Systemic Activity and Phytotoxicity of Fluensulfone in Vegetable Transplants Infected by Meloidogyne incognita

A M. incognita race 3 population, originally isolated from infested cotton in the San Joaquin Valley, CA, was multiplied on pot-grown tomato plants in a greenhouse. Eggs were extracted from heavily infested tomato cv. Red Cherry roots by shaking them for 3 min in a 0.5% NaOCl solution, washing them over one 60 μm pore-size sieve, and then collected on two stacked 25 μm pore-size sieves (Hussey and Barker, 1973). The resulting egg suspension was carefully poured over a filter paper over a dish with tap water and placed at 27ºC. Hatched second-stage juveniles (J2) were collected daily from the third day onwards and kept at room temperature with a slow air flow bubbling through the J2 suspension. After 7 days, the number of J2 in the suspension was counted at 40x magnification under a dissecting microscope and the concentration was estimated.

Tomato cv. Daniela (Osborne Quality Seeds, Mt. Vernon, WA), eggplant cv. Black Beauty (Park Seed, Greenwood, SC), cantaloupe (Cucumis melo) cv. Durango (Seminis, Oxnard, CA), bell pepper cv. Baron (Seminis, Oxnard, CA), chili pepper cv. Thai Dragon (Reimer Seeds, Saint Leonard, MD), and bell pepper cv. Sweet Mini Pepper (Sunworld, Bakersfield, CA) were used in one or more of the trials. All crops were seeded in seedling trays with potting mix (Sunshine Mix 5, Sungro, Vancouver, Canada) in a greenhouse at 27 ± 4 °C under natural light conditions and used when plants had 3 to 4 true leaves.

In a first set of experiments, seedlings of all crop cultivars at the 3 to 4 true leaf stage were removed from the seedling trays, the root systems gently washed with tap water to remove the potting mix, transplanted into 1 liter plastic pots filled with steam-sterilized sand (93% sand, 4% silt, 3% clay, pH 7.1) and then placed on a greenhouse bench for a week. All pots were then removed from the greenhouse into the shade, avoiding direct sunlight on the leaves. Plants were subjected to one of four treatments: 1) foliar spray with tap water (=untreated control), 2) foliar spray with 3 g/liter fluensulfone, 3) foliar spray with 6 g/liter fluensulfone, 4) foliar spray with 12 g/liter fluensulfone, and 5) a soil drench treatment. The amount of fluensulfone added to each pot for the soil drench treatment was based on a pot surface of 78.5 cm2 and the application rate of 2.8 kg fluensulfone per ha. To each pot, 100 ml of a 0.022 g/liter fluensulfone solution was added, which was sufficient to completely saturate the soil in the pot.

Before spraying, the soil of each pot was covered around the plant stem with a layer of plastic and three layers of paper to prevent contact between fluensulfone and the soil surface. For spraying, a pressurized hand-held sprayer was used delivering a fine mist, and spraying was stopped when the leaf surfaces were saturated with liquid. Each treatment had five replicates. Plants were allowed to dry for 10 minutes, paper layers and plastic sheet were removed, and pots were placed in a random order on a greenhouse bench not allowing the plants to touch. Two days after the treatments, all pots were inoculated with 2,000 M. incognita J2 by adding 1 ml of a suspension containing 500 J2/ml to each of four shallow holes around the base of each plant. Pots were watered though an automated drip system, and each pot received 5 g of Osmocote 17-6-10 controlled release fertilizer (Scotts-Sierra Horticultural Products Co, Marysville, OH) 3 days after nematode inoculation. Plants were visually examined for phytotoxic effects of the treatments 2, 7, and 14 days after applying the treatments. Phytotoxic effects were indexed on a 0–5 scale according to 0 = no symptoms, 1 = <20% of leaf surface with damage only visible on youngest leaves, 2 = 20–40% of leaf surface with damage and minor necrosis on the older leaves, 3 = 41–60% of leaf surface with damage and moderate necrosis on the older leaves, 4 = 61–80% of leaf surface with damage and major necrosis on the older leaves, and 5 = 100% of leaf surface with damage (dead plant).

Six weeks after nematode inoculation, all plants were carefully removed from the pots, and the root systems were gently washed free of soil with tap water. The plants were cut at the soil level, and the fresh and dry shoot weights of each plant were determined. The root systems were weighed and visually examined for the presence and severity of galling and indexed on a scale from 0 to 10, representing the presence of root galling on 0 to 100% of the root system. RKN J2 were extracted from each root system in a misting chamber for 5 days (Niblack and Hussey, 1985) and counted at 40x magnification under a dissecting microscope. The number of J2 per gram root and the reproduction factor (Rf) were calculated (where Rf = Pf/Pi, with Pi = the initial inoculum level (J2) and Pf = the final recovery of J2) (Thies, 2011). The experiment was done three times.

To test if a foliar application of fluensulfone had a nematicidal effect in the root rhizosphere, tomato cv. Daniela and pepper cv. Baron seedlings at the 3 to 4 true leaf stage were removed from the seedling trays, and the root systems were gently washed with tap water to remove the potting mix. Plants were placed flat on a table with the leave surface facing upwards and the roots were covered with two layers of filter paper and a sheet of plastic, leaving only the leaves exposed. There were 4 foliar spray treatments, applied as before: a spray with tap water (untreated control, and a spray with fluensulfone at 3, 6, or 12 g/liter. The leaves were allowed to air-dry for 10 min, an absorbent filter paper was then placed on the leaves for another minute, and plants were then transferred to 100 ml plastic vials containing 50 ml of water. Vials were wrapped with aluminum foil and plants were supported with an aluminum foil lid wrapped around the stem. The vials were placed in a random order on a table in a greenhouse, and two days later 1 ml of a suspension containing 500 M. incognita J2 was added to each vial. Five days later, the plants were removed from the vials and the number of dead and live J2 were counted in three 3 ml samples from each vial. J2 that were straight, not moving after prodding with a fine needle and with a disintegrated body content were considered dead. The average of the ratio dead/live J2 from the three subsamples was used for statistical analysis. Each plant-treatment combination had five replicates, and the experiment was done three times. The average greenhouse air temperature during the experiments was 23.3 (range 21.0–26.6), 27.8 (range 23.2–35.8), and 25.9ºC (range 23.8–38.9) in experiments one, two, and three, respectively.

To determine the time between a foliar spray with fluensulfone and the appearance of nematicidal activity in the rhizosphere, two repeated experiments were done. Tomato cv. Daniela and bell pepper cv. Baron were sprayed with fluensulfone (6 g/liter) as described above. A water-only spray served as a control. Immediately after transferring the plants to the vials, 1 ml of a suspension containing 500 M. incognita J2 was added to each vial. The plants were removed from the vials 2, 12, 24, 72, and 120 hr after the treatment and the nematodes in the vials were collected, examined, and J2 mortality in the suspensions was evaluated as described above. Each plant-treatment combination had five replicates, and the experiment was done three times.

Effects of repeated experiments (1, 2, or 3), treatment, and the interaction between experiment and treatment on fresh and dry shoot weights and on nematode root infestation levels were analyzed using ANOVA procedures. Treatment means were separated using an LSD-test. For nematode infestation levels, data were transformed by x¹ = log10 (x + 1) before analysis. For analysis of the ratio dead/live J2, data were transformed by arcsin (√x) before analysis. Non-transformed data are shown. Normality of fresh and dry shoot weight data and of transformed nematode infestation level data was examined using the Shapiro–Wilk test and through examination of Q-Q plots. Effects of the treatments on the categorical variables: the degree of phytotoxicity (scale 0–5) and the degree of root-galling (scale 0–10), were analyzed using the non-parametric Kruskal–Wallis test. The R software package was used for statistical analysis (R Core Team, 2022).

Analysis of variance showed that with almost all crops tested, there was a significant interactive experiment × treatment effect on the fresh and dry shoot weights. Therefore, results were analyzed separately for each experiment. Shapiro–Wilk tests and examination of Q-Q plots showed that fresh and dry shoot weights for individual experiments and crops were normally distributed. For each of the crops the treatment effects on fresh and dry shoot weights were very similar, and therefore, only effects on dry shoot weights are shown.

Tomato fresh and dry shoot weights were affected by the treatments in each experiment. In two of three experiments, plants from the untreated control and the drench treatment resulted in higher fresh and dry shoot weights than plants sprayed with 6 or 12 g/liter fluensulfone. Averaged over the three experiments, fresh and dry shoot weights were significantly reduced by each of the spray treatments (Table 1). Fresh shoot weights were reduced by 24%, 45%, and 70%, and dry shoot weights were reduced by 38%, 63%, and 85% in the 3, 6, and 12 g/liter fluensulfone sprays, respectively.

The phytotoxicity ratings were significantly affected by the treatments in a similar way in the three replicated experiments. Strong phytotoxic effects were observed in the highest spray rate, intermediate effects in the 3 and 6 g/liter spray treatments, and very minor effects after the drench application (Fig. 1; Supplement Figure 1A).

Figure 1:

Average (n=15) phytotoxicity symptom ratings (scale 0-5: 0 = no symptoms, 5 = plant dead) of plants treated with fluensulfone as foliar spray at 3, 6, or 12 g/liter or applied as a soil drench at 5 pints/acre (0.022 g/liter) 2, 7, and 14 days after application. Water-sprayed plants served as untreated controls (UTC). Different letters above bars indicate significant differences at P ≤ 0.05, Kruskal–Wallis test.

A spray with the high rate (12 g/liter) of fluensulfone was highly phytotoxic to eggplant as none of the plants survived this treatment. In experiment two, a spray with the low or intermediate rate also resulted in plant death, but in experiment one and three, plants from these treatments survived. Dry shoot weights of the surviving eggplants were significantly lower than the control after being sprayed with 3 or 6 g/liter fluensulfone. The drench application did not significantly reduce the dry shoot weight (Table 1). On average, the fresh weights were reduced by 71% and 67%, and the dry shoot weights by 77% and 73% after the 3 and 6 g/liter spray treatments. Phytotoxicity ratings reflected the effects on shoot weights. Very strong phytotoxic effects were observed already two days after spraying plants with fluensulfone at all three rates (Fig. 1; Supplement Figure 1B).

Figure 2:

Average (n=15) root galling index (scale 0–10: 0 = no galling, 10 = 100% of roots with galls) of plants treated with fluensulfone as foliar spray at 3, 6, 12 g/liter, or applied as a 100 ml soil drench at 0.022 g/liter. Plants were inoculated with 2,000 Meloidogyne incognita J2 per 1 liter pot 2 days after treatments and evaluated 6 weeks after treatments. Water-sprayed plants served as untreated controls (UTC). Different letters above bars within the same plant cultivar indicate significant differences at P ≤ 0.05, Kruskal–Wallis test. x = missing (dead) plants.

Melon plants also reacted very strongly to fluensulfone foliar sprays. None of the melon plants that were sprayed with fluensulfone survived 6 weeks after nematode inoculation. However, pots with melon that were drenched with fluensulfone all survived, and the dry shoot weights of these plants were not affected by the drench treatments (Table 1). Phytotoxic symptoms on melon after fluensulfone sprays were already apparent two days after the treatments, and one week after these treatments most plants were dead or close to dying (Fig. 1; Supplement Figure 1C).

Pepper plants were slightly more tolerant to fluensulfone sprays than the other crops. Pepper Baron fresh or dry shoot weights were generally not significantly affected by a fluensulfone spray at the lowest rate of 3 g/liter. Effects of fluensulfone sprays on pepper Baron dry shoot weights were slightly different between the three replicated experiments. On average however, a significant reduction in shoot weights occurred after spraying the plants with fluensulfone at rates of 6 or 12 g/liter. The drench application did not affect Baron shoot weights (Table 2). Phytotoxicity symptoms increased with increasing fluensulfone rates of the spray application. The lowest rate resulted only in minor symptoms that did not progress over time, whereas the highest rate caused obvious phytotoxic effects. The drench application did not result in any phytotoxic effects (Fig. 1; Supplement Figure 1D).

Thai Dragon pepper shoot weights were only affected by the treatments in one of the three replicated experiments (experiment 2). Averaged over the three experiments; however, shoot weights were significantly reduced by the fluensulfone spray applications (Table 2). Phytotoxic effects occurred with all three spray applications, with the stronger effects after the high-rate fluensulfone sprays. Plants did, however, somewhat recover over time, as the average phytotoxicity rating of each spray application at Day 14 was lower than at Day 2 or Day 7 (Fig. 1; Supplement Figure 1E).

In the second replicate experiment with Sweet Mini peppers, all plants were lost due to an accidental herbicide spray. Therefore, data are from two replicated experiments only. Effects of the treatments on the shoot weights were different between the two experiments but averaged over the two experiments the treatments did not significantly affect the shoot weights (Table 2). There was a clear correlation between the rate of the fluensulfone spray and the severity of phytotoxic effects. The high rate resulted in severe symptoms, whereas the lower rate only caused minor symptoms which also decreased over time (Fig. 1; Supplement Figure 1F).

Because the treatments affected plant growth, the effects of the treatments on M. incognita infestation levels were corrected for plant root weight (number of J2/g root). Treatment effects on root galling (scale 0–10) were very similar between the three replicated experiments and are shown as averages of the three experiments (Fig. 2).

On tomato, all fluensulfone treatments significantly reduced the root nematode infestation levels compared with the untreated control. Overall, the strongest reductions resulted from the 12 g/liter foliar spray (99% reduction) and the drench application (97%) (Table 3). Results on root galling were similar, with significant reductions resulting from each of the fluensulfone treatments (Fig. 2).

On eggplant, all fluensulfone treatments strongly reduced root nematode infestation levels, although some of the foliar spray treatments also resulted in plant death. On average, foliar sprays at 3 and 6 g/liter and the drench treatment all reduced root infestation levels by ≥98% compared with the untreated control (Table 3). These treatments were similarly effective in reducing root galling on eggplant (Fig. 2).

Only untreated and drench-treated melon plants survived. On average, the drench treatment significantly reduced root infestation by 65% and the average root galling index from 5.7 in the untreated control to 2.4 in the drench treatment (Table 3 and Fig. 2).

There were some differences between the three replicated experiments regarding the efficacy of the fluensulfone treatments on reducing nematode infestation in Baron peppers but on average the 12 g/liter foliar spray and the drench treatment were most effective reducing infestation levels by 99% and 98% compared to the untreated control (Table 4). These two treatments were also most effective in reducing Baron root galling (Fig. 2).

Nematode infestation levels in Thai Dragon peppers were low and variable even in the untreated control plants (Table 4), indicating that this cultivar is a poor host for M. incognita. Consequently, there were no significant differences among the treatments. Correspondingly, only very minimal galling occurred on Thai Dragon pepper roots, and there were no significant treatment effects (Fig. 2).

Nematode infestation levels in Sweet Mini Pepper roots were also variable between experiment one and three, with on average 80 and 881 J2 per g root in the untreated control plants, respectively. Averaged over the two experiments, however, all fluensulfone treatments significantly reduced J2 root infestation levels by more than 90% (Table 4). Galling on Sweet Mini Pepper roots was minor even in the untreated control plants, but on average all fluensulfone treatments were equally effective in reducing root galling (Fig. 2).

Foliar applications with fluensulfone had strong nematicidal effects in the water surrounding the roots 5 days after the applications were made. The arcsin (√x) transformed data (ratio dead/live J2) satisfied normal distribution requirements (Shapiro–Wilk test: P > 0.05). Although ANOVA analysis indicated a significant interaction between the treatments and the replicated experiments, the results were very similar between the three replicated experiments for both the tomato cv. Daniela and pepper cv. Baron test plants. With tomato, the M. incognita J2 mortality levels in the untreated control plants ranged between 9% and 28%, whereas the mortality in the water surrounding the roots of the fluensulfone-treated plants was significantly higher, ranging between 79% and 100%. Results with pepper were very similar, with a significantly higher J2 mortality in the water surrounding the roots of the fluensulfone-treated plants compared with the untreated plants. Except for tomato in experiment 2, each increase in the fluensulfone rate resulted in a significant increase in J2 mortality (Fig. 3).

Figure 3:

Average (n=5) percent mortality, 5 days after treatments, of Meloidogyne incognita J2 in a water suspension surrounding the roots of tomato (A) or pepper (B) plants treated with a foliar spray application of fluensulfone (3, 6 and 12 g/liter). UTC = untreated control. Three replicated experiments were done. Error bars are ±SE. Different letters above bars indicate significant differences within the same experiment at P ≤ 0.05, LSD-test. Percentage mortality data were transformed by arcsin (√x) before statistical analysis. Non-transformed data are shown.

Statistical analysis of the results of the subsequent time-series experiments with tomato and pepper showed that the factor experiment (1, 2, 3) or the “experiment x treatment” interaction was not significant (P>0.05). Therefore, results are presented as averages of the three repeated experiments (n=15). Results showed that treatment effects on J2 mortality became apparent as soon as 12 hr (pepper plants) and 24 hr (tomato plants) after the applications were made. At these times, the mortality of M. incognita J2 in the root solution of fluensulfone-treated plants was significantly greater than in the water-sprayed control plants. Differences between these two treatments increased over time, and at 120 hr after application, the average J2 mortality in fluensulfone-treated tomato or pepper plants was 95%, whereas mortality in untreated tomato or pepper plants was 25% and 14%, respectively (Fig. 4).

Figure 4:

Average (n=15) percent mortality of M. incognita J2 in a water suspension surrounding the roots of tomato (A) or pepper (B) plants treated with a foliar spray application of 6 g/liter fluensulfone at different time intervals after application. UTC = untreated control. Error bars are ±SE, * indicate significant differences within the same time interval at P ≤ 0.05, LSD-test. Percentage mortality data were transformed by arcsin (√x) before statistical analysis. Non-transformed data are shown.