Reduced Sensitivity to Fluopyram in Meloidogyne graminis following Long-Term Exposure in Golf Turf

Some soil and nematodes used in some of these experiments originated from replicated small plots at a single location, and others were from commercial turfgrass sites at differing locations. Soil collected was used for degradation experiments, and nematodes collected were used for resistance experiments.

The small plots were located on bermudagrass (Cyndon dactylon × C. transvaalensis) ‘Jones Dwarf’ at the University of Florida Plant Science Unit in Citra, FL. The plots were 1.5 m² with 0.6 m untreated borders between them. The plots were arranged in a randomized block design with 5 replications of 3 fluopyram histories. The fluopyram histories included plots that never received fluopyram (treatment U), plots that received fluopyram four times annually from 2018 to 2022 (treatment A), and plots receiving fluopyram four times during the single year 2021 to 2022 (treatment B). Fluopyram was applied using the commercial Indemnify formulation, representing the maximum labeled rate for turfgrass use (Bayer Environmental Science, 2016), with two fall applications and two spring applications each year of 500 g fluopyram/ha. All applications were made with a CO²-powered backpack sprayer (Weed Systems, Hawthorne, FL) with an output of 1.2 L/ha using TJ-08 nozzles. After each application the plots were irrigated with 0.65 cm of water to move the fluopyram into the soil profile. Mitochondrial haplotyping (Baidoo et al., 2016) of the root-knot nematode population in the small plot area was conducted prior to the study initiation, and M. graminis was the only Meloidogyne sp. detected in the field.

The commercial field sites included those with a prolonged history of fluopyram use and those with no history of fluopyram use. Most golf course greens in Florida where M. graminis are considered a problem are treated with fluopyram for nematode management. Commercial site soil was thus collected from golf greens for degradation experiments. The sampled greens that had been treated regularly with fluopyram all had experienced chronic problems associated with M. graminis, and the greens that had not been treated with fluopyram were not known to experience damage from it.

Large populations of M. graminis were needed for resistance experiments. Since the golf greens with no fluopyram history used for collecting soil used in degradation experiments contained few M. graminis, different turfgrass field locations were sampled to collect M. graminis for resistance experiments. Due to the high cost of Indemnify, it is seldom used on golf course fairways, athletic fields, or sod farms. Therefore, the fluopyram-exposed nematode populations were M. graminis collected from golf greens with prolonged fluopyram use, and non-exposed populations were collected from fairways, athletic fields, and sod farms.

If enhanced degradation of fluopyram occurs, fluopyram applied to soil originating from locations where it had never been used should be more effective than fluopyram applied to soil originating from locations where fluopyram has been continually used. To test for enhanced degradation of fluopyram, we performed bioassays using either Meloidogyne incognita or M. enterolobii as the test nematode and Lycopersicon lycopersicum ‘Rutgers’ tomato as the test plant.

The first enhanced degradation experiment used soil collected from the small plots. Twelve 5-cm-diam. ×15.2-cm deep cores were collected from each of the small plots using an AMS sediment sampler (AMS, American Falls, ID). The samples were collected from the center of the 1.5 m² plots with care taken to avoid the edge of the plots. Plastic liners were inserted into the steel core sampler to hold the intact sample (Fig. 1) and the sampler was pounded into the soil until it was flush with the turf surface. Cores were collected July 2021 for the first repetition and October 2021 for the second repetition. To keep the cores intact and minimize impacts to the microbial community, the liners containing the intact cores were placed directly into custom-made 5-cm-diam. × 15.2-cm deep PVC tubes with a mesh bottom (Fig. 2), and maintained in greenhouses on the University of Florida campus in Gainesville, FL.

Figure 1:

Figure 2:

Fluopyram solutions were prepared using the commercial Indemnify formulation. After turf cores were placed in the greenhouse, fluopyram treatments were applied by pipetting 6.5 ml of fluopyram solution onto the turfgrass surface of each core. Fluopyram rates were 0 g fluopyram/ha (water control), 250 g fluopyram/ha (1/2 the maximum labeled single-dose rate), 500 g fluopyram (maximum labeled single-dose rate), or 1000 g fluopyram/ha (twice the maximum labeled single-dose rate). Cores were taken down for bioassay at three intervals, 30, 60, and 180 days after application (DAA). Irrigation was applied as needed using an overhead sprinkler system delivering 0.5 cm water with each irrigation event. Nutrients, in the form of Miracle-Gro Osmocote 14-14-14 slow release (Scotts Miracle-Gro Company, Marysville, OH) and Miracle-Gro All Purpose Plant Food 24-8-16 quick release (Scotts Miracle-Gro Company, Marysville, OH), were added as needed.

The intent of the bioassay was to evaluate the efficacy of fluopyram remaining in the soil. To account for any effects the long-term fluopyram may have had on the nematodes in the system, i.e. on their nematicide resistance or tolerance, new nematodes that had never been exposed to fluopyram were used as the bioassay nematodes, and a host plant was selected that was not a host to the root-knot nematodes parasitizing the bermudagrass. Because M. graminis was the only Meloidogyne sp. present in the plot area, M. incognita was used as the bioassay nematode, and ‘Rutgers’ tomato, a non-host to M. graminis, was the bioassay host plant.

Soil from each individual core was separated from the turfgrass and thatch, which were then discarded. The soil was placed into a 12.7-cm-diam. clay pot. Eggs of M. incognita for bioassay inoculum were collected from a greenhouse culture maintained on tomato, as described by Hussey (1973). A ‘Rutgers’ tomato seedling was transplanted into each pot, and a solution containing 500 M. incognita eggs and J2 were pipetted into two 5-cm deep holes in the soil on either side of the transplant and then covered. After inoculation, the pots were arranged in the greenhouse using a randomized complete block design with five replications and left for four weeks. Irrigation was closely monitored to not exceed 0.32 cm of irrigation per day to prevent overwatering.

After four weeks, the pots were taken down, the roots rinsed, and the number of galls per root system were recorded. If enhanced degradation had occurred, more galls would be detected on fluopyram-treated tomato plants growing in soil previously exposed to fluopyram than in those growing in soil with no previous fluopyram exposure.

An additional experiment was conducted using soil collected from commercial turf fields to test for enhanced degradation of fluopyram. In late September through early October 2021, 11 turf fields located in different parts of Florida were sampled; six of these locations reported having used fluopyram multiple times per year for multiple years, and five reported never having used fluopyram. The experimental procedures used were the same as described for the previous experiment except that the bioassay nematode used for this experiment was M. enterolobii.

Statistical analyses from enhanced degradation experiments were conducted using R version 4.1.2 (R Core Team, 2021, Ames, Iowa). A generalized linear mixed-effects model was constructed, and type 2 analysis of variance with a Wald chi-square test were used to confirm statistical significance. The number of galls were designated as the response variables. Fluopyram rate and fluopyram history were the fixed-effect predictor variables. The block locations were treated as a random effect. An additional random effect was created and labeled as “observation.” This random effect takes into account the random effect variation between each observation. The family type for the model was negative binomial. Pairwise comparisons were used to detect differences in gall numbers between fluopyram history as well as an interaction between fluopyram history and fluopyram rate. The emmeans function from the emmeans package (Lenth, 2022) was used with a confidence interval of 95% to determine differences between fluopyram history treatments and differences for treatments across the drench rates. This function provided the estimated marginal mean, upper confidence interval, lower confidence interval, degrees of freedom, and the standard error. The r-squared GLMM function from the MuMIn package was used to calculate the delta-conditional r². The conditional r² accounts for the combined variance of fixed and random effects.

Testing for heterogeneity was conducted using a linear model with repetition as one of the predictor variables. The repetitions were heterogeneous (P < 0.0001) and thus were analyzed separately. Analysis of variance was used on the model to determine if the repetition was significant (P ≤ 0.05). Weak statistical significance was noted at 0.1 ≥ P > 0.05 and statistical difference at P ≤ 0.05. Figures were constructed with the use of the ggplot and tidyverse packages (Wickham, 2016; Wickham et al., 2019). Figures used the estimated marginal mean values from the emmeans function as their data points. The maximum error for each point was the estimated marginal mean value plus standard error. The minimum error for the point was the estimated marginal mean value minus the standard error.

If resistance is the cause of fluopyram failure, nematodes collected from sites where fluopyram had often been applied should exhibit less sensitivity to fluopyram than nematodes collected from sites where fluopyram had never been used. A series of experiments compared the impacts of fluopyram exposure on populations of M. graminis that had either been exposed to field applications repeatedly for multiple years or had never been exposed to fluopyram.

In the first experiment, M. graminis was collected from the small plots with three nematicide history treatments; no fluopyram history (treatment U), a history of fluopyram applied four times annually for 4 years (treatment A), and a history of fluopyram applied four times during a single year (treatment B), with five replications of each nematicide history treatment.

Fifteen 3.8-cm-diam. × 10 cm-deep turf plugs were collected from each plot twice (January 3 2022 and April 9 2022) for independent repetitions. The soil was washed from the roots and the turf plugs, and adhering roots were incubated in a mist chamber for 72 hours for extraction of M. graminis J2 (Crow et al., 2020). After nematode extraction, 1 ml of water containing M. graminis was pipetted into wells of a 24-well cell culture plate (Corning Incorporated, Maine), followed by 1 ml of fluopyram solution. During the first repetition, the M. graminis were standardized so that each well contained ~20 J2/well. However, for the second repetition, the nematode numbers were variable, ranging from 20 to 300 J2/well.

Fluopyram solutions were prepared using the commercial Indemnify formulation containing 2× the desired final desired exposure concentrations, so after being added to the M. graminis solutions, the final exposure concentrations were 8 ppm or 16 ppm. These concentrations were selected because the estimated maximum concentration of fluopyram a nematode would be exposed to is 16 ppm, assuming the labeled rate 500 g of fluopyram/ha was used followed by 3 mm of irrigation water to move it off of the turf surface. Nematode activity was monitored at 24 hours and 72 hours after addition (HAA) of the fluopyram solution. There were 5 replicates (one well for each field plot) of each treatment × concentation × exposure time combination.

Fluopyram causes Meloidogyne spp. J2 to become straight and stiff (Faske and Hurd, 2015). Thirty-two μl of 1 N sodium hydroxide (Fisher Chemical, Pittsburgh, PA) were added to each well to stimulate the nematodes’ movement (Chen and Dickson, 2000). This stimulant is known to cause live nematodes to respond by contorting their bodies or by otherwise moving, while impaired nematodes remain rigid and outstretched. The nematodes were given 15 seconds to respond to the stimulant before observation was initiated. Because the stimulant kills the nematodes, separate wells were used for each of the observation times (24 and 72 HAA). These data were used to calculate the percentage of impaired nematodes.

An additional experiment, using the same procedure described above, tested fluopyram sensitivity of M. graminis from six commercial turf fields (golf courses, athletic fields, sod farms), where fluopyram had never been used, to those from nine golf course putting greens with a reported long-term use of fluopyram. The nematode samples were collected from the fields in the second week of May, 2021. In this experiment each nematode population was a single replication of a historical fluopyram treatment.

A final turf field experiment, using the same testing protocols described above, compared M. graminis populations collected from two golf course locations in the immediately preceding experiment that exhibited reduced fluopyram sensitivity, a population from a turf field with no documented fluopyram use, and a population maintained on bermudagrass in the greenhouse that had never been exposed to fluopyram. In this trial there were 3 replications of every treatment (previous fluopyram exposure and no previous fluopyram exposure).

In the resistance experiments, R version 4.1.2 was used for statistical analysis. For the small plot experiment, a test of heterogeneity was conducted using a linear model with repetition as the predictor variable. The repetitions were heterogeneous (P = 0.004) and, therefore, they were analyzed separately. A generalized linear mixed-effects model was used to evaluate the effects of HAA, exposure rate, and time on the percent impaired. The binomial distribution was used when constructing a model. The fixed-effect predictor variables were fluopyram treatment history, time of exposure, and fluopyram exposure concentration. The theoretical conditional r² was calculated using the r.squaredGLMM() function from the MuMIn package. Data was subset by the time variable at 1 HAA, 24 HAA, and 72 HAA. Data was additionally subset between the rates of 0 ppm fluopyram, 8 ppm fluopyram, and 16 ppm of fluopyram.

A new variable was made in R to account for observation-level random effects due to the initial observations being exposed to fluopyram for a shorter period then the later observations. Type 2 Wald chi-square test in an analysis of variance was used to calculate the x² and P-values. The emmeans function from the emmeans package (Lenth, 2022), was used to conduct a pairwise comparison to determine differences among treatments. P-values were considered weakly significant if 0.1 ≥ P > 0.05 and statistically significant if P ≤ 0.05. Comparisons were made among the different fluopyram history treatments and exposure concentrations, as well as the interaction between the different concentrations at each history treatment and exposure time. A 95% confidence interval was used for the pairwise analysis. The estimated marginal mean values were constructed into figures using the tidyverse and ggplot packages (Wickham, 2016; Wickham et al., 2019).

The data from the experiment on soil collected from small plots were heterogeneous among repetitions (P < 0.1) and, hence, these repetitions were analyzed separately. In the first repetition, fluopyram history had an effect on the quantity of M. incognita galls at 30, 60, and 180 DAA across fluopyram drench rates; P < 0.0001, r² = 0.61. When differences in galls occurred among fluopyram histories, treatment A had fewer galls than treatment U (Fig. 3). The fluopyram rate had no effect on the quantity of galls (Table 1). Fluopyram history had no effect on the quantity of galls across different fluopyram rates (P > 0.1, r² = 0.25), but did have an effect on the number of galls at 30 and 60 DAA (Table 1). Treatment A had fewer galls than treatment U (P = 0.047) at the highest fluopyram rate (Fig. 3) at 180 DAA. Treatment B had more galls than treatment A (Fig. 3).

Figure 3:

Figure 4:

In the second repetition, fluopyram history had no effect on the quantity of galls across drench rates (P > 0.1, r² = 0.73), but did have an effect on the number of galls at 30 and 60 DAA (Table 1). Treatment A had fewer galls than treatment U (P = 0.047) at the highest fluopyram rate (Fig. 5) at 180 DAA. Treatment B had more galls than treatment A (Fig. 3).

Figure 5:

Figure 6:

In the first experiment using soil collected from commercial turf fields, fluopyram history and fluopyram rate influenced the quantity of galls at 60 DAA, P = 0.002, P = 0.033, respectively (Table 2), but there were no effects on galls at 30 and 180 DAA (P > 0.1). At 60 DAA, there were 52% more galls on tomato growing in soil from fields with no history of fluopyram than from fields with frequent use of fluopyram (Fig. 4).

In both repetitions of the experiment using nematodes originating from small plots, fluopyram history affected the percentage of impaired M. graminis, with P = 0.003 and r² = 0.91 in repetition 1 and P = < 0.0001 and r² = 0.82 in repetition 2. When fluopyram history was significant, treatment A had the fewest impaired nematodes (Fig. 5). Differences in nematode impairment between treatments A and U were observed at 16 ppm of fluopyram for the first repetition after 24 hours of exposure, and at both 8 ppm and 16 ppm of fluopyram in the second repetition.

In both experiments using nematodes collected from turf fields, the history of fluopyram use influenced the percentage of nematodes that became impaired after exposure to fluopyram in-vitro. Nematodes from fields with no history of fluopyram had greater percent impairment than nematodes from fields where fluopyram had been used previously; in experiment 1 P < 0.0001 and r² = 0.99, and in experiment 2 P < 0.0001 and r²=0.97 (Fig. 7).

Figure 7:

In experiment 2, nematodes having no previous exposure to fluopyram had 27% greater impairment than nematodes from fields with a history of fluopyram use following 24 hours of exposure to 8 ppm of fluopyram, and after 72 hours of exposure to fluopyram at 8 and 16 ppm.