Nematicidal activity of fipronil against Pratylenchus zeae in sugarcane

Before the greenhouse and field experiments, the root-lesion nematodes present in the sugarcane field soils were confirmed to be predominantly P. zeae using the real-time PCR method (Kawanobe et al., 2015). In the soil for the greenhouse experiment, 100% (n = 8) were P. zeae and in the field experiment, 71% (n = 7) and 100% (n = 8) were P. zeae for Trials 1 and 2, respectively. The other root-lesion nematodes were confirmed to be P. coffeae using unpublished real-time PCR primers.

The fipronil products used in the experiments, Prince® in granular form: 1% active ingredient (a.i.) for the greenhouse experiment and Prince® Bait: 0.5% a.i. for the field experiment, were supplied by BASF Japan (Tokyo, Japan). Prince® Bait (0.5% a.i.) has been registered for pests including the sugarcane wireworm.

A greenhouse experiment was conducted to test nematicidal efficacy of fipronil against the root-lesion nematodes in soil and in sugarcane roots. Additionally, initial growth of sugarcane seedlings was evaluated. The experiment was conducted in a greenhouse for 7 wk on Kitadaito Island (25°56N, 131°17E), Okinawa, Japan. The soil used for the greenhouse experiment was collected in May 2016 from 0 to 30 cm depth in a sugarcane field, which was known to be infested with root-lesion nematodes. On the same day of collection, the soil (sand 0.1%, silt 27.8%, clay 72.1% with total C 16.8 mg/g, total N 2.0 mg/g, pH (H₂O) 4.7, and EC 400 µS/cm) was well mixed. Then, a 2.8 kg subsample was mixed with fipronil at 0.34 g/kg soil (Prince® granular, 0.3 kg a.i./ha; equivalent to 30 kg/ha in the planting furrow) and chemical fertilizer at 0.61 g/kg soil (49-26-50 N-P-K). The soil was put into each plastic pot (inside diam. 17 cm and 15 cm height) with a hole in the bottom. Two single bud setts of sugarcane cv. Ni29, one of the commonly grown cultivars in Okinawa, were planted in each pot, watered when necessary, and grown for 7 wk. Pots without fipronil were also prepared and used as a control. There were three replicates per treatment.

After 7 wk, culm height and the number of fully extended leaves per plant were measured and each plant was removed from the pot. Roots were carefully washed and kept at room temperature for no more than a day until nematode extraction. The roots were cut into 1–2 cm pieces and homogenized in water with a blender (BL143GJP, T-fal, Tokyo, Japan) for 15 sec. From 1 g fresh root subsample, nematodes were extracted using the Baermann funnel method (Kawanobe et al., 2019), at room temperature and collected after 48 h. The soil in each pot was collected and passed through a 5 mm aperture sieve to remove rocks and debris, mixed well and kept at room temperature for no more than a day before nematodes were extracted. Nematodes were extracted from 20 g subsamples of soils using the Baermann funnel method (Ingham, 1994) at room temperature and collected after 72 h. Nematodes were counted and identified based on their morphological characters under a stereo-microscope (SZX10, Olympus, Tokyo, Japan). The root-lesion nematodes extracted from roots were randomly selected (five or more nematodes per experiment) and identified using the real-time PCR method (Kawanobe et al., 2015).

In total, 32 experimental plots (8 m length × 1.35 m width; sugarcane variety cv. Ni22) were established in each of the two trials. Fipronil treated and non-treated control plots were prepared in 16 replicates. The experimental fields were also known to be infested with an economically important insect, the sugarcane wireworm, and clothianidin (Dantotsu® for sugarcane wireworm control, Sumitomo Chemical, Osaka, Japan; 0.5% a.i.) was applied in a granular form at 60 kg/ha (0.3 kg a.i./ha) for controlling the wireworm in the non-treated control in Trial 1 and in the entire experimental field in Trial 2. The soils in the two field sites had the following properties: Trial 1 was a silty clay (sand 3%, silt 40%, clay 57%), with total C 13.5 mg/g, total N 1.0 mg/g, pH (H₂O) 4.3, and EC 98 µS/cm and Trial 2 was a silty clay loam (sand 3%, silt 58%, clay 39%), with total C 13.3 mg/g, total N 1.2 mg/g, pH (H₂O) 3.9, and EC 147 µS/cm.Two-bud sugarcane setts were planted in March 2017 (Trial 1) and March 2018 (Trial 2). Sugarcane plants were grown under the conventional management practice for 11 months and harvested in February 2018 and 2019, respectively. Fipronil (Prince® Bait) was applied in a granular form at 90 kg/ha (0.45 kg a.i./ha), which is the manufacturer’s recommended rate for the sugarcane wireworm (Melanotus sp.) in the planting furrow just before spring planting and incorporated by tillage.

Roots from a randomly selected sugarcane shoots were collected from each of 32 plots at 3- and 6-month to determine numbers of root-lesion nematodes. Root samples were kept at room temperature for no more than a day before the nematodes were extracted using the methods described for the greenhouse experiment.

Soil was collected at 0- (just before cane planting), 1-, 3-, and 6-month after sugarcane planting and 11-month (at harvest; Trial 1 only). The soil was collected with an auger (3 cm diam.) at 0 to 30 cm depth within 10 to 15 cm of the base of one or two randomly selected sugarcane plants in each plot. Four plot samples were combined to make a composite sample; thus there were four replicates per treatment rather than 16 for the soil samples. The soil was passed through a 5 mm aperture sieve to remove rocks and debris, mixed well and kept at room temperature for no more than two days before nematodes were extracted using the methods described for the greenhouse experiment.

After 11 months of growth, the number of canes in each plot was counted. Then, all the millable stalks were harvested from 4 m sections in the middle of each plot. The number of stalks and the total stalk weight per hectare were recorded and average single stalk weight was calculated. Of the stalks harvested from each plot, 12 were randomly chosen to measure length, diameter, and the value of Brix.

The statistical differences were determined by Student’s t-test comparing control and test groups (Greenhouse experiment) or analyzed by ANOVA (field experiment) where the effect of treatment, trial, and treatment × trial on nematode densities and sugarcane yield were included in the model. Statistical analyses were conducted using Microsoft Excel and its add-in software Statcel (3rd ed., OMS, Tokyo, Japan).

Initial root-lesion nematode densities were 21 nematodes/20 g soil. After 7 wk of sugarcane growth, root-lesion nematode densities were 63 and 70 nematodes/20 g soil in control and fipronil treatments, respectively, and no significant difference between the treatments was observed. Root-lesion nematode population densities in roots after 7 wk were greater (P < 0.01) in the control (1,550/g root) than the fipronil treatment (764/g root). Root-lesion nematodes (100%; n = 5) extracted from sugarcane roots at the end of the greenhouse experiment were confirmed to be P. zeae by real-time PCR. Culm height and numbers of fully extended leaves after 7 wk of sugarcane growth did not differ between the treatments.

Free-living, root-lesion, and spiral nematodes were found throughout 2017 and 2018 spring-planted crops (Trials 1 and 2). Population densities of free-living and lesion nematode tended to increase after planting, whereas densities of the spiral nematode tended to decrease or remain the same (Fig. 1). However, densities of these nematodes did not differ between the treatments. Root-lesion nematode densities in roots after three months of sugarcane growth in the fipronil treatment were lower (P < 0.01) than in the control by 44% and 73% in Trials 1 and 2, respectively (Fig. 2A). There was an interaction between treatment and trial (P < 0.05) for the densities of P. zeae in roots at three months. Nevertheless, in both trials, root-lesion nematode densities were lower (P < 0.01) in the fipronil treatment than in the control, although the magnitude of the difference between treatments was greater for Trial 2 than for Trial 1 (Fig. 2A). Root-lesion nematodes (100%; n = 5 and 9) extracted from sugarcane roots at three months in Trials 1 and 2, respectively, were confirmed to be P. zeae by real-time PCR. The population densities after six months of sugarcane growth were almost equivalent between the treatments (Fig. 2B).

Figure 1:

Figure 2:

The millable stalk weight in Trials 1 and 2 were more (P < 0.05) in the fipronil treatment than in the non-treated control by 6% and 8%, respectively (Fig. 3A) and these results were consistent among trials (no treatment × trial interaction). The numbers of millable stalks were greater (P < 0.05) in the fipronil treatment than in the non-treated control by 5% and 6% in Trials 1 and 2, respectively (Fig. 3B). The average single stalk weight in Trials 1 and 2 were greater in the fipronil treatment than in the non-treated control by 1% and 3%, respectively, but not statistically significant (Fig. 3C). Stalk length was 5% longer (P < 0.05) in the fipronil treatment than in the non-treated control in Trial 1 but not in Trial 2 (Fig. 3D). Millable stalk diameter was not different between treatments in both Trials 1 and 2 (Fig. 3E). The Brix in Trial 1 was higher (P < 0.05) in the fipronil treatment than in the non-treated control by 2%, although there was no difference in Trial 2 (Fig. 3F). For single stalk weight, stalk length, diameter, and the value of Brix, no significant difference was observed between in the fipronil treatment and the non-treated control in the combined analysis of Trials 1 and 2.

Figure 3: