Efficacy of succinate dehydrogenase inhibitor nematicides fluopyram and cyclobutrifluram against Hoplolaimus galeatus in Golf Turf

1.1. Microwell mortality bioassay

The effects of fluopyram and cyclobutrifluram on lance nematode were evaluated using mortality bioassays in 5 mL Falcon® 12-well clear multi-well plates (Corning, Rochester, NY) and were repeated non-concurrently in 2025. Nematodes were exposed to cyclobutrifluram and fluopyram at concentrations of 0, 10, and 50 ppm a.i.; for fluopyram, the commercial nematicide formulation Indemnify was used, and for cyclobutrifluram, the commercial nematicide formulation Trefinti was used.

Lance nematode inocula were collected from cultures maintained on ‘Latitude 36’ bermudagrass (Cynodon dactylon × C. transvaalensis) grown in 1-liter clay pots filled with golf course top-dressing sand under greenhouse conditions. Nematode inocula were extracted using a modified Baermann method, as described by McSorley and Frederick (1991), to ensure healthy nematodes were used. Nematode solutions were prepared to contain at least 25 nematodes/mL, and 1 mL of nematode solution was pipetted into each of thirty microwells. Fluopyram and cyclobutrifluram solutions were prepared at three concentrations (0 ppm, 20 ppm, and 100 ppm a.i.), double the desired final concentrations. One milliliter of nematicide solution was then pipetted into the microwells containing the nematode solution to achieve final exposure concentrations of 0, 10, and 50 ppm a.i.

Nematode activity was assessed after 24 and 72 hr of exposure using an Olympus CK30 microscope. Following the addition of 70 µL of 1 N NaOH (Fisher Chemical, Pittsburgh, PA), as described by Chen and Dickson (2000), the numbers of active (moving) and inactive (non-moving) nematodes were recorded. This stimulant induced a response in living nematodes, causing them to twist their bodies, while dead nematodes remained rigid and straight. Following the application of the stimulant, a thirty-second interval was allowed for the nematodes to react before counting, ensuring sufficient time to elicit a response but not long enough for the NaOH to kill the nematodes. The percent mortality was calculated by dividing the number of active nematodes by the total number of nematodes and multiplying by 100. Because NaOH is toxic to nematodes, separate untreated control treatments were used for each exposure time, so there were a total of six treatments (three concentrations and two observation times). The experiment was set up in a randomized complete block design with five replications for each treatment.

1.2. Greenhouse experiment

A greenhouse trial compared the efficacy of cyclobutrifluram to fluopyram using field-labeled application rates for the management of lance nematode on bermudagrass turf. The experiment was originally conducted from June to October 2021 and repeated during the same period in 2025. The experiment was conducted in “pipe pots” constructed of PVC pipes measuring 5-cm diameter by 20 cm deep. The bottom of each pot was covered with screen and landscape fabric to allow drainage, and each was filled with 440 cm³ of sterilized sand. Each pot was sprigged with Latitude 36 bermudagrass and allowed to establish root systems for 30 days before inoculation. Lance nematodes were extracted from Latitude 36 bermudagrass as described above using a modified Baermann funnel technique (McSorley and Frederick, 1991) to ensure healthy inoculum was used. Inoculum solution was concentrated to approximately 400 nematodes per mL. Following root establishment, each pot was inoculated with 400 mixed life stages of lance nematodes by pipetting 1 mL of nematode solution into a 2-cm-deep hole made in the sand of each pot that was closed after inoculation. 2 weeks after inoculation, treatment applications commenced.

The experiment was arranged in a randomized complete block design with 5 blocks of 5 treatments. The treatments included fluopyram at 500 g a.i./ha, cyclobutrifluram applied at 125, 250, and 500 g a.i./ha, and untreated control. Treatments were applied 4 times at 4-week intervals as described by the curative spot-treatment rates on the Indemnify (fluopyram) and Trefinti (cyclobutrifluram) labels. Treatment solutions were prepared to deliver the desired amount of nematicide in 15 mL of solution, while untreated control pots received 15 mL of water. Treatments in the 2021 greenhouse trial were applied using the same spray equipment described for the field trial, whereas treatments in the repeated 2025 greenhouse trial were applied as a soil drench. All pots were maintained under consistent greenhouse conditions (18–33°C), received biweekly applications of Miracle-Gro® (Scotts Miracle-Gro, Marysville, OH), and were routinely trimmed to promote uniform turf growth.

4 weeks after the final treatment application, all pots were taken down for nematode extraction. Lance nematodes were extracted from the soil using centrifugal flotation (Jenkins, 1964). Lance nematode per 100 cm³ soil were compared among treatments to determine the efficacy of each treatment in suppressing nematode populations.

1.3. Field trials

Turfgrass field trials were conducted in 2021 and 2022 to evaluate the cyclobutrifluram nematicide Trefinti as a treatment for management of lance nematodes on golf greens compared to the standard fluopyram nematicide Indemnify. The 2021 field trial was conducted on ‘Sunday’ bermudagrass infested with H. galeatus, and the 2022 trial on ‘TifEagle’ bermudagrass infested with H. galeatus along with the grass root-knot nematode Meloidogyne graminis. Both sites were located at the University of Florida’s Plant Science Unit in Citra, Florida, and were maintained as golf greens. Plots were 1.5 m² with 0.62 m untreated borders between adjacent plots.

Treatments in both trials were (i) untreated control, (ii) four monthly applications of 500 g fluopyram/ha as per the curative spot-treatment rate for turfgrass use allowed by the Indemnify label, (iii) four monthly applications of cyclobutrifluram at 250 g a.i./ha, and (iv) four monthly applications of cyclobutrifluram at 500 g a.i./ha as allowed by the spot-treatment rate on the Trefinti label. Applications of all treatments were made using a CO²-powered backpack sprayer with TJ-08 nozzles delivering 2445 l/ha of solution. All plots, including the untreated controls, were treated with a modified alkylated polyol soil penetrant (Revolution®, Aquatrols, Paulsboro, NJ) at each treatment application date, a standard golf management practice to facilitate even movement of water into the soil profile. After each application, treated and untreated plots were irrigated with 0.32 cm of irrigation to move the nematicides off of the turf surface and into the soil. The experiments used a randomized block design with five replications, with blocking based on the initial H. galeatus population density in individual plots.

Measurements evaluated were (i) population density of H. galeatus in soil, (ii) root health, (iii) turf quality, and (iv) normalized difference vegetative index (NDVI). Nematode samples consisted of nine 1.9-cm-diam. plugs taken 9- to 10-cm-deep from each plot. Nematodes were extracted from a 100 cm³ soil subsample using the centrifugal-flotation method (Jenkins, 1964). Nematode samples were collected before the initial treatment application (Pi), 2 weeks after the second treatment application (Pm), and 2 weeks after the final treatment application (Pf). Root samples were two 3.8-diam. cores taken 15.25 cm deep from each plot (175 cm³ of soil from each core). Soil was washed from the roots, inspected visually, and rated for root depth, root density, and root quality. Each root parameter used a 1-5 scale, with 1 being the worst and 5 being the best. The three individual root measurements were used to calculate percentage overall root health using the formula [((length × 20) + (density × 20) + (quality × 20))/3] × 100. Root samples were collected before the initial treatment applications (initial), 2 weeks after the second treatment application (mid), and 2 weeks after the final treatment application (final). Turf quality is a subjective 1-9 scale widely used by the turfgrass industry, with 9 being the best quality and 6 the minimum acceptable quality color (Krans and Morris, 2007). NDVI was measured using a handheld GreenSeeker (Trimble, Westminster CO). Turf quality and NDVI were measured at approximately 2-week intervals starting the day of the initial treatment application and ending 2 weeks after the final treatment application.

1.4. Statistical analysis

Statistical analyses for microwell and greenhouse experiments were conducted using R version 4.5.1. All microwell mortality bioassays had the response variable represented as counts of dead and alive nematodes. A generalized linear model (GLM) with a logit link was fitted using the glm () function. Variables such as nematicide, concentration, and time were converted to factors when needed due to their categorical nature. For greenhouse trials, a generalized linear mixed model (GLMM) was fitted using the glmmTMB package in R (version 4.5.1; Brooks et al., 2017) to analyze the effect of treatment on final nematode counts.

The model included treatment as a fixed effect and block as a random effect to account for variability among experimental blocks. Data were modeled using a negative binomial distribution (nbinom2) with a log-link function. Model assumptions were assessed with DHARMa residual diagnostics, including QQ plots (KS test) and dispersion tests. A Type II Wald Chi-square analysis of deviance was used to test for the significance of predictors (exposure time, concentration, and nematicide) on nematode mortality in the in vitro assay and on final nematode counts in the greenhouse experiments. Data from the two greenhouse trials were tested for heterogeneity and were not different from each other. Therefore, data from both trials were combined into a single dataset for mean separation according to Tukey’s HSD test (P ≤ 0.05).

For the field experiments, data were subjected to analysis of variance, and treatment means were separated according to Tukey’s HSD test (P ≤ 0.05) in SAS 9.4 (SAS Institute, Cary NC).

2.1. In vitro mortality bioassay

A GLM model with main effects terms was tested, and concentration 0 was removed to prevent complete separation in the model, which manifested as convergence warnings. Diagnostic plots showed no evidence of overdispersion (first experiment P = 0.688, repeated experiment P = 0.632), outliers (first experiment P = 1.0, repeated experiment P = 1.0), and non-uniform residual patterns (KS test for first experiment P = 0.894, repeated experiment P = 0.905).

Tests of significance revealed that nematicide had the greatest effect on mortality in both experiments (P < 0.001), followed by exposure time (P < 0.001) and concentration (P < 0.01). No significant two-way or three-way interactions were observed in either experiment. Estimated marginal means indicated that cyclobutrifluram consistently caused higher mortality in lance nematodes than fluopyram. In the first experiment, at 24 hours, exposure to 10 ppm resulted in about 11% mortality with cyclobutrifluram compared to less than 1% with fluopyram. At 50 ppm, cyclobutrifluram caused 20% mortality, whereas fluopyram resulted in only 1% mortality (Figure 1a). At 72 hours, the difference became more pronounced. Cyclobutrifluram caused 54% mortality at 10 ppm and nearly 69% at 50 ppm, while fluopyram produced only 4% and 8% mortality at 10 ppm and 50 ppm, respectively (Figure 1a).

Figure 1

The repeated experiment showed a similar pattern. At 24 hours, exposure to 10 ppm resulted in approximately 26% mortality with cyclobutrifluram compared to 3% with fluopyram. At 50 ppm, cyclobutrifluram caused 38% mortality, whereas fluopyram resulted in only 6% (Figure 1b). By 72 hours, the difference was more pronounced. Cyclobutrifluram caused 44% mortality at 10 ppm and nearly 58% at 50 ppm, while fluopyram produced only 8 and 13% mortality at 10 ppm and 50 ppm, respectively (Figure 1b).

2.2. Greenhouse experiment

DHARMa residual diagnostic plot showed that the model fit the data well; hence, no major assumption was violated (KS test P = 0.76; dispersion P = 0.43; outlier P = 1). Final nematode counts differed significantly among treatments (Type II Wald χ ² = 107.81, P < 0.001). Population densities of H. galeatus in the fluopyram treatment (500 g/ha) and the lowest rate of cyclobutrifluram (125 g/ha) did not differ from the untreated control. However, significantly (P ≤ 0.05) lower population densities were observed at the higher cyclobutrifluram rates (250 and 500 g/ha; Figure 2).

Figure 2

2.3. Field trials

Neither fluopyram nor cyclobutrifluram reduced population densities of H. galeatus in either field trial ((P > 0.05); Table 1). However, in 2022, the fluopyram treatment had greater H. galeatus Pf than the untreated control. Both SDHI nematicides had positive impacts on turf health in both trials. Percent root health was improved compared to the untreated (P ≤ 0.05) by the highest rate of cyclobutrifluram in 2021, and by all SDHI nematicide treatments in 2022 (Table 2). NDVI was improved (P ≤ 0.05) compared to the untreated by fluopyram and the lower rate of cyclobutrifluram on 1 date and by the higher rate of cyclobutrifluram on 4 dates in 2021 (Table 3). In 2022, fluopyram improved NDVI compared to the untreated on 4 dates, and compared to both cyclobutrifluram rates on 7 dates (Table 3). In 2021, turf quality was improved compared to the untreated only by the higher rate of cyclobutrifluram (Table 4). In 2022, fluopyram improved turf quality on 5 dates and the cyclobutrifluram treatments on 6 dates (Table 4).