Insecticidal Effects of Fumigants (EF, MB, and PH3) towards Phosphine-Susceptible and -Resistant Sitophilus oryzae (Coleoptera: Curculionidae)

This study was conducted to evaluate the insecticidal effects of ethyl formate (EF), methyl bromide (MB), and phosphine (PH3) fumigants against PH3-susceptible and -resistant strains of the rice weevil (Sitophilus oryzae), a major rice pest. The highest lethal concentration time 50 (LCt50) values of the PH3-susceptible strains were 255.797, 21.104, and 6.171 mg h L−1 for EF, MB, and PH3, respectively, at pupal stage. The highest LCt50 values of the PH3-resistant strains were 149.028 and 48.170 mg h L−1 for EF and PH3, respectively, at late larval stage, and 43.520 mg h L−1 for MB at pupal stage. In comparison to the susceptible strains, the PH3-resistant strain collected in South Korea had a resistance level 4 to 56 times higher. Use of the major quarantine fumigants EF, MB, and PH3 indicated the existence of PH3-resistant rice weevils in South Korea for the first time.


Introduction
Stored pests are known to cause damage to stored products, such as reduced grain weight, quality, commercial value, and seed survival [1]. More than 600 species of insects infesting stored grains belong to Coleoptera, among which the genus Sitophilus includes several important grain pest species [2][3][4][5]. Sitophilus oryzae (L.) (Coleoptera: Curculionidae) reduces the amount and quality of stored grains and causes economic losses [1,6]. S. oryzae females make holes and lay an egg, and larval and pupal period is spent hidden in the stored grain [6]. S. oryzae larvae feed on proteins and vitamins by consuming the germ of cereals, and adults largely reduce the carbohydrate content by consuming the endosperm of the grains [1,7].
Methyl bromide (MB) and phosphine (PH 3 ) fumigants are mainly used for fumigation of grain at storages and silos to control pests damaging stored grains [8]. MB is an old fumigant discovered in 1932 that has been used in virtually every field of disinfection and disinfestation, such as fruit, grains, wood, and necessities [9]. However, it has been designated as an environmentally destructive substance causing ozone depletion [10], and its use has been completely prohibited except for quarantine and pre-shipment purpose under the Montreal Protocol [11,12]. PH 3 is known to spread rapidly during fumigation, has low fumigant residue after treatment, and has no side effects on seed survival [13,14]. However, PH 3 resistance has been found in India, China, Vietnam, Morocco, Brazil, and Australia. In each country, S. oryzae has been reported to be phosphine-resistant [15][16][17][18][19][20][21].
As an alternative to these fumigants, the efficacy of ethyl formate (EF) against stored product insects have been investigated in a wide range of studies [22][23][24]. EF is a low-molecular-weight volatile material characterized by little residue [25] and occurs naturally in a variety of products such as beer, rice, and beef [26]. It has been used for dry fruit insect control since 1927 and has also been used for the disinfection of cereals, stored dry beans, tomatoes, and preserved foods [27,28]. In the United States, pesticides have been registered [29] for the control of pests in storage products such as Tribolium confusum and Ephestia figuliella in raisins [30].
S. oryzae is an important pest in the management of postharvest agricultural products. Phosphine resistance of S. oryzae has risen sharply to 75% in developing countries by 2000 [20], requiring the management of resistance. Therefore, this study was conducted to investigate the resistance levels of the newly discovered PH 3 -resistant rice weevils of South Korea and compare these resistance levels to those of susceptible strains. EF, MB, and PH 3 fumigants were used to provide baseline data on rice weevil control through a comparison of treatments currently recommended by quarantine.

Insects
PH 3 -susceptible strain (control strains) of S. oryzae was reared at Murdoch University (WA, Australia), and the PH 3 -resistant strain of S. oryzae was reared at Chungbuk National University (Chungcheongbuk-do, South Korea). All tested insects were brought to the Plant Quarantine Technology Center before experiment (Gyeongsangbuk-do, South Korea). The breeding conditions were maintained at a temperature of 27 ± 1 • C and relative humidity of 50-60% in a brown rice feed at the Plant Quarantine Technology Center. The presence or absence of S. oryzae eggs were confirmed by thinning the area covered with gelatinous fluid near the rice grain ( Figure 1). S. oryzae eggs were used in the experiment within 2-3 days after oviposition of S. oryzae adults. The larvae within 1-2 and 22-25 days after hatching were used as early and late larvae, respectively. Pupae were collected within 3-4 days after pupation. Adult of S. oryzae were grown for 10-20 days after emergence. The inside of the rice was checked to determine whether larvae or pupae were present. It was difficult to observe the inside of the brown rice, so a stereomicroscope (MDG33, Leica, Wetzlar, Germany) was used. All insects were inoculated on an insect breeding dish (Φ 5.5 cm × 1.5 cm, SPL, Pocheon, Gyeonggi-do, Korea) and fed 2 g of brown rice.
Insects 2019, 10, x FOR PEER REVIEW 2 of 12 volatile material characterized by little residue [25] and occurs naturally in a variety of products such as beer, rice, and beef [26]. It has been used for dry fruit insect control since 1927 and has also been used for the disinfection of cereals, stored dry beans, tomatoes, and preserved foods [27,28]. In the United States, pesticides have been registered [29] for the control of pests in storage products such as Tribolium confusum and Ephestia figuliella in raisins [30]. S. oryzae is an important pest in the management of postharvest agricultural products. Phosphine resistance of S. oryzae has risen sharply to 75% in developing countries by 2000 [20], requiring the management of resistance. Therefore, this study was conducted to investigate the resistance levels of the newly discovered PH3-resistant rice weevils of South Korea and compare these resistance levels to those of susceptible strains. EF, MB, and PH3 fumigants were used to provide baseline data on rice weevil control through a comparison of treatments currently recommended by quarantine.

Insects
PH3-susceptible strain (control strains) of S. oryzae was reared at Murdoch University (WA, Australia), and the PH3-resistant strain of S. oryzae was reared at Chungbuk National University (Chungcheongbuk-do, South Korea). All tested insects were brought to the Plant Quarantine Technology Center before experiment (Gyeongsangbuk-do, South Korea). The breeding conditions were maintained at a temperature of 27 ± 1 °C and relative humidity of 50-60% in a brown rice feed at the Plant Quarantine Technology Center. The presence or absence of S. oryzae eggs were confirmed by thinning the area covered with gelatinous fluid near the rice grain ( Figure 1). S. oryzae eggs were used in the experiment within 2-3 days after oviposition of S. oryzae adults. The larvae within 1-2 and 22-25 days after hatching were used as early and late larvae, respectively. Pupae were collected within 3-4 days after pupation. Adult of S. oryzae were grown for 10-20 days after emergence. The inside of the rice was checked to determine whether larvae or pupae were present. It was difficult to observe the inside of the brown rice, so a stereomicroscope (MDG33, Leica, Wetzlar, Germany) was used. All insects were inoculated on an insect breeding dish (Φ 5.5 cm × 1.5 cm, SPL, Pocheon, Gyeonggi-do, Korea) and fed 2 g of brown rice.

Fumigation System
Fumigant treatment was performed at the Plant Quarantine Technology Center. The fumigant treatment was applied in a fumigation chamber at a temperature of 20 ± 1 °C and a relative humidity of 60 ± 10%, and fumigation was carried out in a 12 L glass desiccator (DWK Life Sciences, Mainz, Germany). Tested insects were put in 100 × 40 mm size insect breeding dish (SPL, Pocheon, Gyeonggido, Korea) before fumigation. More than 30 insects were carried in each dish and three dishes were used for each test as replicates.
PH3-susceptible S. oryzae was treated at a concentration of 0.01 to 4.00 mg/L for 20 h, but in the case of PH3-resistant S. oryzae, a high concentration exceeding 4 mg/L is required, so concentration was fixed to 0.50 mg/L and the treatment time was varied from 24 h to 504 h. EF was applied at 10 to 150 mg/L for 4 h, and MB was applied at 3 to 25 mg/L for 4 h. Test of PH3 resistance was performed according to the Food and Agriculture Organization (FAO) criteria [31]. According to FAO test, a population was judged to have resistance when more than 2 individuals survived for 20 h at PH3 0.04 mg/L. EF and MB treatments were observed at 24 h after fumigation, and PH3 treatment was observed at 72 h after fumigation due to the knock-down phenomenon. The insecticidal rate was determined by touching an insect body with a microscopic needle. It was judged as dead if there was no movement using a microscope (MDG33, Leica, Wetzlar, Germany).

Measurement of Fumigant Concentrations
To investigate the actual concentration in the treated samples, 60 mL of sample gas was extracted from all experimental groups using a 60 mL syringe and 1 L Tedlar bag (SKC, Dorset, United Kingdom). EF and MB samples were taken at 30 min and 1, 2, and 4 h, and PH3 samples were taken at 30 min and 1, 4, and 20 h after fumigation. If fumigation time was longer than 20 h, samples were extracted at 24-h intervals until the completion of the experiment.
The concentrations of EF and MB were measured using an Agilent GC 7890A equipped with a flame ionization detector (FID) after separation on an Rtx-5 column (15 m × 250 μm × 1 μm, RESTEK,

Fumigation System
Fumigant treatment was performed at the Plant Quarantine Technology Center. The fumigant treatment was applied in a fumigation chamber at a temperature of 20 ± 1 • C and a relative humidity of 60 ± 10%, and fumigation was carried out in a 12 L glass desiccator (DWK Life Sciences, Mainz, Germany). Tested insects were put in 100 × 40 mm size insect breeding dish (SPL, Pocheon, Gyeonggi-do, Korea) before fumigation. More than 30 insects were carried in each dish and three dishes were used for each test as replicates. PH 3 -susceptible S. oryzae was treated at a concentration of 0.01 to 4.00 mg/L for 20 h, but in the case of PH 3 -resistant S. oryzae, a high concentration exceeding 4 mg/L is required, so concentration was fixed to 0.50 mg/L and the treatment time was varied from 24 h to 504 h. EF was applied at 10 to 150 mg/L for 4 h, and MB was applied at 3 to 25 mg/L for 4 h. Test of PH 3 resistance was performed according to the Food and Agriculture Organization (FAO) criteria [31]. According to FAO test, a population was judged to have resistance when more than 2 individuals survived for 20 h at PH 3 0.04 mg/L. EF and MB treatments were observed at 24 h after fumigation, and PH 3 treatment was observed at 72 h after fumigation due to the knock-down phenomenon. The insecticidal rate was determined by touching an insect body with a microscopic needle. It was judged as dead if there was no movement using a microscope (MDG33, Leica, Wetzlar, Germany).

Measurement of Fumigant Concentrations
To investigate the actual concentration in the treated samples, 60 mL of sample gas was extracted from all experimental groups using a 60 mL syringe and 1 L Tedlar bag (SKC, Dorset, United Kingdom). EF and MB samples were taken at 30 min and 1, 2, and 4 h, and PH 3 samples were taken at 30 min and 1, 4, and 20 h after fumigation. If fumigation time was longer than 20 h, samples were extracted at 24-h intervals until the completion of the experiment.
The concentrations of EF and MB were measured using an Agilent GC 7890A equipped with a flame ionization detector (FID) after separation on an Rtx-5 column (15 m × 250 µm × 1 µm, RESTEK, Bellefonte, PA, USA) operating in split mode (10:1). The PH 3 concentration was determined using an Agilent GC 7890A equipped with a flame photometric detector (FPD) and HP-PLOT/Q (30 m × 530 µm × 40 µm, Agilent, Santa Clara, CA, USA) operating in split mode (10:1). The injector and oven temperature were 200 • C. The detector temperature was 250 • C. The injection volumes and flow rates of EF, MB, and PH 3 were 60, 60, and 20 µL and 1.5, 1.5, and 5 mL/min, respectively. The concentrations of EF, MB, and PH 3 were calculated based on peak areas against external standards.

Determination of the Concentration × Time (Ct) of the Fumigants
Concentration is an important factor to determine efficacy of chemical insecticide, including fumigants. However, fumigation time also is an important factor particularly for the fumigant, although, importance can be varied by the kind of fumigant [32]. To examine the effect of both concentration and time, exposure of the fumigant was expressed as concentration × time (Ct) product. The Ct of fumigants was calculated using the equation of Monro [33].

Statistical Analysis
All experiments were performed with 3 replicates. Percent mortality and standard error (SE) were determined via an Excel v. 2007. The lethal concentration time 50 (LCt 50 ) and 99 (LCt 99 ) values of S. oryzae were obtained using the Probit analysis [34] program (CSIRO 1998), with logarithmic transformation of data.

Fumigation of Phosphine-Susceptible S. oryzae
The PH 3 susceptibility-related effects of PH 3 , EF, and MB fumigants on S. oryzae eggs, early larvae, late larvae, pupae, and adults were evaluated. PH 3 showed a 100% insecticidal rate against eggs, early larvae, and adults at 1.00 mg/L and an 85.56% insecticidal rate in late larvae at 1.00 mg/L ( Figure 2). Pupae treated with 4.00 mg/L, the highest treatment concentration, showed an insecticidal rate of 86.04%. As a result of treatment with EF, eggs, early larvae, and adults showed insecticidal rates of 90% or more at an EF concentration of 70 mg/L ( Figure 3). However, in the case of late larvae treated with 120 mg/L, the insecticidal rate was 94.43%. In the pupae, the highest treatment concentration of 150 mg/L was applied, but the insecticidal rate was as low as 73.30%. MB showed a 100% insecticidal rate at 10 mg/L for eggs, 8 mg/L for adults, and 7 mg/L for early larvae but 25 mg/L for pupae and 20 mg/L for late larvae (Figure 4). These results indicate that PH 3 -susceptible S. oryzae is most tolerant in the pupal and late larval stages. Among PH 3 , EF, and MB fumigants, LCt 50 was the lowest (0.295 mg h/L) when treated with PH 3 , and the highest LCt 50 (255.797 mg h/L) was obtained by treating the pupae with EF. PH 3 showed a high insecticidal rate at a low concentration but had to be treated for a long time, and EF had to be treated at a high concentration, though the treatment time was short.

Evaluation of Phosphine-RESISTANT S. oryzae by Fumigation Time of Phosphine
Assessment of PH 3 resistance was conducted according to the FAO criteria. S. oryzae collected from South Korea was treated with 0.04 mg/L PH 3 for 20 h and compared with PH 3 -susceptible S. oryzae distributed from Australia. The PH 3 -susceptible S. oryzae showed a 98.3% insecticidal rate, and S. oryzae in South Korea was 21.0%. According to the FAO resistance evaluation criteria, S. oryzae in South Korea is considered to have resistance. The effect of PH 3 on PH 3 -resistant S. oryzae was evaluated by setting the concentration to 0.5 mg/L and then by treatment time. As a result, the eggs, early larvae, late larvae, pupae, and adults showed 100% insecticidal rates when treated for 72, 120, 168, 336, and 504 h, respectively ( Figure 5). Insects 2019, 10, x FOR PEER REVIEW 5 of 12 . Figure 2. Mortality of phosphine susceptible S. oryzae exposed to phosphine (PH3) fumigant for 20 h at 20 °C in 12 L desiccator.

Alternative Fumigants for PH3-Resistant S. oryzae
The effect of EF and MB fumigants on PH3-resistant S. oryzae eggs, early larvae, late larvae, pupae, and adults was evaluated. EF showed insecticidal rates of 100% at 70 mg/L for egg, early larvae, and pupae, 100% at 90 mg/L for adults, and 88.90% at 90 mg/L for late larvae ( Figure 6).
MB treatment showed a 100% insecticidal rate at 8 mg/L for eggs, 10 mg/L for early larvae and adults, whereas late larvae and pupae showed a 98.9% insecticidal rate at 20 mg/L (Figure 7). The LCt values were analyzed based on the effect of the fumigants on PH3-resistant S. oryzae. The LCt50 value of EF was the highest (149.028 mg h/L), and the LCt50 of MB was the lowest (25.840 mg h/L). The LCt50 of EF was 5.76 times higher than that of MB.

LCt Analysis of the PH3-Susceptible and PH3-Resistant Rice Weevils
The LCt50 and LCt99 values of the PH3-susceptible and PH3-resistant strains of rice weevils were analyzed based on the results of the effects of the three fumigants.
The LCt analysis results for EF showed that the LCt50 values of EF were confirmed to be the highest at pupal stage (255.797 mg h/L) and the lowest at early larval stage (60.110 mg h/L). The LCt50 values of EF on the PH3-resistant strains showed a different aspect with the previous one, that egg stage was the lowest (60.034 mg h/L) and late larval stage was the highest (149.028 mg h/L). PH3resistant strain showed higher LCt50 than PH3-susceptible strain at early larval stages, but lower LCt50 at late larval and pupal stages, which are tolerant stages (Table 1).

Figure 5.
Mortality of phosphine resistant S. oryzae exposed to PH 3 fumigant for 0.5 mg/L at 20 • C in 12 L desiccator.

Alternative Fumigants for PH 3 -Resistant S. oryzae
The effect of EF and MB fumigants on PH 3 -resistant S. oryzae eggs, early larvae, late larvae, pupae, and adults was evaluated. EF showed insecticidal rates of 100% at 70 mg/L for egg, early larvae, and pupae, 100% at 90 mg/L for adults, and 88.90% at 90 mg/L for late larvae ( Figure 6).
MB treatment showed a 100% insecticidal rate at 8 mg/L for eggs, 10 mg/L for early larvae and adults, whereas late larvae and pupae showed a 98.9% insecticidal rate at 20 mg/L (Figure 7). The LCt values were analyzed based on the effect of the fumigants on PH 3 -resistant S. oryzae. The LCt 50 value of EF was the highest (149.028 mg h/L), and the LCt 50 of MB was the lowest (25.840 mg h/L). The LCt50 of EF was 5.76 times higher than that of MB.

LCt Analysis of the PH 3 -Susceptible and PH 3 -Resistant Rice Weevils
The LCt 50 and LCt 99 values of the PH 3 -susceptible and PH 3 -resistant strains of rice weevils were analyzed based on the results of the effects of the three fumigants.
The LCt analysis results for EF showed that the LCt 50 values of EF were confirmed to be the highest at pupal stage (255.797 mg h/L) and the lowest at early larval stage (60.110 mg h/L). The LCt 50 values of EF on the PH 3 -resistant strains showed a different aspect with the previous one, that egg stage was the lowest (60.034 mg h/L) and late larval stage was the highest (149.028 mg h/L). PH 3 -resistant strain showed higher LCt 50 than PH 3 -susceptible strain at early larval stages, but lower LCt 50 at late larval and pupal stages, which are tolerant stages (Table 1).
The LCt 50 values of MB for the PH 3 -susceptible strain were the highest at pupal stage (21.104 mg h/L) and the lowest at egg stage (9.997 mg h/L), and the LCt 50 value of MB for the PH 3 -resistant strain were the highest at pupal stage (43.520 mg h/L) and the lowest at early larval stage (14.900 mg h/L). PH 3 -resistant strain showed higher LCt 99 than PH 3 -susceptible strain at all stages except adult, but did not exceed 2.06-fold ( Table 2).
As a result of LCt analysis for PH 3 , the LCt 50 values of the PH 3 -susceptible strain were the highest at pupal stage (6.171 mg h/L) and the lowest at adult stage (0.295 mg h/L). The LCt 50 values of the PH 3 -resistant strain were the highest at late larval stage (48.170 mg h/L) and the lowest at egg stage (6.595 mg h/L). The highest concentration needed to control the late larvae was different from that needed to control the susceptible strain. The concentration needed to control the eggs was 57.206 mg h/L, the concentration needed to control the early larvae was 107.914 mg h/L, the concentration need to control the late larvae was 241.311 mg h/L, the concentration need to control the pupae was 91.760 mg h/L, and the concentration needed to control the adults was 25.938 mg h/L. Control of rice weevil late larvae and pupae using PH 3 should be considered (Table 3).  The LCt50 values of MB for the PH3-susceptible strain were the highest at pupal stage (21.104 mg h/L) and the lowest at egg stage (9.997 mg h/L), and the LCt50 value of MB for the PH3-resistant strain were the highest at pupal stage (43.520 mg h/L) and the lowest at early larval stage (14.900 mg h/L). PH3-resistant strain showed higher LCt99 than PH3-susceptible strain at all stages except adult, but did not exceed 2.06-fold ( Table 2).
As a result of LCt analysis for PH3, the LCt50 values of the PH3-susceptible strain were the highest at pupal stage (6.171 mg h/L) and the lowest at adult stage (0.295 mg h/L). The LCt50 values of the PH3-resistant strain were the highest at late larval stage (48.170 mg h/L) and the lowest at egg stage (6.595 mg h/L). The highest concentration needed to control the late larvae was different from that needed to control the susceptible strain. The concentration needed to control the eggs was 57.206 mg h/L, the concentration needed to control the early larvae was 107.914 mg h/L, the concentration need to control the late larvae was 241.311 mg h/L, the concentration need to control the pupae was 91.760 mg h/L, and the concentration needed to control the adults was 25.938 mg h/L. Control of rice weevil late larvae and pupae using PH3 should be considered (Table 3).  The LCt50 values of MB for the PH3-susceptible strain were the highest at pupal stage (21.104 mg h/L) and the lowest at egg stage (9.997 mg h/L), and the LCt50 value of MB for the PH3-resistant strain were the highest at pupal stage (43.520 mg h/L) and the lowest at early larval stage (14.900 mg h/L). PH3-resistant strain showed higher LCt99 than PH3-susceptible strain at all stages except adult, but did not exceed 2.06-fold ( Table 2).
As a result of LCt analysis for PH3, the LCt50 values of the PH3-susceptible strain were the highest at pupal stage (6.171 mg h/L) and the lowest at adult stage (0.295 mg h/L). The LCt50 values of the PH3-resistant strain were the highest at late larval stage (48.170 mg h/L) and the lowest at egg stage (6.595 mg h/L). The highest concentration needed to control the late larvae was different from that needed to control the susceptible strain. The concentration needed to control the eggs was 57.206 mg h/L, the concentration needed to control the early larvae was 107.914 mg h/L, the concentration need to control the late larvae was 241.311 mg h/L, the concentration need to control the pupae was 91.760 mg h/L, and the concentration needed to control the adults was 25.938 mg h/L. Control of rice weevil late larvae and pupae using PH3 should be considered (Table 3).

Discussion
In most species, egg and pupal stages are reported to be the most tolerant developmental stages to the fumigants, whereas S. oryzae pupae were the most tolerant [35]. In Phthorimaea operculella, pupal resistance was the strongest under EF treatment [36]. Rhyzopertha dominica and Tribolium castaneum showed 100% mortality under 24 h EF treatment, while S. oryzae survived in the immature stages [37]. In another study of S. oryzae and T. castaneum, the tolerance of eggs was the lowest and that of the pupae was the highest [22]. In this study, the PH 3 -sensitive S. oryzae and PH 3 -resistant S. oryzae were treated with PH 3 , EF, and MB fumigants, and the PH 3 -resistant S. oryzae was treated with EF. The lowest tolerance was confirmed in eggs.
There is a growing worldwide problem of PH 3 resistant grain pests, and PH 3 resistant S. oryzae has also been reported in various countries. As a result of the FAO test (0.04 g/m 3 ) on S. oryzae, R. dominica, and T. castaneum for the detection of PH 3 resistance in Morocco, 18 of 19 groups of S. oryzae showed PH 3 resistance [18]. As PH 3 -resistant S. oryzae is increasing, FAO test was performed to confirm the PH 3 resistant S. oryzae of South Korea. Here, we report the first confirmed the existence of PH 3 -resistant S. oryzae in South Korea. In general, exposure time of pests to PH 3 takes three to five days to have an insecticidal effect, while exposure time of Sitophilus sp. to PH 3 requires 12 days or more [13,38]. Chaudhry [13] found that resistant pests did not meet sufficient resistance levels at past disinfection sites, even with the long-term use of phosphine to control S. oryzae, but under insufficient disinfection time, some surviving insects have rebuilt their population. Insecticides must be applied repeatedly at overdose levels to cause resistance. Therefore, the airtightness of fumigation chamber should be maintained and fumigation time should be considered when applying PH 3 in the field. Efficacy of PH 3 was found to be affected by the exposure time more than the concentration [39]. Therefore, in this study, the concentration was fixed and the treatment time was varied to control PH 3 -resistant S. oryzae. PH 3 -resistant S. oryzae treated with 0.5 mg/L PH 3 for 502 h was able to achieve 100% mortality. Therefore, PH 3 should be exposed for approximately 21 days to control PH 3 -resistant S. oryzae. PH 3 could be used to treat the PH 3 resistant S. oryzae for 100% mortality rate, but a long exposure time is needed. It cannot be used in the field because of long exposure time. Therefore EF, which shows effect in short time, can used to control PH 3 resistant S. oryzae instead of PH 3 . EF was first reported to be effective in box-car grain fumigation in Australia, and the use of EF in grains has been proposed for the last 40 years, but it has been difficult to apply in the field because of the disadvantages of low insecticidal activity and high flammability compared to other fumigants [40]. In this study, EF was treated at 20 • C, and 90 mg/L or more had to be added to control all of the PH 3 -susceptible and -resistant S. oryzae. Since EF has a low explosion threshold (85 g/t), more studies should be conducted to increase the efficacy of EF at lower concentration to avoid explosion.

Conclusions
EF was used as a substitute to treat PH 3 -susceptible and -resistant S. oryzae, but EF required a higher concentration of fumigant than that of other fumigants. To use EF to control S. oryzae, it is necessary to carry out studies to increase the insecticidal effect at a low concentration.