A new strategy for durable control of late blight in potato by a single soil application of an oxathiapiprolin mixture in early season

Root treatment with oxathiapiprolin, benthiavalicarb or their mixture Zorvec-Endavia [ZE (3+7, w/w)] was shown to provide prolonged systemic protection against foliar oomycete pathogens attacking cucumber, tomato and basil. Here we report that these fungicides can effectively protect potato plants against late blight when applied to the soil in which such potato plants are grown. In two field experiments, performed in 2019 and 2020, potato plants grown in 64 L containers were treated with a soil drench of oxathiapiprolin, benthiavalicarb or ZE at 12.5, 25 or 50 mg ai/five plants in a container. Artificial inoculations with Phytophthora infestans revealed that such treated plants were protected against late blight in a dose-dependent manner all along the season. Interestingly, oxathiapiprolin persisted in the treated soil for at least 139 days, providing systemic protection against late blight to the following potato crops grown in that treated soils. Potato plants grown in loess soil in the field were either sprayed or drenched with ZE. Plants treated via the soil were significantly better protected against late blight compared to the plants treated by a spray. The data demonstrate a new strategy for season-long protection of potato against late blight by a single soil application of ZE. The systemic nature of oxathiapiprolin and benthiavalicarb composing ZE assures the translocation to the foliage of two fungicides with different modes of action. This shall minimize the risk of developing resistance against either fungicide in the treated crops.

Resistance against OXPT was induced in Phytophthora capsici by UV irradiation [7,8]. These authors reported on three point-mutations that confer resistance to OXPT in P. capsica and P. sojae. Mutants with any of these mutations may have survival potential in the field [7].
According to FRAC (http://www.frac.info/), the resistance risk of OXTP is medium to high. Therefore, resistance management is required, including mixing with another fungicide with a different mode of action. Oxathiapiprolin mixtures (with azoxystrobin, mandipropamid or mefenoxam) were reported to be highly effective against P. infestans in tomato [5] and Pseudoperonospora cubensis in cucumber [6]. OXPT+mefenoxam was effective against mefenoxam-resistant isolates of these pathogens [5,6].
Benthiavalicarb belongs to the carboxylic acid amides (CAA) group of fungicides (FRAC code 40). CAA mode of action involves inhibition of cell wall synthesis of oomycetes by blocking the activity of cellulose synthase Ces3A [9]. Benthiavalicarb inhibits mycelia growth, zoosporangia germination cystospore germination, and sporulation of Phytophthora infestans at very low concentration [10]. The fungicidal and disease-controlling activities of benthiavalicarb are characterized by its preventive, curative, translaminar, systemic movement, residual activity and inhibitory activity toward lesion development [10].
Sensitivity monitoring studies over several years revealed that in populations of the late blight pathogen, P. infestans, all isolates were fully sensitive to CAA fungicides. However, resistant isolates occur in populations of the grape downy mildew pathogen Plasmopara viticola and the cucurbit downy mildew pathogen P. cubensis (FRAC, annual meeting 2018). With P. infestans, we were able to artificially mutate sporangia for stable resistance to the phenylamide fungicide mefenoxam but failed to select mutants with stable resistance to CAAs [11], suggesting a low risk of resistance developing in this pathogen in field populations against CAAs. No laboratory or field resistance to oxathiapiprolin have been reported in P. infestans.
In a recent study [3] we demonstrated that a single application of oxathiapiprolin, benthiavalicarb, or their mixture (3+7, w/w) to the root system of nursery plants grown in multi-cell trays provided prolonged systemic protection against late blight and downy mildews in growth chambers and in the field. Soil application of 1mg active ingredient per plant provided durable protection for up to four weeks in tomato against late blight, cucumber against downy mildew and basil against downy mildew. Other researchers demonstrated efficacy of soil drench with oxathiapiprolin against Phytophthora black shank in tobacco [12] Phytophthora blight in pepper [13] and Phytophthora root rot in citrus [14].
These findings provided the conceptual basis for the developing of a new strategy to control late blight in potato by fungicidal soil application. The new fungicidal mixture Zorvec Endavia© by Corteva looked ideal for this purpose as it was proven highly effective when applied as a root treatment to tomato against late blight [5]. It is composed of two systemic fungicides, oxathiapiprolin and benthiavalicarb (3+7, w/w) that can protect each other from resistance development due to their different modes of action. The fact that no resistance occurs in P. infestans population against either fungicide in nature encouraged us in developing this strategy.

Field experiments
Four field experiments were conducted: three at Bar-Ilan University (BIU) Farm, Ramat Gan, Israel, and one at Kibbutz Magen, Western Negev, Israel In Experiment 1 at BIU Farm, tubers cv Nicola were sown (1.10.2019) in 64 L polystyrene containers (80x40x20 cm) filled with peat: perlite (10:1, v/v) soil mixture, 5 tubers (50-60 mm in size) per container. Containers were placed a 50x6 m net house covered with white, 50 mesh, insect-proof plastic screens. At 7 weeks after sowing (18.11.2019), 1L fungicide suspension containing 12.5, 25 or 50 mg ai of a fungicide was applied to the soil surface of each container. Five fungicides were used: OXPT, BENT, ZE, MFX and Infinito. Control plants were treated with 1L of water. Three containers were used per fungicide per dose treatment. At 1h and 5h after soil drench, 1L of water was added to each container to facilitate the uptake of the fungicides by the root system. Thereafter, plants were drip irrigated 4 times a day with 0.5 L per container each time. NPK fertilizer (0.5%) was supplied twice a week.
In Experiment 2 at BIU farm, potato cv Sifra were sown in new containers on 26.12.2019 and the fungicides were applied to the soil surface as above at 8 weeks after sowing (19.2.2020). Three fungicides were used: OXPT, BENT and ZE. MFX and Infinito were excluded due to their inefficacy in the previous experiment. Experiment 3 was performed to determine whether the fungicides applied in experiment 1 at BIU Farm on 18.11.2019 persisted in the soil so to provide protection against late light to the new potato crop in the subsequent season. Potato tubers cv Nicola were sown on 7.1.2020 in the same containers of Experiment 1, two days after tuber harvest. At 10 weeks after planting (15.3.2020), 118 days after soil treatment, the plants grown in the containers were inoculated with sporangia of P. infestans as described below. Experiment 4 was done at Kibbutz Magen in the Western Negev. Four plots (4x12m each, 240 plants per plot in 4 rows of 60 plants per row) in a commercial field of potato Sifra were used. At 10 weeks after planting (9.12.2019), plots were treated as follows: untreated control; spray with ZE at the recommended dose of 40 g ai/ha; spray with ZE of 150 g ai/h which was immediately washed down with overhead sprinkling irrigation equivalent to 20 mm rain; spray with ZE of 500 g ai/h which was immediately washed down with 20 mm rain. The two last treatments were considered as a soil drench treatment.

Estimation of disease control in field-grown plants
At various time intervals after soil drench (in Experiment 1, at 1-20 days; in Experiment 2, at 4-16 days) leaves were detached from the control and the treated plants (1 compound leaf per plant) and brought to the laboratory for inoculation with P. infestans. Leaves were placed on a wet filter paper in sealed transparent boxes (60x30x10 cm), upper surface uppermost, and spray-inoculated with sporangial suspension (5x10 3 sporangia /ml) of P. infestans isolate 164. This isolate was collected in March 2016 from potato at Nirim, Western Negev, Israel. It is resistant to mefenoxam (MFX) and belongs to the 23_A1 genotype. The inoculated leaves were placed in a dew chamber at 18˚C overnight and then in a growth chamber at 20˚C, 14h/ day. Percent leaf area blighted was visually estimated at 7 days post inoculation (dpi), unless stated otherwise. At 21 and 25 days after soil drench in Experiment 1 and 2, respectively, all plants in the net-house were spray-inoculated with of P. infestans (5x10 3 sporangia /ml) isolate 164. Plants in Experiment 3 were inoculated on the same day as the plants in Experiment 2. Plants in Experiment 4 were sampled at 10, 21 and 43 days after treatment; one compound leaf was sampled from every 6th plant in a row. Leaves were brought to the laboratory and inoculated with P. infestans as described above. Disease development was visually estimated (% infected leaf area) in the detached leaves at 6 or 7 dpi.

Data analysis
Effective dose values (ED90, the dose of a fungicide(s) required to reduce % leaf area blighted by 90% relative to the control) were derived from log-probit regression curves using SPSS software. SF (synergy factor) was calculated using the Wadley formulae as described before [15]. ttest was performed using SPSS software to determine significant differences between means at α = 0.05.

Experiment 1: Efficacy of soil drench in cultivar Nicola
Results presented in Fig 1 show the response to inoculation in growth chambers of the leaves which were detached from control and fungicide-treated plants at 1,3,7,9,15 and 20 days after soil drench. Leaves that were detached at 1 day from plants treated with ZE produced significantly less disease compared to leaves that were taken from plants treated with BENT or OXPT, demonstrating rapid uptake and synergistic interaction (SF = 4.31) between OXPT and BENT, the components of ZE (Fig 1A). Leaves that were taken from plants treated with MFX or Infinito produced as much disease as leaves that were taken from control plants (Fig 1A). Efficacy of OXPT and BENT increased at �3 days after soil drench, indicating on enhanced translocation to the leaves, reaching a similar level of protection as ZE ( Fig 1B). OXPT, and ZE, and to a lesser extent BENT, maintained high control efficacy in leaves that were detached at 7, 9, 15 and 20 days after soil drench (Fig 1C-1F). MFX and Infinito were totally ineffective. The appearance of the disease in artificially inoculated leaves detached at 7 days after soil drench is shown in Fig 2. At 21 days after soil treatment, plants grown in all containers were spray-inoculated in situ with sporangial suspension of P. infestans isolate 164. Disease development as was visually estimated at various time intervals after inoculation is shown in Fig 3A-3F. OXPT, BENT and ZE were protective against the blight in a dose dependent manner all along the epidemic period ( Fig 3A-3C and 3F). The higher was the dose applied the better was the protection against the blight, except for OXPT in which no significant difference was seen between the highest two doses. MFX and Infinito were ineffective (Fig 3D-3F).

Experiment 2: Efficacy of soil drench in cultivar Sifra
Results presented in Fig 5 show the response to inoculation with P. infestans in growth chambers of the leaves which were detached from control and fungicide-treated plants at 4, 6, 12 and 16 days after soil drench. OXPT, BENT and ZE contributed a high and significant protection against the blight in all sampling days. High level of protection was already seen as early as 4 days after treatment (data for 1 and 3 days are unavailable due to technical reasons). At this time, ZE provided better protection compared to OXPT (Fig 5A), but at later sampling times, all fungicides performed in a similar effective manner (Fig 5B-5D).
At 25 days after soil drench, plants in all containers were spray inoculated in situ with sporangial suspension of P. infestans isolate 164. Late blight progress was visually estimated for 21 days after inoculation. The results are shown in Fig 6A-6D. All three fungicides were highly effective in protecting against the blight. At 50 mg ai per container, all fungicides were fully protective but at the lowest dose of 12.5 mg ai per container OXPT outperformed BENT and ZE. At 25 mg ai per container, ZE performed better that BENT (Fig 6A-6D). The appearance of the plants at 10 dpi (days post inoculation) is shown in Fig 7.

Experiment 3: Persistence of fungicides in soil
The data presented in Fig 8A and 8E show that OXPT applied to the soil in the previous season at a dose of 25 or 50 mg ai per container provided in the present season excellent control of the disease for 21 days after inoculation, suggesting its persistence (or the persistence of its degradation products) in the treated soil for at least 139 days. BENT, MFX and Infinito provided no control at all (Fig 8B and 8E) whereas ZE provided partial control of the disease at 50 mg ai per container (Fig 8C and 8E).

Experiment 4: Comparative efficacy of ZE applied as a foliar spray or as a soil drench
The data in Table 1 show that all three treatments (a foliar spray and two soil drenches) were highly and significantly effective in controlling the blight in the leaves that were collected at 10 days after treatment. Control efficacy in leaves collected at 21 and 43 days after treatment was poor in the foliar spray treatment but very high in the two soil drench treatments, suggesting that the sprinkling irrigation applied after the spray was effective in enabling the wash down of ZE to the soil and facilitating its uptake to the foliage. The appearance of infected leaves (at 6 dpi) which were collected at 10 days after treatment is shown in Fig 10A-10D and of the leaves which were collected at 21 days after treatment is shown in Fig 10E-10G.

Discussion
In a recent study [3] we showed that a single application of oxathiapiprolin, benthiavalicarb, or their mixture Zorvec Endavia (3+7, w/w) to the root system of nursery plants grown in multicell trays provided prolonged systemic protection against late blight and downy mildews in growth chambers and in field tests. Soil application of 1mg ai per plant provided durable protection of up to four weeks in tomato against late blight, cucumber against downy mildew and basil against downy mildew.
Others showed efficacy of soil drenches with oxathiapiprolin against black shank in tobacco [12], Phytophthora root rot in citrus [14] and Phytophthora blight in bell pepper [13]. No soil drench tests with the above fungicides were reported for potato.
The present research proves that a single application of oxathiapiprolin, benthiavalicarb or their mixture ZE to the soil in which potato plants are grown was highly protective against late blight caused by mefenoxam-resistant P. infestans in the field. ZE, composed of the two   systemic fungicides OXPT [2,3] and BENT [10], was the only commercial oxathiapiprolin mixture that with soil application to potted potato plants could provide effective systemic control of late blight caused by mefenoxam-resistant P. infestans. Dose response experiments showed ED90 values of 0.04, 7.52, 10.79, 12.18, 13.50 and 15.00 mg ai per pot/plant for OXPT +Benthiavalicarb, OXPT+Mefenoxam, OXPT+Azoxystrobin, OXPT+Mandipropamid, OXPT +Famoxadone, and OXPT+ Zoxamide, respectively (Cohen, unpublished data). In such experiments, a dose of 0.1 mg ai ZE applied to the root system of 12-leaf potted potato plant pro-vided~90% protection against the disease. Protection of the foliage was already detected at 1 day after root treatment, suggesting a rapid acropetal translocation of the compounds from the root. Qu et al [13] reported that when oxathiapiprolin was applied to the roots of bell pepper plants grown in hydroculture, the compound was detected in the second true leaf within 8 h and in the top new leaf 48 h after application to the root. In potted plants, translocation to the top new leaf was slower, within 3 days after application. Our data show that ZE reached the top leaf of 5 leaf tomato plants grown in soil, within one hour after soil drench (Cohen, unpublished).
Our data showed that ZE is a synergistic mixture, namely it provides a significant better disease control than the combined control efficacies of its two components [5,6]. OXPT was proved to act synergistically with mefenoxam in controlling P. infestans in tomato [5]. It also acts synergistically with mefenoxam and/or Bion in controlling downy mildew in sunflower [4]. In the present field experiments, a single soil drench of ZE or OXPT provided detached leaves of potato with 20 days of high protection against P. infestans. BENT was moderately effective while Infinito (serving as a control) was ineffective. Mefenoxam was ineffective due to the resistance of P. infestans used to the fungicide. Treated plants in the field remained protected for additional 20 days after in situ inoculation, totaling about 6 weeks after root treatment. A similar long protective period P. infestans was reported in tomato [3].
ii. A single soil application of ZE provides full season protection against late blight caused by mefenoxam resistant P. infestans.