Evaluation of nematicides for Meloidogyne enterolobii management in sweetpotato

The population of nematode used in this study was originally isolated from black nightshade (Solanum nigrum) in Lufeng county (22°55′57.44″N, 115°33′10.31″E), Guangdong Province, China (7). Based on the morphological, molecular analyses of mtDNA cytochrome c oxidase I (COI) and D2-D3 regions of 28S rDNA and detection using species-specific primers, the nematode was identified as M. enterolobii. M. enterolobii was maintained on potted sweetpotato (Ipomoea batatas (L.) Lam ‘Long 9’) plants in a growth cabinet for three months prior to use. Eggs of M. enterolobii were extracted from infected roots of sweetpotato with 0.5% sodium hypochlorite solution(Hussey & Barker, 1973). The whole infested root systems were put into 500 mL screw-top flasks and shaken in 0.5% sodium hypochlorite solution for 5 min prior to being washed with water over a No. 500 sieve (25 μm) and transferred to the beaker with about 30 mL of water. To obtain second-stage juveniles, the egg suspension was poured over a Baermann funnel and incubated in sterile water for 5 days at 28°C. Emerging J2s were collected daily by a sieve, and only those collected on the fifth day of incubation were used in the experiments.

Cyclobutrifluram [45% suspension concentrate, SC] was obtained from Syngenta group China and Fluopyram [41.7% suspension concentrate, SC] was provided by Bayer Crop Science, Greater China. Fluensulfone [40.0% emulsifiable concentrate, EC] was obtained from ADAMA Agricultural Solutions Ltd, Beijing, China. Hymexazol [70.0% water power, WP] was obtained from Weifang Huanuo Biotechnology Co., Ltd., Weifang, China.

To determine the effect of nematicide concentration on J2s motility of M. enterolobii, a 24-well microplate motility assay was performed. In the preliminary phase, we first conducted a pre-experiment to determine the approximate range of EC₅₀ for each chemical. Based on the result of the preliminary experiment, we designed distinct application rates for each chemical accordingly. Plate wells were filled 1ml aqueous solutions of cyclobutrifluram at 2.5, 1.25, 0.63, 0.31 and 0.16 mg L⁻¹, Fluopyram at 1.25, 0.63, 0.31, 0.16 and 0.08mgL⁻¹, Fluensulfone at 80, 40, 20, 10 and 5 mgL⁻¹, and Hymexazol at 640, 320, 160, 80 and 40 mg L⁻¹, or tap water (control; PH 7.3). Approximately 100 J2s suspended in 10 μL of tap water were introduced into each well. At 48 h post inoculation, 25 μL of mol L⁻¹ sodium hydroxide (NaOH) was added to the appropriate wells as an irritant to distinguish between motile and immotile nematodes (8). Percent motility was recorded 30 seconds after the addition of the NaOH using a stereoscope (Nikon SMZ745). Each treatment was replicated three times, and all experiments were conducted twice.

To determine the effect of nematicide concentration and exposure time on eggs hatching of M. enterolobii, a 24-well microplate egg hatching assay was performed. The treatment concentration of cyclobutrifluram was 2.5, 0.63 and 0.16 mg L⁻¹; Fluopyram was 1.25, 0.31 and 0.08 mg L⁻¹, Fluensulfone was 80, 20 and 5 mg L⁻¹, and Hymexazol was 640, 160 and 40 mg L⁻¹. Eggs were exposed to tap water (PH 7.3) as the control. Approximately 100 eggs suspended in 10 μL of tap water were introduced into each well. At 24, 48 or 72 h post inoculation, the number of hatched nematodes in each treatment was observed and counted by a stereomicroscope. Each treatment was replicated three times, and all experiments were conducted twice. Inhibitory hatching rate (IHR) was calculated for each nematicide treatment using the following formula: IHR=number of hatched nematodes in control − number of hatched nematodes in treatmentnumber of hatched nematodes in control × 100% {\rm{IHR}} = {{{\rm{number}}\,{\rm{of}}\,{\rm{hatched}}\,{\rm{nematodes}}\,{\rm{in}}\,{\rm{control}}\, - \,{\rm{number}}\,{\rm{of}}\,{\rm{hatched}}\,{\rm{nematodes}}\,{\rm{in}}\,{\rm{treatment}}} \over {{\rm{number}}\,{\rm{of}}\,{\rm{hatched}}\,{\rm{nematodes}}\,{\rm{in}}\,{\rm{control}}\, \times \,100\% }}

A growth cabinet experiment was conducted to assess the impact of applying nematicides at the recommended dosage on the reproductive capacity of M. enterolobii infecting sweet potato root systems. In simple terms, 20 cm wide square pots were filled with 1.5 L of dry heat sterilized sandy loam soil, which was augmented with organic peat. The control group consisted of pots inoculated with M. enterolobii but not treated with any nematicides. Three days after the sweetpotato seedlings (‘Long 9’) were transplanted, nematodes were inoculated into the potted soil. Each pot was inoculated with 1000 J2s of M. enterolobii. Nematicide treatments were applied to each pot except for fluensulfone, which was applied 3 days prior to planting to avoid phytotoxicity issues. Each treatment was as follows: (1) Cyclobutrifluram (0.03 mL pot⁻¹), (2) Fluopyram (0.03 mL pot⁻¹), (3) Fluensulfone (0.2 mL pot⁻¹), (4) Hymexazol (1 mg pot⁻¹), and (5) Nema⁺ (nematodes with no nematicide). Each treatment consisted of three replicates, each containing six sweetpotato plants, with one 15 cm tall sweetpotato seedling per pot. All experiments were conducted twice. Pots were watered as necessary throughout the experiment to maintain adequate soil moisture. After 50 days of growth in a temperature-controlled growth cabinet set at 28±1°C with 12 hours of light per day, the experiment was terminated.

A disease measurement was obtained in the pot experiments. The first was disease incidence, which was the number of diseased plants out of the total number of plants in each replicate and was reported on a percentage basis. The sweetpotato seedlings whose root systems exhibited visible galls were identified as diseased plants. The root gall index (GI) was calculated as the number of root galls on an individual plant divided by the total weight of the plant roots, and then multiplied by 100. The egg mass index (EMI) was calculated as the number of egg masses on an individual plant divided by the total weight of the plant roots, and then multiplying the result by 100. The GI and EMI can effectively reflect the nematode's reproduction status in plant and the severity of damage to the plant. Counting egg masses was performed using a stereoscope. A disease severity was determined by rating the roots on each plant on a 0 to 5 scale (Zhou et al., 2016), in which 0= less than 10% root-knot; 1= very slight root-knot, between 11%–20%; 2= obvious root-knot, percentage was between 21%–50%; 3, percentage was between 51%–80% root-knot; 4, percentage was between 81%–90% root-knot; 5= root knots exceeded 91%. A disease index (DI), whose values ranged from 0 to 100 was calculated for each nematicide treatment using the following formula: DI=Σ(the number of diseased plants in the pot × rating i)total number of plants in the pot× 5×100 {\rm{DI}} = {{\Sigma ({\rm{the}}\,{\rm{number}}\,{\rm{of}}\,{\rm{diseased}}\,{\rm{plants}}\,{\rm{in}}\,{\rm{the}}\,{\rm{pot}}\, \times \,{\rm{rating}}\,i)} \over {{\rm{total}}\,{\rm{number}}\,{\rm{of}}\,{\rm{plants}}\,{\rm{in}}\,{\rm{the}}\,{\rm{pot}} \times \,5}} \times 100

Where i is 1–5.

Analysis of variance was performed on data from individual experiments using IBM® SPSS® Statistics (Version 20). Data were subjected to analysis of variance (ANOVA) and following significant results, means were separated according to the Student's Protected LSD tests. A significance level of α = 0.05 was used in all analyses. Median effective concentration (EC₅₀) values for each nematicide were calculated by probit analysis of the nematodes' motility after 48h of incubation.

Non-motile J2s of M. enterolobii did not recover their motility after NaOH was add to the wells, indicating that cyclobutrifluram, fluopyram, fluensulfone and hymexazol were nematicidal. The EC₅₀ (48h) for cyclobutrifluram, fluopyram, fluensulfone and hymexazol was 0.423, 0.204, 22.335 and 216.622 mg L⁻¹, respectively (Table 1). Based on the J2s motility assays, J2s of M. enterolobii were more sensitive to fluopyram and cyclobutrifluram than fluensulfone and hymexazol.

After incubating M. enterolobii eggs in each of the nematicide treatments, the inhibitory hatching rate of eggs increased over time at the same concentration, and it also increased with concentrations at same time point (Fig. 1). Incubating M. enterolobii eggs in each nematicide concentration for 24 hours resulted in inhibitory hatching rate of 67.19%, 72.53%, 76.39% and 49.90% for cyclobutrifluram (2.5 mg L⁻¹), fluopyram (1.25 mg L⁻¹), fluensulfone (80 mg L⁻¹) and hymexazol (640 mg L⁻¹), respectively. After incubating M. enterolobii eggs in each nematicide concentration for 48 hours, inhibitory hatching rates were observed as follows: cyclobutrifluram (2.5 mg L⁻¹)- 88.28%, fluopyram (1.25mg L⁻¹)- 83.81%, fluensulfone (80 mg L⁻¹)- 88.08% and hymexazol (640 mg L⁻¹)- 63.51%. Following an incubation period of 72 hours, inhibitory hatching rates were recorded as cyclobutrifluram (2.5 mg L⁻¹)-90.73%, fluopyram (1.25mg L⁻¹)- 96.78%, fluensulfone (80 mg L⁻¹)-87.45% and hymexazol (640 mg L⁻¹)-67.39%. The highest concentration among all tested nematicides after 72 hours of exposure showed that cyclobutrifluram, fluopyram and fluensulfone achieved an inhibitory hatching effect exceeding 85%, while hymexazol only reached a rate of 67%.

Figure 1:

In the growth cabinet studies conducted on sweetpotato infected with M. enterolobii, all the nematicide treatments exhibited lower disease incidence compared to the control, except for hymexazol. Among these treatments, fluopyram demonstrated the lowest disease incidence (50%), which was significantly different from the control (100%) (Table 2). However, each nematicide treatment resulted in a significantly lower DI of sweetpotato compared to the control. The ascending order of DI under each treatment was as follows: fluopyram (7), cyclobutrifluram (13), fluensulfone (25) and hymexazol (38). Cyclobutrifluram and fluopyram showed significantly difference from the other treatments. Similarly, the GI of sweetpotato under each nematicides was significantly lower than that of the control. The ascending order of GI was as follows: fluopyram (64), cyclobutrifluram (69), fluensulfone (157) and hymexazol (238). Cyclobutrifluram and fluopyram were significantly different from other treatments. The EMI of sweetpotato in each of the nematicide treatments was significantly lower than the control except for hymexazol. The EMIs of sweetpotato under each treatment were fluopyram (36), cyclobutrifluram (51), fluensulfone (150) and hymexazol (238) in ascending order, with cyclobutrifluram and fluopyram being significantly different from other treatments.