Soybean (
Numerous chemical methods have been used to control SCN; nematicide is an important management tool, but effective control of SCN has been a challenging problem (Raddy et al., 2013). Increasing concerns to human and environment safety led to the widespread deregistration of several agronomic important nematicides; thus, new and safer nematicides are urgently needed to date (Lai et al., 2014). Fosthiazate, an organophosphorus (OP) nematicide developed by Ishihara Sangyo Kaisha, Ltd., was registered and marketed in Japan in 1992; it is widely used for controlling
Fosthiazate can act on the nervous system of targeted nematode pest, inhibit acetylcholinesterase (AChE), and block normal nerve impulse conduction (Xu, 2007). The efficacy of fosthiazate in controlling pests on tomato (
Root-knot nematode is the most frequently target nematode in nematicidal activity determination of fosthiazate; however, limited relevant research focused on the effect of fosthiazate on cyst nematodes; only potato cyst nematode (
This study aimed to investigate the following: nematicidal activity of fosthiazate on second-stage juvenile (J2)
Fosthiazate (85.1%) was obtained from the Zhengbang Bio-chemical Co., Ltd. (Nanchang, China). In bioassay, five fosthiazate concentrations (2.18, 3.44, 5.45, 8.61, and 13.62 mg/l) were used in experiments. The used concentrations were based on the relationship of fosthiazate and nematode mortality assessed in a previous experiment. When the five concentrations were increased by 1.58 times, nematode mortality ranged from 10 to 90% at 12 hr after exposure in the experiment. Concentrations of 2.18, 5.45, 13.62, 34.04, and 85.10 mg/l were used in the pot test according to lethal concentrations (LC90) at 12 hr in bioassay. Dimethyl sulfoxide (DMSO, Tianjin, China) was used as a dissolvent. The controls included 0.5% DMSO and distilled water in all experiments.
Pot with 200 ml autoclaved fine sand (diameter 850 µm), and 50 cysts were inoculated, and susceptible soybean (cultivar, Ludou 4) was sowed in pots, with three seeds each pot, and grown in intelligent illumination incubator SPX–250B–G (Shanghai Boxun Industry and Commerce Co., Ltd, Shanghai, China) at 25°C with a 14 hr/10 hr (light/dark) photoperiod and 50–75% relative humidity. The cysts were harvested from 41-day cultures and extracted with sieving-decanting method, as described by Liu (1995). All cysts were transferred into a 50 µm pore diameter hatching sieve, surface-sterilized using 70% ethanol for 3 min, and then washed four times with distilled water. Subsequently, the hatching sieve with cysts was placed in a petri dish added with 2 ml distilled water and incubated at 25°C in darkness. Fresh infective J2 were collected from the bottom of petri dishes on the day of the experiment.
For each fosthiazate concentration, a total of 200 ml were dispensed into a 96-well plate with 40–50 nematodes in each well (four replicates). The process was repeated three times for each concentration, and the assay plates were kept at 25°C. Nematodes in plates were observed at 0, 2, 4, 6, 8, 10, and 12 hr after exposure under an inverted compound microscope XD30A (Sunny Optical Technology (Group) Co., Ltd, Yuyao, China) and the immobile nematodes were recorded.
At each observation time point, knockdown rates of J2 were calculated: Knockdown rate (%) = (number of knockdown J2)/(total number of J2) × 100%. In this study, the knockdown J2 indicates the ceased motor behavior of nematodes after treatment with different concentrations of fosthiazate. Then, mortality rate of J2 was calculated using the equation: mortality rate (%) = (number of dead J2)/(total number of J2) × 100%. In the present study, mortality rate of J2 in each concentrations at different exposure times were evaluated. In order to judge the nematode was really dead or fake dead, when the respective fosthiazate concentration was replaced with distilled water, the touch test occurred after 12 hr, J2s were considered really dead if nematodes could not recover upon immersion in water and showed no movement when touched with a hair needle.
During the study, 13.62 mg/l of fosthiazate showed significant effect on
Cysts were collected from the roots 41 days after inoculation. Fresh eggs were released and treated with different concentrations of fosthiazate as described above. Distilled water and 0.5% DMSO solutions were used as control. The eggs were dispensed in a 96-well plate and subjected to five concentrations of fosthiazate, with four replicates, and the process was repeated three times. The assay plates were incubated at 25°C and kept in the dark. The J2 that hatched from free eggs were recorded under an inverted compound microscope at 0, 3, 6, 9, 12, 15, and 18 days after exposure to different concentrations of fosthiazate. The accumulative hatching rate was calculated using the following formula:
Nematode reproduction on soybean in different treatments was investigated in pot experiments using a completely randomized design with two replicates. Seeds were placed in a petri dish with a filter paper after rinsing for five times in sterile water and germinated at 25°C. The germinated seeds with the same shoot length were retrieved after 2 days. One germinated seed was sowed in a pot (7 × 7 × 8 cm) with autoclaved fine sand. Seedling plants were inoculated with 3,000 eggs per plant at 3 days after transplanting by injection. Three holes (1 cm depth) and about 1 cm from the main stem of the plant were made in each pot. The egg suspension was transferred using a pipet which was subsequently covered with surface sand. Soybean seedlings were grown at 25°C under a 14 hr light and 10 hr dark photoperiod in an intelligent illumination incubator, and the moisture was kept constant. A total of 2 ml of each fosthiazate concentration was applied to each pot at 4 days after sowing, and water and 0.5% DMSO used as control with four replicates. Nematode reproduction was checked at 35 days after inoculation. The whole roots and all sand in the pot were collected, the root system was washed with tap water, and cysts on the roots and sand were extracted by sieving-decanting method. The cysts were collected on a 177 µm mesh sieve and hand-picked with a dissecting needle under a stereomicroscope. All cysts on the roots and sand were counted, eggs were released from the cysts and average eggs per cyst were calculated. In this study, there was no significant difference in the average eggs per cyst among different treatments. Nematode reproduction was expressed as the total number of cysts per one pot with soybean plant. The decline rate of cysts was calculated as follows:
Statistical analysis was performed with SPSS 12 (SPSS Inc., Chicago, IL). Least significant difference tests were performed, and differences with
Fosthiazate exhibited a strong toxic activity against
Mortality rate (%) of
Exposure time (hr) | ||||||
---|---|---|---|---|---|---|
Concentration (mg/l) | 2 | 4 | 6 | 8 | 10 | 12 |
13.62 | 2.86 ± 0.74 a | 9.80 ± 1.15 a | 19.48 ± 2.29 a | 32.96 ± 3.81 a | 73.04 ± 2.52 a | 86.35 ± 1.33 a |
8.61 | 1.97 ± 0.86 ab | 7.96 ± 0.85 a | 13.54 ± 0.48 b | 26.32 ± 2.04 b | 67.23 ± 3.14 b | 79.77 ± 2.72 b |
5.45 | 1.55 ± 0.61 abc | 5.07 ± 0.90 b | 10.90 ± 0.86 bc | 22.64 ± 1.22 b | 39.15 ± 1.68 c | 66.82 ± 3.26 c |
3.44 | 2.24 ± 0.70 ab | 5.17 ± 0.99 b | 8.10 ± 1.16 cd | 11.18 ± 1.62 c | 21.35 ± 1.88 d | 48.39 ± 1.93 d |
2.18 | 0.89 ± 0.60 bc | 4.29 ± 1.20 bc | 6.41 ± 0.98 d | 6.41 ± 0.98 cd | 10.01 ± 0.65 e | 13.43 ± 0.57 e |
0.5% DMSO | 0.52 ± 0.52 bc | 1.93 ± 1.00 cd | 1.90 ± 0.73 e | 2.27 ± 0.68 de | 2.27 ± 0.68 f | 3.23 ± 0.80 f |
Water | 0.00 ± 0.00 c | 0.00 ± 0.00 d | 0.00 ± 0.00 e | 0.00 ± 0.00 e | 0.00 ± 0.00 f | 0.00 ± 0.00 f |
Mortality rate (%) of
Nematicidal activity of fosthiazate was evaluated by comparing the median lethal concentrations (LC50) for different concentrations on
Toxicity of fosthiazate to
Exposure time (h) | Slope(±SE) | Correlation coefficient | LC50 (95%CI) | LC90 (95%CI) |
---|---|---|---|---|
4 | 0.54 (± 0.10) | 0.95 | 3912.59 (317.10−48275.85) | 941773.86 (9,541.28−92957,985.01) |
8 | 1.38 (± 0.18) | 0.98 | 25.00 (16.53−37.80) | 212.43 (83.90−537.87) |
12 | 2.65 (± 0.44) | 0.97 | 4.41 (3.53−5.50) | 13.41 (9.36−19.20 ) |
The shape of J2 killed by fosthiazate (curved; Figs 2C 2D) differed from the straight appearance of natural-death J2 (Fig. 2A 2B). Fosthiazate can shorten the body length of J2s. The dead nematodes cannot move with a twisted body shape and shrunk. The length of nematode body, stylet, and the distance from the esophageal glands to head were significantly shorter than that in the control (
Lengths of
Length (µm) | ||||
---|---|---|---|---|
Treatment | Stylet | Esophageal glands to head | Hyaline region | Entire body |
Fosthiazate | 24.16 ± 0.41 b | 155.11 ± 3.74 b | 57.98 ± 1.13 a | 371.77 ± 10.97 b |
Water | 25.77 ± 0.33 a | 199.48 ± 2.03 a | 27.99 ± 0.59 b | 501.04 ± 8.44 a |
The shape of
Free eggs of
Cumulative hatching percentage of
Fosthiazate can reduce the number of SCNs. Application of 2 ml fosthiazate at 5.45, 13.62, 34.04, and 85.10 mg/l concentrations significantly affected SCN reproduction in this test. The number of cysts decreased by 43.64, 53.00, 67.21, and 97.94% at 31 days after application, respectively. Application of 0.0272 mg fosthiazate (available ingredient) per pot can reduce the population by 53.0% (Table 4).
Effect of fosthiazate on control of
Treatment | Amount of fosthiazate (a.i.) (mg) | Average number of cyst | Population decrease (%) |
---|---|---|---|
Water | 0 | 412 a | |
0.5% DMSO | 0 | 432 a | |
2.18 mg/l | 0.0044 | 389 ab | 5.43 |
5.45 mg/l | 0.0109 | 232 bc | 43.64 |
13.62 mg/l | 0.0272 | 194 c | 53.00 |
34.04 mg/l | 0.0681 | 135 cd | 67.21 |
85.10 mg/l | 0.1702 | 9 d | 97.94 |
OP compounds have been widely used in controlling agriculture and sanitary insects for their high-efficiency broad spectrum (Jokanović et al., 2011). Fosthiazate, a non-fumigant nematicide, is effective in controlling a wide range of plant parasitic nematodes, including root-knot nematodes, cyst nematodes, and root lesion nematodes (Kimpinski et al., 1997; Pullen and Fortnum, 1999; Zasada et al., 2010).
The nematicidal activity of fosthiazate is not influenced easily by the chemical and physical characteristics of soil (Koyanagi et al., 1998; Wada and Toyota, 2008). Fosthiazate exerts a moderately long residual effect against J2 of plant parasitic nematodes (
A previous study showed that graphite, graphite oxide nanoplatelets, and graphene quantum dots can significantly inhibit the effects on the body length of nematodes after exposure in a concentration-dependent manner (Li et al., 2017). In the present study, fosthiazate reduced nematode mobility and shorten the body length of J2 probably due to the influence of AChE. As far as we know, it is the first report of fosthiazate effect on the nematode morphology. However, the stylet of nematode exposed in fosthiazate is shorter than that of control, which phenomenon cannot be explained, because the stylet is ossific. Fosthiazate solution also exhibited a pronounced effect on
This study demonstrated that fosthiazate exhibits strong toxicity against SCN, including increasing of J2 mortality, and reducing egg hatching and reproduction rates, thus providing evidence to support the use fosthiazate in further studies against