose of the houstoria of P. cubensis in a cucumber leaf treated with 1 mg/l oxathiapiprolin at 5dpi. Leaf discs were removed at 7 dpi and stained with basic aniline blue for callose (yellow) and calcofluor for cellulose (blue). Images were taken with an epifluorescent microscope under mixed UV-incandescent light illuminations. Bar = 30 μm. Note in C-the dark sporangia and blue sporangiophores, and in D-the callose-encased haustoria (yellow). Bar = 30 μm. to the hypocotyl resulted with no protection of the leaves relative to the control plants but a spray of 100 mg/l provided full protection against the disease (Fig 11C). Oxathiapiprolin applied as a soil drench to the root system (1 ml, 100 mg/l) supplied full protection against the disease (Fig 11D).
Seed coating. Seed coating had no impact on percent germination or growth rate of cucumber plants compared to uncoated seeds. Downy mildew development was dramatically suppressed in plants developed from coated seeds relative to plants developed from regular seeds. Number of lesions per plant in control untreated-inoculated plants at the 1-leaf, 2-leaf and 3-leaf stage was 70±25, 180±54 and 375±28, respectively as against 0, 0 and 1.0±1.7 in coated-seed plants, respectively. All lesions in 3-leaf plants developed in leaf 3; no lesions developed in leaves 1 or 2.
Oxathiapiprolin is a new oomycide, with a novel mode of action. As shown here, it is capable of controlling isolates of P. cubensis resistant PA, QoI and CAAs. We have examined the inhibitory effects of oxathiapiprolin on almost every stage in the asexual life cycle of P. cubensis. Oxathiapiprolin allowed the cleavage of the zoospores inside the sporangium during zoosporogenesis (not shown) but suppressed their discharge from the sporangium. Similar inhibition of zoospore release was observed with P. capsici [30]. Inhibition of zoospore release was almost complete both in vitro and in vivo on leaf surface. When added to motile zoospores in vitro oxathiapiprolin had a weak negative effect on the speed or duration of their motility, suggesting that it does not hamper the energy supply required by the locomotive machinery of the The most pronounced effect of oxathiapiprolin was its inhibitory effect on cystospore germination. Very low doses of the compound were required to completely avoid germination of the cystospores either in vitro or in vivo. Similar inhibition of cystospore germination was observed in vitro with P. capsici [29,30]. The biochemical mechanism(s) responsible for this inhibition is not known. CAA fungicides, which target the cellulose synthase CesA3 protein of oomycetes [19] are also strong inhibitors of cystospore germination but the dose required is x100 higher compared to oxathiapiprolin [42]. Oxathiapiprolin targets oxysterol-binding proteins in oomycetes [25]. Oxysterol-binding protein (OSBP)-related proteins (ORPs) are lipid-binding proteins implicated in many cellular processes including signaling, vesicular trafficking, lipid metabolism, and non-vesicular sterol transport [43]. It might occur that oxathiapiprolin affects cellulose micro-fibrils trafficking towards the tip membrane of the germinating cystospore, thus preventing germ-tube emergence.
The effect of oxathiapiprolin on downy mildew development was strongly dependent on the time of its application relative to the inoculation event. When applied preventively before inoculation, oxathiapirolin totally prevented disease production whereas when applied after inoculation it enabled disease development but reduced lesion expansion and sporangiophore/ sporangia production, probably due to the inhibition of mycelium growth.
Because cystospore germination is inhibited in vivo, no stomatal penetration could take place and no lesions of downy mildew were produced in leaves treated preventively with oxathiapiprolin. All 26 isolates used in this study exhibit similar high sensitivity to the compound, regardless of the host used for inoculation, suggesting on high expected efficacy against a variety of pathotypes known to prevail in various locations around the globe [4,33]. Very low doses of the compound were required to avoid lesion formation, the lowest dose known in the oomycides world. This is a great advantage of this oomycide but at the same time may turn it into a high-risk oomycide in terms of resistance. Oxathiapiprolin possess curative effects against P. cubensis. When applied to alreadyinfected leaf tissue it induces callose encasement of the haustoria. Callose encasement of haustoria was implicated in genetic resistance of melon against P. cubensis [35] and in resistance induced by DL-β amino-butyric acid (BABA) against Bremia lactucae in lettuce [44]. Such encasement may starve the pathogen, lead to cessation of mycelium growth and restrict lesion expansion. Lesion expansion arrest was also reported for metalaxyl, but it lasted for only one or two days after inoculation [45] as compared to 3 days with oxathiapiprolin.
Oxathiapiprolin also exhibited anti-sporulative effect against P. cubensis. When applied to artificially inoculated leaves at the terminal phase of disease development it strongly suppressed sporangiophore emergence from stomata and therefore no sporangia were formed. When applied in the laboratory to naturally-infected leaves a strong suppression of sporangial production was obtained with 0.0001 mg/l of the compound. Infected plants treated in the field with 3 mg/l of oxathiapiprolin failed to sporulate, suggesting a "knock-down" effect of the compound. A strong anti-sporulation activity of oxathiapiprolin was reported for P. capsici in vitro [29,30]. Other oomycides (e.g. phenylamides, CAAs) exhibit anti-sporulation effects but the doses required are higher compared to oxathiapiprolin [42,45].
Oxathiapiprolin is shown here to translocate acropetally in cucumber plants. When applied to the root system or to the hypocotyl, leaves were protected against downy mildew. When was applied to leaf 1, leaf 2 was protected. However, relatively high doses were required to achieve such protection (50-100 mg/l) indicating on partial mobility (or degradation) of oxathiapiprolin in the plant. Systemic protection by soil drench was also observed in potted tomato plants against late blight and in basil plants against downy mildew (Y. Cohen, unpublished data). Soil application of oxathiapiprolin was effective against P. nicotianae in tobacco [46].
Oxathiapiprolin exhibited excellent activity against P. cubensis when applied as seed dressing (~40 μg per seed). Plants developed from such seeds were fully protected against downy mildew at the 1, 2, and 3 true leaf stage, suggesting on effective mobility from the embryo to the developing seedling. Using radiolabeled oxathiapiprolin may provide better insight on the uptake, mobility and degradation of the compound in the plant (e.g. see [47] for 14 C-cymoxanil). Oxathiapiprolin applied as seed treatment was already reported to control downy mildew in basil [27].
Oxathiapiprolin exhibits remarkable translaminar movement across the leaf lamina. At concentration of 0.001 mg/l disease was completely controlled regardless of whether the inoculum was applied to the treated or the untreated leaf surface. Only at the lowest dose used of 0.0001 mg/l one could distinguished that translaminar movement of the compound was partial.
In conclusion: oxathiapiprolin, a novel oomycide with a new mode of action, is highly effective in controlling downy mildew in cucurbits caused by P. cubensis. It was effective against various pathotypes, mating types, and oomycide-resistant isolates. It is effective against all stages in the asexual life cycle of the disease. It possesses preventive and curative properties. It shows excellent translaminar mobility and partial acropetal systemicity.
The availability of oxathiapiprolin to the farmers may now improve significantly their ability to combat this devastating downy mildew disease of cucurbits. To avoid possible resistance build up in P.cubensis, oxathiapiprolin should be mixed with unrelated oomycide (s) before applied in the field.
Supporting Information S1 File. Efficacy of oxathiapiprolin applied via the root system.