Evaluation of fluopyram for the control of Ditylenchus dipsaci in sugar beet

The Ditylenchus dipsaci population used in all experiments was extracted using Oostenbrink dishes (European and Mediterranean Plant Protection Organization, 2013) from infested sugar beets collected in the Seeland region in Switzerland. Using a fine needle, 2,000 fourth-stage juveniles (J4) and adult nematode stages were hand-picked and transferred to sterile centrifuge tubes. After suspending the nematodes in an antibiotic solution containing 0.1% streptomycin sulfate (w/v) and 0.1% amphotericin-B (w/v), nematodes were inoculated on carrot cylinders (2.5 × 5 cm) and incubated at 20°C in the dark for 50 days (Kühnhold et al., 2006). After extraction from the carrot cylinders, nematodes were stored at 6 to 8°C in the dark up to 20 days until further use.

The nematode population for the inoculation of in vitro, growth chamber, and glasshouse experiments consisted of J2, J3, J4 juveniles, and adults, with a predominance of adults and J4 juveniles.

A suspension concentrate formulation, containing fluopyram at 500 g a.i./L (MOON PRIVILEGE), was supplied by Bayer CropSciences Schweiz AG (Zollikofen, Switzerland). This formulation was already registered for use on tree crops, floriculture crops, and vegetable crops in Switzerland (FOAG, 2019).

To evaluate the direct effect of fluopyram on Ditylenchus dipsaci, nematodes were incubated for 96 h at 22°C in water containing of 50, 30, 10, 5, 3, 2, 1, and 0 µg/ml of fluopyram. The experiment was conducted in six-well polystyrene plates with each well receiving 2.5 ml of a 2 × concentrated test solution and 2.5 ml of distilled water containing 1,000 D. dipsaci (Dutta et al., 2012). Each treatment was replicated three times, and the experiment was conducted four times. After exposure for 24, 48, 72, and 96 h, motile and immotile nematodes were counted using an optical microscope at 100 × magnification. Motile D. dipsaci were characterized by their serpentine shape, while immotile nematodes were straight with no visible movement. Additionally, after 72 h exposure to fluopyram, nematodes were washed with water over a 20 µm sieve, and after further 24 h incubation in water, the number of motile/immotile D. dipsaci was recorded to determine their recovery in motility (Faske and Hurd, 2015).

The impact of fluopyram on the penetration rate of D. dipsaci into sugar beet was investigated in a growth chamber. The treatments were two application methods, either at planting or post-planting (cotyledons horizontally unfolded, BBCH 10), and five concentrations (10, 5, 1, 0.5, and 0.0 µg/ml). The susceptible cultivar Dorena (KWS Saat SE, Germany) was sown into 3-cm-diameter plastic pots filled with 80 ml of soil. The soil was a loam that was steamed, passed through a 2 mm sieve, and mixed with sand (1/1,v/v). Each plastic pot contained one sugar beet seed sown 2 cm deep. Fluopyram was applied to the soil surface of the pots receiving the at-planting application. At 7 days post-planting (dpp), 1 ml fluopyram solution was applied 3 h before nematode inoculation to the sugar beet leaves and soil surface of the pots receiving the post-plant application. Because BBCH 11 (first pair of leaves visible, not yet unfolded) seedlings allow optimal staining of the nematodes in the plant, fluopyram was applied at BBCH 10 instead of BBCH 12 (two leaves unfolded), as done in the glasshouse and field experiments. At BBCH 10, approximately 200 nematodes per seedling were inoculated in 10 µl of 1% carboxymethylcellulose (w/v), which increases the adhesion of nematodes to the leaf-axils. The inoculation droplet was placed between the first pair of true leaves of the seedlings (Kühnhold et al., 2006). At 7 days post-inoculation (dpi), the seedlings were removed from the pots, gently washed, transferred to a 100-ml plastic beaker containing a 0.1% acid fuchsin/lactic acid solution and boiled twice in a microwave oven for 1 min. Stained seedlings were then rinsed to remove the staining solution. The total number of nematodes per seedling was counted using a stereomicroscope at 10 × magnification after maceration in 30 ml tap water using an Ultra Turrax blender (T25 basic, IKA Labortechnik, Germany). The experimental design was a factorial design arranged in randomized complete blocks, each treatment was replicated ten times, and the experiment was conducted twice. The experiment was conducted at 15°C with a 16-h photoperiod and covered with a transparent plastic foil to ensure 95% relative humidity.

The impact of fluopyram on the reproduction rate of D. dipsaci in sugar beet was investigated in a glasshouse. The treatments were two application methods, either at planting or post-planting (two leaves unfolded, BBCH 12), and four concentrations (5.0, 1.0, 0.5, and 0.0 µg/ml). The susceptible cultivar Dorena (KWS Saat SE, Germany) was sown in 1 L plastic pots filled with 800 ml soil. The soil was a loam that was steamed, passed through a 2 mm sieve, and mixed with sand (1/3, v/v). The aim to obtain well-developed roots motivated the higher rate of sand in the soil mixture compared to the experiment conducted in the growth chamber. Each plastic pot contained one sugar beet seed sown 2 cm deep. Fluopyram was applied to the soil surface of the pots receiving the at-planting application. At BBCH 10, approximately 200 nematodes per seedling were inoculated in 10 µl of 1% carboxymethylcellulose (w/v). The inoculation droplet was placed between the first pair of true leaves of the seedlings (Kühnhold et al., 2006). At BBCH 12, 1 ml fluopyram solution was applied to the sugar beet leaves and soil surface of the pots receiving the post-plant application. The experiment was started in a growth chamber at 15°C with a 16-h photoperiod and covered with a transparent plastic foil to ensure 95% relative humidity to ensure nematode infection. At 7 dpi, the plants were transferred for higher temperatures to a glasshouse at 20 ± 1°C with an 18-h photoperiod. At 50 dpi, the plants were removed from pots, washed, weighed, and cut in 0.5 cm pieces. Nematodes were extracted from the sliced plant material using Oostenbrinck dishes (European and Mediterranean Plant Protection Organization, 2013). The number of D. dipsaci per sugar beet plant was determined by counting 3 × 1 ml aliquots from 200 ml total volume using a stereomicroscope. The experimental design was a factorial design arranged in randomized complete blocks, each treatment was replicated 10 times, and the experiment was conducted twice.

The field experiment, conducted in 2016, was set up as randomized complete block designs with four treatments: fluopyram applied in-furrow at planting at 250 g a.i./ha, in-band at BBCH 12 at either 125 or 250 g a.i./ha, and an untreated control. According to the Swiss standard, fluopyram was applied with 300 L/ha. The timing of the post-plant application (BBCH 12) was 28 dpp. The two widely used cultivars Samuela (KWS Saat SE, Germany) and Hannibal (Strube GmbH, Germany) were planted at two locations in Kappelen and Bargen, respectively. Both cultivars are similar in their susceptibility to D. dipsaci compared to the cultivar Dorena. Sugar-beet plant samples (10 plants/plot and sampling date) were randomly collected 27 and 62 dpp. Plants collected at 27 dpp were analyzed by staining with 0.1% acid fuchsin (see above), whereas the Oostenbrink dish technique was used to determine nematode numbers in plants collected 62 dpp.

Population densities of D. dipsaci (Pf/Pi) in these field sites were investigated by taking random soil samples seven days before planting and at harvest. In all, 10 subsamples/plot of harvested sugar beets were randomly collected, and the number of D. dipsaci in 100 g sugar beet hypocotyl tissue was determined by extraction using Oostenbrink dishes.

The effect of fluopyram on root-rot symptom development on sugar beets was determined by de-heading 20 beets/plot down to the crown surface and slicing them vertically until the bottom of the root. The percentage of root volume showing rot symptoms was determined using an index with 0 = no rot visible, 1 = ≤ 10% of rotten root, 2 = 11 to 33% of rotten root, 3 = 34 to 66% of rotten root, and 4 = 67 to 100% of rotten root.

In 2017, field experiments were repeated, but warm and dry conditions in spring were unfavorable to D. dipsaci development. Consequently, nematode populations were low and did not lead to any damage symptoms. Therefore, we did not use the data for the year 2017.

Data for nematode motility, infectivity (D. dipsaci/seedling), reproduction under glasshouse conditions (D. dipsaci/plant 50 dpi), soil populations (Pf/(Pi + 1)), reproduction in the field (D. dipsaci/100 g beet crown tissue), and root rot symptoms were tested for normality (Shapiro-Wilk normality test) and homogeneity of variances (Bartlette and Levene Test). Data obtained in the growth chamber and in the glasshouse were subjected to a two-way analysis of variance. The data from repeated experiments were pooled after confirming the absence of variation between the experiment replications. Data from field experiments were analyzed individually for each site and subjected to a Friedman test. Tukey multiple comparisons of means were performed as a post hoc test (Lenth, 2016). Statistical analysis, as well as graphs, were performed with R.

A logistic dose-response curve was used in the motility bioassay to describe the relationship between inactive D. dipsaci after 24, 48, 72, 96 h, and 72+rinse (y) and the concentration (x) of the active ingredient fluopyram (Hutchinson et al., 1999; Kiewnick and Sikora, 2006). The logistic curve can be expressed as follows: (1) y = c + [ d − c 1 + ( x EC 50 ) ] b , (1)where c and d are the lower and upper limits of the curve, respectively. The EC₅₀ is the concentration reducing the response (y) 50% between d and c, i.e., the point of inflection. The parameter b is the proportional slope of the curve around the point of inflection (Ritz and Streibig, 2005). The four-parameter logistic model (Equation (1)) was reduced to a three-parameter model: (2) y = d / [ 1 + ( x E C 20 ) b ] \text{(2)} & y=d/[1+{(\frac{x}{E{C}_{20}})}^{b}]\end{array}\end{eqnarray}?> (2)where d is the upper limit of response (upper asymptote) (Ritz and Streibig, 2005; Kiewnick and Sikora, 2006). Curve fitting was performed by non-linear regression using the extension package drc in R (Ritz et al., 2015).

When exposed to fluopyram, the percentage of immotile D. dipsaci increased with exposure time and concentration (Fig. 1). After 24 h, only 58% of D. dipsaci were immotile at the maximum concentration of 50.0 µg/ml fluopyram, resulting in an EC₅₀ value of 46.73 µg/ml (Table 1). After exposure for 48, 72, and 96 h to fluopyram, EC₅₀ values were 9.32, 3.98, and 2.02 µg/ml, respectively. After 72 h, only concentrations of 30 and 50 µg/ml fluopyram resulted in 100% immotile D. dipsaci. However, after washing D. dipsaci free of fluopyram and further incubation in water for 24 h, no increase in motility was observed. The EC₅₀ value for the 72 h + rinse was 3.00 µg/ml (Table 1). The percentage of immotile D. dipsaci continued to grow with the exposure time for each fluopyram concentration, except for 0 µg/ml. After a further 24 h incubation in water (72 h + rinse), the percentage of immotile D. dipsaci continued to grow compared to 72 h exposure to fluopyram. At 3.00 µg/ml a.i., the percentage of immotile nematodes was 40, 76, and 58% after exposure for 72 h, 96 h, and 72 h + rinse, respectively.

Figure 1:

In the growth chamber experiment, there was a significant effect of the fluopyram concentration (p < 0.001), but no effect of the application time (p = 0.597) and no interaction between fluopyram concentration and application time (p = 0.224) on the number of penetrated D. dipsaci in sugar beet seedlings at 7 dpi (Fig. 2). When fluopyram was applied to the leaves and soil surface at BBCH 10 with 10.0 µg/ml or 5.0 µg/ml per plant, the number of D. dipsaci penetrating sugar beet seedlings was significantly lower compared to a concentration of 0.5 µg/ml (p < 0.001). Fluopyram application did not affect the rate of D. dipsaci penetration compared to the untreated plants (water control).

Figure 2:

In the glasshouse experiment, where the reproduction of D. dipsaci in sugar beet plants was determined at 50 dpi, population densities of 12,344 to 27,681 nematodes per plant were found in the untreated control plants (Table 2). Fluopyram treatment did not affect the final number of D. dipsaci/plant. No significant effect of the fluopyram concentration and application time were found (p > 0.05). Fluopyram at rates of 0.5, 1.0, or 5.0 µg a.i./plant did not reduce D. dipsaci densities in plants, neither when applied at planting nor at BBCH 12 (Table 2).

At 27 dpp, fluopyram applied at planting at 250 g a.i./ha showed a significant reduction in the number of D. dipsaci per seedling compared to the non-treated control at the Kappelen site (Table 3). Similar numbers of D. dipsaci per plant were found in all untreated plots (untreated control and plots designated for post-planting application at BBCH 12), indicating a homogenous infestation throughout the field. At 62 dpp, all fluopyram applications in Kappelen resulted in a significantly lower number of D. dipsaci/100 g plant (Fig. 3). At the second site, in Bargen, sugar beet plants from untreated plots tended to show higher numbers of D. dipsaci per plant at 62 dpp compared to fluopyram treated plants, but differences were not significant (Fig. 3). At harvest at both sites, fluopyram was not effective in reducing the final population densities of D. dipsaci in the soil and in the plant at any rate or application time. All treatments showed a Pf/Pi > 2 of D. dipsaci in soil. The root-rot index, determined at harvest, was high in Kappelen with 3.4 for untreated plots. Only the high application rate of fluopyram (250 g a.i./ha), either at planting or post-planting (BBCH 12), reduced the root-rot index significantly (Fig. 4). In Kappelen, crown rot symptoms caused by Rhizoctonia solani (AG-2IIIB) were observed in all four treatments, with a trend to higher symptoms expressions on untreated plants.

Figure 3:

Figure 4: