Differential Systemic Translocation of Oxathiapiprolin, Benthiavalicarb, and Their Mixture to Tomato Leaves and Fruits as Evidenced by Their Differential Protection from Late Blight Caused by Phytophthora infestans

Abstract

The fungicides oxathiapiprolin, benthiavalicarb, and their mixture (Zorvec Endavia) provided excellent protection for tomato fruits against Phytophthora infestans when applied directly to the fruits or to the fruit stem scar. High levels of protection were also recorded when the fungicides were applied to the root system of fruit-bearing plants grown in a greenhouse. The objective of this study was to follow the translocation of oxathiapiprolin and benthiavalicarb into the fruits of tomato. We discovered that while soil drenching conferred strong protection to leaves, it failed to provide good protection for the fruits. Similarly, a fungicidal spray applied to plants while their fruits were bagged during spraying provided full protection to the leaves but failed to protect the fruits. These results indicate differential systemic translocation of the fungicides to leaves versus fruits. LC–MS/MS analyses revealed translocation of oxathiapiprolin and benthiavalicarb to leaves but not to fruits in soil-treated plants. Thus, while fruits may be less protected, they may also pose a lower risk of pesticide residues to consumers. This is the first study to compare fruit versus leaf infection and demonstrate differential systemic translocation of systemic fungicides to leaves versus fruits.

1. Introduction

Tomato (Solanum lycopersicon L.) is the second most widely consumed vegetable worldwide, following potato, with an annual global production of nearly 189 million tons in 2021 (The Food and Agriculture Organization, FAO, Rome, Italy; http://www.fao.org accessed on 1 January 2024). Tomato and potato are major hosts of the oomycete pathogen Phytophthora infestans (Mont.) de Bary, which requires frequent application of anti-oomycete fungicides for its control. While fungicide residues reaching tomato fruits are crucial for effective management of late blight, they may also pose potential health risks to consumers.

Several studies have examined fungicide residues in tomato fruits, with residues primarily detected in fruits directly exposed to the fungicides. However, no studies have investigated the presence of residues in tomato fruits from plants treated with systemic fungicides via the roots.

Warneke et al. [1] provided a general overview of the movement of commonly used pesticides from their initial point of deposition to other parts of the plant, where they maintain activity against target pathogens. They categorized anti-oomycete fungicides as follows: (a) non-translocated: copper, chlorothalonil, mancozeb, captan; (b) translaminar: azoxystrobin, mandipropamid, cyazofamid; (c) xylem-translocated: propamocarb-HCl, metalaxyl, mefenoxam, fluopicolide, oxathiapiprolin; (d) phloem-translocated: fosetyl-Al, phosphorous acid, and salts.

Metalaxyl (mefenoxam) was once considered one of the most potent anti-oomycete fungicides until the development of resistant strains reduced its efficacy. When applied to tomato roots, metalaxyl effectively protected leaves and fruits against P. infestans, but no systemic protection occurred when it was applied to the leaf lamina [2].

Oxathiapiprolin (FRAC code 49) is a piperidinyl-thiazole isooxazoline fungicide that targets oxysterol-binding proteins (OSBPs) of oomycetes (https://www.frac.info accessed on 1 January 2024). OSBPs are involved in lipid trafficking between the plasma membrane and endoplasmic reticulum, membrane maintenance, and synthesis of lipids essential for cell survival (FRAC, 2022). Oxathiapiprolin inhibits all developmental stages in the asexual life cycle of oomycete pathogens [3,4,5] including oospore germination [6]. It is highly effective when applied as a foliar spray [3,7,8], soil drench [9,10], or seed treatment [11].

The systemic movement of oxathiapiprolin from roots to foliage has been documented in several species, including cucumber, basil, and tomato [10]. Moreover, it has been shown to provide season-long protection for potato against late blight [9]. Oxathiapiprolin is ambimobile, capable of both acropetal (upward) and basipetal (downward) translocation, including transfer from treated to untreated plants via interconnected root systems [7].

Despite its importance for food safety, no studies have investigated the systemic translocation of oxathiapiprolin, benthiavalicarb (Bent), or their mixture (Zorvec Endavia) into tomato fruits. Several reports have quantified the dissipation and residues of oxathiapiprolin and benthiavalicarb in fruits, vegetables, and soil [12,13,14], but all involved direct fungicide contact with the fruits. Qu et al. [15] reported systemic translocation of oxathiapiprolin to the foliage of bell peppers but not to the fruits.

According to the manufacturer Corteva, field studies demonstrated protection of grape bunches that were bagged during spraying with oxathiapiprolin, suggesting sufficient systemic movement from foliage to bunches to protect against downy mildew Plasmopara viticola. Also, no downward movement to potato tubers was observed (G. Jean-Luc, personal communication). To date, no published data exists regarding the translocation of these systemic fungicides to tomato fruits.

The objective of the present study was to investigate the systemic translocation of oxathiapiprolin, benthiavalicarb a systemic CAA fungicide [16], and their mixture Zorvec Endavia to tomato fruits. To test this, fungicides were applied to the roots or leaves of green-fruit-bearing tomato plants. Fruits were then detached and inoculated with P. infestans. The level of protection conferred by the fungicides against late blight served as an indirect measure of their systemic translocation to the fruits.

2. Materials and Methods

2.1. Plants

The tomato cultivar Roter Gnom (RG; a gift from Syngenta, Stein, Switzerland; determinate growth type) was used in all experiments. Plants were grown either in 0.1 L pots, filled with peat/perlite (10:1, v/v) mixture, or in the soil in the greenhouse. Plants were fertilized with 0.05% NPK solution twice a week.

2.2. Fruits

All experiments were performed with green fruits, 3–5 cm diameter, as they are more susceptible to late blight infection than red fruits and their symptoms are easily recognized. The calyx remained attached to the fruits in all experiments, except in cases where it was deliberately removed to allow fungicide application to the abscission zone (fruit stem scar).

2.3. Fungicides

Oxathiapiprolin (Oxa; 100 OD, oil dispersion, 100 g/L oxathiapiprolin) was kindly provided by DuPont, France. Benthiavalicarb (Bent; 98% technical grade) was a gift from Syngenta Crop Protection, Basel, Switzerland. The commercial mixture of oxathiapiprolin + benthiavalicarb (Zorvec Endavia, ZE; 70 g/L benthiavalicarb + 30 g/L oxathiapiprolin) was provided by Corteva via its local producer Agrochem Ltd. (Petach Tikva, Israel). All fungicides (except Bent) were suspended in water and diluted into a series of 10-fold concentrations ranging from 0.01 to 1000 μg active ingredient (ai) per mL Bent was dissolved in DMSO (100 mg/10 mL) and subsequently suspended in water. For experiments with the fungicide mixture, the indicated dose represents the combined doses of both ingredients.

2.4. Fungicide Application

Fungicides (1–100 µg ai per mL) were applied to detached fruits in two ways: (i) by direct spraying onto the whole fruit; (ii) by applying ~10 µL to the fruit stem scar using a cotton swab.

Potted tomato plants were drenched to the soil with 1 mL of fungicide suspension containing 0.01–10 mg ai. For intact plants bearing green fruit, fungicides were applied in one of the following ways: (i) as a soil drench (1–10 mg ai per plant) or (ii) as a whole-plant spray (200 µg ai per mL) while the fruits were protected in plastic bags.

2.5. Pathogen and Inoculation

Phytophthora infestans isolate 164 (collected in March 2016 from potato at Nirim, Western Negev, Israel), resistant to mefenoxam and belonging to genotype 23A1, was used in all experiments. The pathogen was propagated on detached tomato leaves in moistened 14 cm Petri dishes kept in a growth chamber at 18 °C (14 h light/day; 100 μmol m⁻² s⁻¹).

For inoculation, fresh sporangia were harvested from sporulating detached leaves into ice-cold distilled water, adjusted to 1 × 10⁶ sporangia/mL, and sprayed with a glass atomizer onto tomato plants, detached leaves, or detached green fruits.

Inoculated material was kept wet overnight in a dew chamber (18 °C, darkness) and subsequently transferred to a growth chamber under the same conditions. Disease severity was recorded 7 days post inoculation (dpi) unless stated otherwise.

Detached leaves were placed in 20 × 20 × 2 cm plastic trays on moistened filter paper (lower surface facing upwards) and spray-inoculated as described above. Detached fruits were placed in 25 × 20 × 15 cm plastic boxes on wet filter paper for inoculation. Disease severity on leaves was assessed by visually estimating the percent infected area, while disease severity on fruits was determined by calculating the proportion of infected fruits at 7–10 dpi.

2.6. Assessment of Systemic Translocation

Systemic translocation of fungicides to fruits was studied using intact plants treated by soil drenching or foliar spraying (with fruits bagged to prevent direct contact). Following an appropriate lapse period (depending on the specific experiment), fruits were excised with the calyx attached, placed in plastic boxes, and inoculated as described above.

2.7. LC–MS/MS Analysis

LC–MS/MS was used to quantitate oxathiapiprolin and benthiavalicarb in leaves and fruits of tomato plants. Calibration curves were obtained by using pure oxathiapiprolin and pure benthiavalicarb provided by Syngenta. The compounds were identified by their specific retention time (RT) on the column, the mass of the precursor ion, and the ionic fragments obtained upon breakdown of the molecule in a collision cell. Extraction of the pesticides and their analyses were performed as described earlier [7].

2.8. Data Analysis

In experiments with soil drenches and bagged fruits, 3–20 plants were used per dose per fungicide. For direct fruit spraying, 10 fruits were used per dose, while 6–10 fruits were used per dose in experiments involving fungicide application to the fruit stem scar. Data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s HSD test to detect significant differences between treatments. Statistical analyses were performed using XLSTAT software v27.1.3.0 (Lumivero Denver, CO, USA).

3. Results

3.1. Proneness of Green Fruits to Infection

The data in Figure 1A show that 5 h of wet inoculation period allowed infection in 83.3% of the fruits and 86.5% of the plant’s leaf area. Increasing the duration of the wet period increased infection only slightly. Inoculation with 1 × 10¹ sporangia per mL produced 7 and 15% infection in detached leaves and intact plants, respectively, but no infection occurred in green fruits. A gradual increase in the inoculum concentration gradually increased % infection. A dose of 1 × 10⁶ sporangia per mL produced 100% infection in intact plants and detached leaves and 88% infection in detached green fruits (Figure 1B). The appearance of fruits inoculated with 1 × 10¹, 1 × 10³, and 1 × 10⁷ sporangia per mL is shown in Figure 1C. In the following experiments, leaves and fruits were inoculated with 1 × 10⁶ sporangia per mL and thereafter maintained for 15 h in moist conditions.

3.2. Fungicide Efficacy—Spray Application to Green Fruits

Oxa, Bent, and ZE were highly effective in controlling late blight when preventively sprayed onto detached green tomato fruits. Thus, with 1 µg ai/mL, Oxa, Bent, and ZE enabled 90, 95, and 66% control of the disease, respectively, while 10 µg ai/mL enabled 100, 95, and 94% control, respectively. At 100 µg ai/mL, all fungicides enabled full control of the disease (Figure 2).

3.3. Application of Fungicides to the Fruit Stem Scar

Fruits were treated on their stem scar with ~20 µL suspension containing 0.02–20 µg ai of a fungicide with the aid of a cotton swab (Figure 3A) and inoculated 3 h later with P. infestans. The percentage of fruits infected at 7 dpi declined gradually as the dose of the fungicides applied to such fruits increased (Figure 3B). Thus, fruits treated with 2 µg ai of Oxa, Bent, or ZE showed 75, 94, and 77% control of the disease, respectively, whereas fruits treated with 20 µg ai were all fully protected.

In the following experiment, 10 µg ai of Oxa, Bent, or ZE was applied to the stem scar of 13–14 fruits. A week after inoculation 2, 1, and 0 fruits became infected compared to 13 out of 14 fruits in the control untreated fruits (Figure 4), indicating the high control efficacy of the treatment.

3.4. Systemic Translocation of Fungicides from Foliage to Fruits

Fruit clusters (three per plant) were each wrapped with a plastic bag, and the foliage of the plants (n = 3), grown in soil in a greenhouse, was sprayed with a fungicide suspension containing 200 µg ai Oxa, Bent, or ZE until run-off was observed. Bags were removed after 3 h, and leaves and fruit clusters were excised after 4 days and inoculated with P. infestans. At 7 dpi, control leaves taken from untreated plants showed 100% infection, whereas leaves taken from fungicide-treated plants showed zero infection. The percentage of fruits showing late blight symptoms in the control and Oxa-, Bent-, and ZE-treated plants was 100, 72.7 ± 24.1, 100, and 100%, respectively, indicating poor systemic translocation of the fungicides from the treated foliage to the untreated fruits.

3.5. Systemic Translocation of Fungicides from Roots to Leaves and Fruits

The systemic translocation of Oxa, Bent, and ZE from the root system of tomato plants to their foliage was first tested in potted plants. As shown in Figure 5, all three fungicides were highly effective in protecting the foliage against late blight. Thus, 1 mg ai per plant of Oxa, Bent, and ZE provided 95, 100, and 100% protection, respectively.

To evaluate the systemic translocation of these fungicides to leaves versus fruits, five experiments were conducted with greenhouse-grown fruit-bearing plants.

In Experiment 1, various doses of ZE (1–10 mg ai per plant) were applied to the soil of greenhouse grown tomato plants (n = 3), and the systemic protection against late blight was evaluated at various time intervals after treatment. The results presented in Figure 6A confirm dose-dependent control of late blight in the leaves of the treated plants at 2, 3, and 4 weeks after soil drenching. However, poor and inconsistent control of the disease was observed in the fruits at 5 weeks after drenching (Figure 6B).

In Experiments 2, 3, and 4, leaves and fruits were detached and inoculated at 2, 3, or 4 days after soil drenching. Data in

Table 1. Systemic protection against late blight in detached leaves and fruits taken from greenhouse-grown tomato plants at 2, 3, or 4 days after soil drenching with 10 mg ai of Oxa, Bent, or ZE per plant (n = 3). Figures denote SD% infected leaf area or mean ± SD% infected fruits. Different letters following figures in columns indicate significant differences between means at α = 0.05 (Tukey’s HDS test). nt, not tested.

	Experiment 2		Experiment 3		Experiment 4
	2 Days		3 Days		4 Days
	Leaves	Fruits	Leaves	Fruits	Leaves	Fruits
Control	89.5 ± 14.2 a	89.9 ± 7.8 a	56.5 ± 26.4 a	69.2 (n = 52)	97.5 ± 3.5 a	95.5 ± 6.4 a
Oxa	nt	88.1 ± 3.2 a	6.5 ± 16.5 b	52.4 (n = 103)	nt	nt
ZE	4.9± 4.4 b	62.7 ± 15.9 b	0.5 ± 0 bc	44.9 (n = 89)	12.8 ± 12.5 b	62.5 ± 15.9 ab
Bent	1.7 ± 2.9 b	79.2 ± 10.9 aba	nt	nt	nt	nt

show that all three fungicides, Oxa, ZE, and Bent, provided good protection against late blight to the leaves but poor protection to the fruits, indicating efficient systemic translocation of the fungicides from roots to the leaves but not to the fruits.

In Experiment 5, potted plants were drenched with 10 mg ai of Oxa or ZE (n = 20) and 3 days later were planted in soil in the greenhouse. Control plants were planted untreated. Leaves and fruits were detached after various time periods and inoculated with P. infestans. The results presented in Figure 7 and

Table 2. Ten-leaf tomato plants (n = 20) grown in 100 mL pots were soil-drenched with 10 mg ai per plant of Oxa or ZE. After 3 days, plants were transplanted to soil in the greenhouse. Leaves were detached after 30, 41, 50, 68, and 78 days, and fruits were detached at 68 and 78 days and inoculated with P. infestans. Disease records were taken from inoculated leaves and fruits at 7 and 10 dpi, respectively. Different letters following figures in columns indicate a significant difference between means at α = 0.05 (Tukey’s HDS test). nt, not tested.

			LEAVES
		% infected leaf area
	30 d	41 d	50 d	68 d	78 d
Control	95.0 ± 4.2 a	97.8 ± 8.3 a	100 ± 0 a	100 ± 0 a	100 ± 0 a
Oxa	0 b	1.1 ± 4.7 b	4.3 ± 8.6 b	4.3 ± 6.2 b	5.6 ± 9.2 c
ZE	0 b	1.1 ± 4.7 b	8.0 ± 16.0 b	4.3 ± 3.5 b	28.1 ± 15.3 b
			FRUITS
		% infected fruits
	30 d	41 d	50 d	68 d	78 d
Control	nt	nt	nt	98.3 ± 3.7 a	95.6 ± 3.9 a
Oxa	nt	nt	nt	67.6 ± 9.1 b	88.0 ± 14.5 a
ZE	nt	nt	nt	65.9 ± 17.6 b	88.2 ± 3.9 a

show excellent protection of the leaves but poor or no protection of the fruits.

The seeds that were collected from the treated plants produced seedlings that were as susceptible to late blight as the seeds that were collected from the control plants, suggesting no accumulation of Oxa or ZE in the seeds.

Forty-nine days after soil drenching, leaves and fruits were detached from the treated plants (n = 3), weighed, and extracted with acetonitrile formic acid as described earlier [7]. The results shown in Figure 8 confirm the occurrence of oxathiapiprolin and benthiavalicarb in leaves but not in fruits. The results show that benthiavalicarb was found in higher amounts in leaves as well as in fruits (Figure 8F) as compared to oxathiapiprolin (Figure 8A), suggesting its better systemic translocation.

The last experiment was conducted in the growth chamber with potted plants carrying small fruits. ZE was applied (n = 4) to the soil at 4 mg ai/plant whereas control plants were left untreated, and plants were inoculated 4 days later. As shown in Figure 9, ZE provided full control of late blight in the leaves but not in the fruits.

4. Discussion

Tomato and potato are major hosts of Phytophthora infestans, with all aerial parts susceptible to infection. In tomato, fruits inoculated with sporangia of both mating types (A1 + A2) produced infected seeds containing oospores in their coat and embryo [17]. Such seeds generated infected seedlings upon germination [18], thereby serving as an initial, genetically recombined inoculum for subsequent seasons.

While extensive research has been conducted on the infection process of potato leaves and tubers (reviewed by Evangelisti & Govers [19]), comparatively little is known about the infection of tomato leaves and fruits. As tomato fruits lack stomata, infection must occur directly through the epidermis. Recent mechanobiological studies demonstrated that Phytophthora species employ a slicing mechanism—termed naifu invasion—in which the hyphal tip acts as a sharp microscopic blade that cuts through the plant cell wall [19].

In this study, we used green tomato fruits, known to be highly susceptible to late blight infection. Red fruits are less susceptible, likely due to the protective properties of lycopene. Using whole-fruit spray inoculation, green fruits required higher sporangial concentrations for infection compared to detached leaves or intact plants and exhibited a longer incubation period before symptoms appeared. Importantly, the stem scar of the fruit represents an efficient entry point for systemic fungicides. Our study confirmed that P. infestans sporulates abundantly on tomato fruit surface despite the absence of stomata. Sporulating hyphae may emerge through cracks or natural pores in the skin [20]. This evidence suggests that the poor systemic translocation of fungicides to tomato fruits is not attributable to reduced fruit susceptibility to P. infestans.

The oomyceticides oxathiapiprolin (OSBPI, FRAC Group 49), benthiavalicarb (CAA, FRAC Group 40), and their mixture (Zorvec Endavia = ZE) have demonstrated excellent field efficacy against late blight in potatoes and tomatoes, as well as against downy mildew in various crops [2,3,4,5,6,8,9,10,11,15,16,21,22,23]. These fungicides have been effective when applied as foliar sprays, soil drenches, or seed treatments. Notably, a single early-season foliar application of oxathiapiprolin or ZE protected potato crops for approximately 30 days, while a single soil drench provided season-long protection.

Here, we show for the first time that oxathiapiprolin and ZE translocate efficiently from the root system to tomato leaves but poorly to the fruits, as evidenced by the differential development of late blight following artificial inoculation with P. infestans. This was found to be true in multiple, though fragmented, experiments. LC–MS/MS analyses of tomato plants at 49 days post soil drench (according to the protocol of Cohen and Weitman [7]) confirmed the translocation of oxathiapiprolin (applied alone) and benthiavalicarb (applied together with oxathiapiprolin) into leaves but only in trace or small amounts to the fruits. Benthiavalicarb was found in higher amounts in leaves as well as in fruits compared to oxathiapiprolin, suggesting its better systemic translocation into both organs.

The reasons for this differential translocation remain unclear. It may be related to the source–sink dynamics of plant physiology and/or the markedly lower transpiration rates of tomato fruits, which lack stomata [20]. Systemic fungicides that move acropetally in the xylem follow the transpiration stream, leading to higher accumulation in transpiring leaves than in fruits. Further research is required to clarify the underlying mechanisms.

Our findings have practical implications for tomato growers:

(a)

Soil application of systemic fungicides effectively controls foliage blight but not fruit blight.

(b)

Fruits, while less protected, may consequently retain lower fungicide residues, enhancing consumer safety.

(c)

Efficient foliar protection may indirectly prevent fruit infection unless external inoculum sources introduce the pathogen.

(d)

Reduced fungicide accumulation in fruits may lower the selection pressure on P. infestans, potentially delaying the development of fungicide resistance.

Unfortunately, resistance to oxathiapiprolin and to the CAA fungicide mandipropamid has recently been reported in Europe [24]. Of greater concern, double resistance to both fungicide classes was recently identified among P. infestans isolates in Europe [25], posing significant challenges in future late blight management.