Effect of spirotetramat and fluensulfone on population densities of Mesocriconema xenoplax and Meloidogyne incognita on peach

Populations of M. incognita and M. xenoplax were originally isolated from peach orchards in Georgia and maintained on eggplant (Solanum melongena cv. ‘Black Beauty’) and peach (Prunus persica cv. ‘Lovell’ and ‘Nemaguard’), respectively, in a greenhouse. Mixed stages of M. xenoplax and eggs of M. incognita were collected from the culture medium and eggplant roots using the centrifugal-flotation method (Jenkins, 1964) and a 0.5% NaOCl solution (Hussey and Barker, 1973), respectively, and used as inoculum in both in vitro assays and greenhouse trials.

The seedlings of peach rootstocks, including ‘Lovell’ and ‘Nemaguard’ that are susceptible to M. incognita and M. xenoplax, respectively, were grown from seed provided by the Southeastern Fruit and Tree Nut Research Laboratory of the USDA-ARS. Briefly, the seeds of both rootstocks were germinated and planted into 28 × 56 × 6 cm planting trays containing 9.4 L of pasteurized Fafard germinating mix (Fafard, Sun Gro Horticulture), and allowed to grow for 4 wk in a greenhouse at 27 ± 5 °C until used in the trials.

Commercial formulations of spirotetramat (Movento™) and fluensulfone (Nimitz™) were obtained from Bayer CropScience (Bayer AG, Rhine, Germany) and ADAMA (Agricultural Solutions Ltd., Raleigh, NC), respectively. Their efficacies were evaluated against M. incognita and M. xenoplax in both in vitro assays and greenhouse trials during 2011 to 2012. Spirotetramat and fluensulfone were tested in a single trial against M. xenoplax, but two trials were conducted for the evaluation of spirotetramat and fluensulfone against M. incognita. Initially, fluensulfone and spirotetramat were both evaluated for efficacy through foliar applications against M. incognita, but the foliar application of fluensulfone lead to phytotoxicity among all plants treated (data not shown). Thus, a separate M. incognita trial was conducted with fluensulfone alone as a soil drench application, where no phytotoxicity was observed (phytotoxic data not shown). Therefore, fluensulfone was applied as soil drench and spirotetramat was applied as foliar application against both M. incognita and M. xenoplax nematodes in all the trials presented here.

An in vitro assay was conducted in 24-well plates to evaluate the efficacy of spirotetramat and fluensulfone against M. incognita second-stage juveniles (J2) and M. xenoplax at room temperature (25 ± 2°C). This assay was comprised of four treatments, including two rates of spirotetramat at 0.017 and 0.026 kg a.i./ha, one rate of fluensulfone at 3.92 kg a.i./ha, and a control (sterile tap water). There were six replications per treatment and the assay was repeated once. Rates were determined from the most efficacious rates used in previously published trials (McKenry et al., 2009, 2010, 2011; Csinos et al., 2010). For spirotetramat treatments, 92 and 140 µl of spirotetramat (0.017 and 0.026 kg a.i./ha, respectively) were mixed with 50 ml of sterile water. For fluensulfone, 1.67 µl of fluensulfone (3.92 kg a.i./ha) was mixed with 100 ml of sterile water. From each prepared stock solution, 1 ml of each concentration was placed in each well and a 1 ml suspension of approximately 1,000 M. xenoplax mix stages were added to each well. For the M. incognita trials, a 1 ml suspension of 500 J2 was prepared for the first in vitro assay and 1,000 J2 were used for the repeated in vitro assay. The percentage of nematode mortality was determined 24, 48, and 72 hr after initial exposure to the treatments. To determine nematode mortality, a 500 μl of thoroughly mixed sub-sample from each well, containing on average 105 M. xenoplax nematodes and 130 M. incognita juveniles, was added to 5-cm-diameter glass dishes containing 3 ml of sterile water, and allowed to diffuse into the solution for 1 hr. The percentage of nematode mortality was determined by counting the numbers of all mobile and non-mobile nematodes under a stereomicroscope. The non-mobile nematodes were considered alive if there was a response to probing with a fine probe. Each bioassay was repeated once for each nematode species and data combined for analysis.

Due to phytotoxicity of fluensulfone to ‘Lovell’ rootstock when foliarly applied, a spirotetramat foliar treatment was evaluated in a separate trial from a soil application of fluensulfone, under greenhouse conditions during 2011. For both M. incognita trials, four-weeks old seedlings of Lovell rootstock were transplanted into 20-cm-diameter standard clay pots containing 3.4 L of pasteurized loamy sand soil mixture of 25% field soil, 50% sand, and 25% Fafard germinating mix (Fafard, Sun Gro Horticulture), and allowed to establish for two weeks in a greenhouse at 27 ± 5 °C. Two weeks after transplanting, each seedling was inoculated with 20,000 M. incognita eggs/pot. Then, 10 d after nematode inoculation, the chemical treatments were applied in each trial with the total number of treatments varying among trials. For the spirotetramat trial, there were four treatments including: (i) spirotetramat at 0.017 kg a.i./ha; (ii) spirotetramat at 0.026 kg a.i./ha; (iii) methylated seed oil blend (Drexel – MES-100) at 2.6 ml/L as an adjuvant control; and (iv) an untreated control. Foliar application rates were based on a spray coverage of spirotetramat at 76.6 L/ha. In the fluensulfone trial, there were only two treatments: (i) fluensulfone at 3.92 kg a.i./ha; and (ii) an untreated control. The fluensulfone rate 3.92 kg a.i./ha was converted from a volumetric rate of 4 mg a.i./L (4 ppm) of soil. For the spirotetramat trial, the low and high rate of spirotetramat, 0.92 ml/L and 1.4 ml/L, respectively, was separately mixed with MES-100 (2.6 ml/L solution) in 1,000 ml of tap water and applied till runoff as a foliar application to each plant using a spray bottle, as an adjuvant control treatment. Similarly, a 1,000 ml solution of MES-100 was applied as a foliar application to each plant, until runoff. In the fluensulfone trial, 14 mg of a.i. of fluensulfone was mixed in 200 ml of tap water and drench applied in four holes (10-cm-deep) made in the soil surface around each plant in each pot. Plants were not watered for a few days after drenching. Treated pots of each of the spirotetramat and fluensulfone trials were arranged in a randomized complete block design with six replications in the same greenhouse and conditions listed above. All plants were watered and fertilized with 1 ml of 13-13-13 fertilizer as needed until termination of trials.

The final population (pf) density of M. incognita in the soil and reproduction factor (Rf) were assessed at 40 and 70 d after inoculation (DAI) for both trials. Both trials were terminated 70 DAI and dry weights of both shoots and roots were recorded. To assess soil nematode population density in each pot, four soil cores (2.5-cm-diam. × 15-cm-deep) were collected randomly, 40 and 70 DAI from the area around each plant, and a composite soil sample was prepared. M. incognita J2 were extracted from a 100-cm³ soil sub-sample using soil sieves and centrifugal-flotation technique (Jenkins, 1964) and counted using a stereomicroscope. Also, the entire root system was washed free of soil and M. incognita eggs were collected from all the roots using a 0.5% NaOCl solution (Hussey and Barker, 1973). Then, the total population of M. incognita in each pot was determined by combining total numbers of J2 in soil and eggs extracted from the respective root system. Furthermore, the nematode reproduction factor (Rf = Pf/Pi) for both trials was also calculated by dividing total numbers of nematodes per pot (Pf = final population) by the number of nematodes added (Pi = initial inoculum) (Roberts and May, 1986). The spirotetramat and fluensulfone trials on M. incognita were repeated in 2012 under similar greenhouse conditions with the same treatments and statistical design, except that there were eight replications per treatment instead of six replications.

In 2012, a third trial was conducted to compare the effects of both spirotetramat and fluensulfone against M. xenoplax as single foliar and drench applications, respectively. This trial was conducted under similar greenhouse conditions with the same treatments, statistical design, and replications as described for both spirotetramat and fluensulfone trials on M. incognita, the only exception being that the efficacy of both compounds were directly compared to each other in a single trial. For this trial, seedlings of ‘Nemaguard’ rootstock, planted as a susceptible host, were planted and prepared as described previously. After establishment, 1,000 mixed stages of M. xenoplax were used as inoculum, and the nematode population density in the soil and reproduction factor were assessed at 30, 60, and 90 DAI. This trial was repeated once as described above, except there were seven replications per treatment instead of six replications.

Two separate trials using a dual application of spirotetramat were also conducted with M. incognita and M. xenoplax. Due to restrictions to peach seedling growth within the greenhouse, only two applications of spirotetramat were evaluated against both nematodes. Protocols were similar to the previous single application trials expect that the treatments were applied twice, the first spirotetramat and (MES-100) adjuvant application occurring 10 DAI followed by a second application at 40 DAI. The treatments for both M. incognita and M. xenoplax trials included: (i) two applications of spirotetramat at each rate of 0.017 and 0.026 kg a.i./h, (ii) an untreated control, and (iii) two applications of MES-100 as an adjuvant control at 2.6 ml/L. The M. incognita trial was terminated 70 DAI, whereas the M. xenoplax trial was terminated at 90 DAI. The observations on nematode density and reproduction factors of M. incognita were recorded at 40 and 70 DAI, and of M. xenoplax at 30, 60, and 90 DAI. These trials were repeated once with similar greenhouse conditions and methods in 2012.

All statistical analyses were performed using a generalized mixed model (GLIMMIX PROC, SAS Institute, Cary NC) to evaluate interactions between trials and if no significant interaction was detected, data were combined for analysis. Nematode and egg counts for each treatment were transformed using log₁₀(x + 1). Means were separated by Fisher’s t-test using LSD α = 0.05.

In the first M. xenoplax assay, both rates of spirotetramat suppressed mobility compared to the untreated control after 24, 48, and 72 hr of exposure. Fluensulfone significantly decreased M. xenoplax mobility to a lower level than both rates of spirotetramat and the untreated control after 24, 48, and 72 hr of exposure as well (Table 1). Similar results were observed in the second M. xenoplax in vitro assay, with both rates of spirotetramat and fluensulfone significantly suppressing nematode mobility compared to the control after 24, 48, and 72 hr of exposure. The higher rate of spirotetramat provided greater suppression than the lower rate at 24 hr, but the effect was lost at 48 and 72 hr (Table 1).

For the first and second M. incognita assays, neither rate of spirotetramat was found to be effective in significantly reducing J2 mobility at the 24, 48, and 72 hr sampling times compared to the control. In contrast, fluensulfone significantly reduced J2 mobility on all three sampling times compared to both spirotetramat treatments and the untreated control (Table 1).

For the M. incognita trials, a single application of spirotetramat at the lower rate reduced the J2 population and eggs of M. incognita J2 in soil and from roots, respectively, compared to the untreated control at 40 DAI, but not at 70 DAI. In contrast, when spirotetramat was applied once at the higher rate, it showed no detrimental effects on nematode population densities at either 40 or 70 DAI (Table 2). The adjuvant control was analogous to the untreated control with no distinction between M. incognita populations sampled (Table 2). Fluensulfone was found effective in lowering the M. incognita population compared to the untreated control at 40 DAI. However, like spirotetramat at the lower rate, the nematode suppressive effect of fluensulfone was lost at 70 DAI (Table 3). No significant differences were observed between treatments in Rf levels of M. incognita, calculated at the completion of the trials (Tables 2 and 3).

For the M. xenoplax trials, both rates of spirotetramat, applied once, were ineffective in suppressing M. xenoplax population densities compared to the untreated control at 30, 60, and 90 DAI. In contrast, fluensulfone was effective in suppressing the M. xenoplax population as compared to the untreated control at 30, 60, and 90 DAI. Fluensulfone also had a significantly lower Rf levels of M. xenoplax compared to the untreated control (Table 4), however, no significant differences in Rf levels of M. xenoplax were observed for the spirotetramat treatments compared to the water control. No significant differences in plant growth parameters were observed among treatments for both M. incognita and M. xenoplax trials.

When spirotetramat was applied twice at the lower rate, M. incognita population densities were significantly reduced compared to the untreated control at 40 DAI (Table 5). However, as with the spirotetramat single application trials, this suppression was not detected in the second sampling at 70 DAI, and 30 d after the second application, as compared to the untreated control. Similarly, the dual application of spirotetramat at the higher rate significantly reduced the M. incognita population densities compared to the untreated control at 40 DAI but not at 70 DAI (Table 5). No differences were observed in Rf levels, calculated at the completion of the trials, of M. incognita among each treatment (Table 5).

The dual application of spirotetramat at both rates showed no difference in M. xenoplax population densities or Rf levels when evaluated against the untreated control (data not shown). No differences in plant growth parameters were observed among treatments for both M. incognita and M. xenoplax trials.