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Article

Development of a Concurrent Treatment Technique of Ethyl Formate and Mixtures (Nitrogen, Phosphine) to Control Citrus Mealybug (Planococcus citri)

by
Bongsu Kim
1,
Min-Goo Park
2,
Gil-Hah Kim
3 and
Jeong-Oh Yang
4,*
1
Plant Quarantine Technology Center, Animal and Plant Quarantine Agency (APQA), Gimcheon 39660, Republic of Korea
2
Department of Bioenvironmental Chemistry, Jeonbuk National University, Jeonju 54896, Republic of Korea
3
Department of Plant Medicine, College of Agriculture, Life and Environment Science, Chungbuk National University, Cheongju 28644, Republic of Korea
4
Youngnam Plant Pest Surveillance and Control Centre, Animal and Plant Quarantine Agency (APQA), Busan 48943, Republic of Korea
*
Author to whom correspondence should be addressed.
Insects 2023, 14(9), 720; https://doi.org/10.3390/insects14090720
Submission received: 15 June 2023 / Revised: 13 August 2023 / Accepted: 17 August 2023 / Published: 22 August 2023
(This article belongs to the Section Insect Pest and Vector Management)
Versions Notes

Abstract

:

Simple Summary

Mealybugs (Hemiptera: Pseucoccidae) are frequently detected pests when importing fresh commodities. Ethyl formate, a methyl bromide alternative fumigant, has been applied to imported bananas in Korea to eliminate mealybugs, but it represents a considerable cost for production and management. A liquid ethyl formate treatment technology was newly developed to reduce the cost, and the efficacy of this technology against Planococcus citri, a highly important species of mealybug in Korea, was evaluated in this study. In addition, the effect of enhancing the control against P. citri was evaluated through the simultaneous treatment of ethyl formate and phosphine. The results show that, for the treatment with ethyl formate + nitrogen, the control effect did not decrease with the addition, and fruit damage did not occur as the nitrogen concentration increased. In the case of the combined treatment with phosphine, the control effect was greatly increased even when a small amount (0.1 g/m3) was applied. The results indicate that the new technology can be used to control mealybugs on banana fruits, and a combined treatment of ethyl formate and phosphine can also enhance the efficacy against mealybugs.

Abstract

Currently, the fumigant ethyl formate (EF) is stored as a liquified gas in metal cylinders mixed with carbon dioxide (CO2), but this product type is expensive to manufacture, transport, and maintain in cylinders. To address these problems, we developed a new EF fumigation technique with a nitrogen (N2) carrier. In this report, the susceptibility of citrus mealybugs, one of the most resistant mealybugs to fumigants, to EF was assessed; the phytotoxicity of an EF + N2 concurrent treatment applied to banana fruit was examined to evaluate the efficacy compared to the current EF + CO2 product; and the increased efficacy with a phosphine (PH3) addition to EF + N2 was also assessed. Concurrent treatment of EF and N2 was performed at an LC50 concentration of EF. N2 was applied in seven doses from concentrations of 79% to 95%. The phytotoxicity of EF to bananas was assessed by applying EF at 35 mg/L with N2 at 79%, and the color, sugar content, and weight loss of bananas were measured for 14 days after treatment. The EF with N2 treatment resulted in more than 50% mortality at all growth stages of the mealybug, and there was no significant difference between the untreated and treated banana fruits. EF mixed with PH3 showed a higher efficacy than treatment with EF alone, but only a slight increase in efficacy was observed when the PH3 concentration increased. These results indicate that concurrent treatment with EF and N2 can be used to control mealybugs on banana fruits, and combined treatment with EF and PH3 can also enhance the efficacy against mealybugs.

1. Introduction

Dysmicoccus neobrevipes (Beardsley), Aspidiotus excisus (Green), and Dysmicoccus brevipes (Cockerell) have been detected frequently when importing bananas to Korea. Therefore, the development of a proper treatment method using a fumigant, a gas-type insecticide, is essential for eliminating mealybugs in commodities. Planococcus citri Risso (Hemiptera: Pseucoccidae) is known as a highly important species of mealybug in Korea [1]. In addition, when testing fumigants against mealybugs, P. citri was reported to have the highest insecticide resistance and has been previously studied [2]. For these reasons, P. citri has been commonly used to test the efficacy of fumigants in Korea.
A fumigant is a synthetic chemical that, at the required temperature and pressure, exists in a gaseous state in sufficient concentrations to be lethal to a target pest [3]. Methyl bromide (MB), which has been used as a plant quarantine fumigant, has been designated as an ozone-layer-depleting substance under the Montreal Protocol enacted in 1989 [4]. In 2008, the IPPC (International Plant Protection Convention) adopted a recommendation for the use of an alternative to MB for plant quarantines [5]. MB not only causes the destruction of the ozone layer, but also causes damage to fresh plants such as imported and exported fruits, and related problems with disinfection have been documented [6,7]. MB causes chronic poisoning in people who perform the disinfection treatment, as well as environmental destruction and toxicity, raising the issue of human safety [8]. Due to problems such as the destruction of the ozone layer by MB, plant damage, and human safety, research on the development of alternative fumigants to MB has been actively conducted worldwide [8,9]. As replacement fumigants for MB, many studies have been conducted on ethyl formate (EF) and phosphine (PH3) in Korea.
Currently, EF fumigants (Vapormate, BOC Ltd., Wagga, Australia) used in Korea are in the form of a mixture of carbon dioxide (83.3%) and ethyl formate (16.7%). EF is a plant volatile substance that has insecticidal effects [10,11,12]. Many synergistic effects have been reported when the ratio of carbon dioxide is increased [13]. The EF fumigant is a fumigant that shows an insecticidal effect when pests breathe after being treated with EF in a gaseous state in an enclosed space. PH3 has been extensively used against insects, especially storage pests [8,14]. PH3 can control pests with a long-term treatment [15,16,17,18], has good penetration without affecting the quality of various grains, and is adsorbed onto plant or leaf residues. Because it does not cause contamination, PH3 is used worldwide for storage pest control [15,19,20].
These two fumigants have been set as the standards for the disinfection treatment of fruits upon import in Korean plant quarantine, and are used in the field according to import requirements [21]. Since both fumigants contain highly explosive substances such as active ingredients, they are manufactured in the form of a high-pressure cylinder containing a large amount of carbon dioxide (83.4% CO2 in EF fumigants, 98% CO2 in PH3 fumigants). That is, the currently used EF fumigant is applied by adding CO2 into a high-pressure gas, spraying liquid EF using a dip tube in a gas pipe, and then vaporizing it through a vaporizer, and a PH3 fumigator uses a similar method. Generally, these fumigants are stored in cylinders in the form of a high-pressure gas because they contain CO2 [22]. Such a high-pressure gas cylinder represents a considerable cost for their production, management and safety during movement of the high-pressure gas. Therefore, it is necessary to compensate for the disadvantages of the existing method and diversify the use of fumigants.
In this study, to supplement the existing cylinder method used to replace MB and diversify plant quarantine fumigants, liquid EF, which was recently developed, was vaporized in a vaporizer, and nonflammable gases, such as nitrogen (N2), were connected to the outside of the vaporizer and dosed. Using this technology [23], efficacy against P. citri was evaluated, and the phytotoxicity was evaluated on imported bananas. In addition, the effect of enhancing the control against scale insects was evaluated through the simultaneous treatment of EF fumigants and PH3 fumigants.

2. Materials and Methods

2.1. Insects and Bananas

Stock colonies of the citrus mealybug, P. citri, were successively reared on eyed potatoes at 25 ± 1 °C at a relative humidity of 60% and a photoperiod of 16:8 h (L:D) at the Plant Quarantine Technology Center in South Korea.
Six boxes of banana (13 kg per box, Dole Food Company, Dublin, Ireland) samples imported from the Philippines were stored at 13 °C until use. The average size of each banana was 500 ± 10 g.

2.2. Chemicals

EF (97%, Sigma–Aldrich, St. Louis, MO, USA) was of analytical grade and purchased from Aldrich Chemical Company Inc. (St. Louis, MO, USA). PH3 was obtained as ECO2Fume™ (2% PH3 + 98% CO2) from Cytec (Sydney, Australia). N2 was of high purity grade and purchased from SJ GasTech Inc. (Gumi, Republic of Korea).

2.3. Fumigants and Nitrogen Treatments

EF and PH3 were used to evaluate fumigant susceptibility to P. citri. EF was in the form of liquid EF (EF, 97%) and PH3 was in the form of ECO2Fume (2% PH3 + 98% CO2).
In the EF and N2 mixture treatment performed to investigate the effect of N2 during EF treatment, an OXYBABY gas analyzer (Wittgas, Witten, Germany) was used to measure and adjust the N2 concentration in a small-scale fumigation chamber (55 L), and a medium-scale (0.5 m3) N2 mixture processor (SafeFUME, Daegu, Republic of Korea) was used for liquid EF vaporization and diffusion in fumigation when evaluating banana toxicity. The N2 mixture processor was composed of a metal mesh net and heater for vaporizing the mixture of liquid EF and nitrogen gas, a controller for adjusting the heating temperature and heating time, and an outlet for discharging the vaporized mixed gas.

2.4. Fumigation

A 55 L fumigation system (UBNC, Incheon, Republic of Korea) was used to evaluate the fumigant efficacy for P. citris. The 55 L fumigation system consisted of a gas injection, sampling port, gas exhaust valve and pressure gauge, and a handle stopper for sealing was installed on the outside. Inside the fumigator, a miniature fan (Φ 10 cm, UBNC, Incheon, Republic of Korea) was placed for the diffusion and circulation of the fumigant.
A 0.5 m3 (0.5 × 1.25 × 0.8 m3) stainless steel fumigation chamber was used to investigate the weakening of bananas according to EF and N2 mixture treatment, and the 0.5 m3 stainless steel fumigation chamber was installed in a temperature-controllable container (13 ± 1 °C) and then carried out.

2.5. Inspector Preparation, Fumigator Treatment, and Insecticidal Rate Investigation

The fumigant efficacy of P. citri was evaluated on eggs, nymphs, and adults. Filter paper (Advantec, Tokyo, Japan) was laid in the insect breeding dish (Φ 5.5 cm × 1.5 cm, SPL, Pocheon, Republic of Korea), distilled water was applied for moisture supply, and potatoes were sliced thinly and then inoculated for each brood.
Fumigant treatment was performed at the Plant Quarantine Technology Development Center of the Agriculture, Forestry and Livestock Quarantine Headquarters. In a fumigation room, at a temperature of 13 ± 1 °C and a relative humidity of 60 ± 10%, the prepared mealybugs were placed in a 55 L desiccator (UBNC, Incheon, Republic of Korea) and then fumigated. The insecticidal rate was observed after 72 h of breeding under the same conditions as the subculture after treatment with a fumigant. The insecticidal rate was investigated using a stereomicroscope (MDG33, Leica, Wetzlar, Germany), and if there was no movement or only very weak movement when touched with a fine needle, the specimen was considered dead. All experiments were performed in triplicate for 4 h.
To measure the state change of bananas according to the fumigant treatment, a fumigant toxicity study was conducted. For fumigant resistance, bananas (Dole Food Company) were placed in a 0.5 m3 stainless fumigation chamber, and 35 mg/L EF and N2 (80, 83, 85, 90, 93, 95%) were mixed and treated. At 1, 7, and 14 days after fumigant treatment, the internal (peeled body) and external (outer skin) color, sugar content, and weight loss rate according to weight change in bananas were investigated.

2.6. Measurement of Fumigant Concentration

To determine the actual concentration of the fumigation treatment, monitoring was conducted for all experimental groups, and 60 mL samples were extracted per experimental group using a 60 mL syringe in a 1 L Tedlar bag (SKC, Dorset, UK). The samples were taken and monitored at 30 min and 1, 2, and 4 h after fumigation.
The concentration of EF was 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 the split mode (10:1). The PH3 concentration was determined using an Agilent GC 7890A that was equipped with a flame photometric detector (FPD) and HP-PLOT/Q (30 m × 530 μm × 40 μm, Agilent, Santa Clara, CA, USA) operating in a 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 and PH3 were 60 and 20 μL and 1.5 and 5 mL/min, respectively. The concentrations of EF and PH3 were calculated based on peak areas against external standards.

2.7. Measurement of Fumigant Concentration × Time (CT)

The fumigant concentration was calculated using an equation outlined by Monro [24], based on the monitoring results performed at regular intervals.
CT = ∑(Ci + Ci + 1) (ti + 1 − ti)/2
where
  • C is the fumigant concentration (mg/L);
  • t is the time of exposure (h);
  • i is the order of measurement;
  • Ct is the concentration × time (mg h/L).

2.8. Statistical Analysis

Based on the results of EF, EF + N2 and EF + PH3 fumigant susceptibility targeting P. citris, the significance test by concentration was performed using an ANOVA with Tukey’s HSD test method using the SPSS program (IBM Corporation, Armonk, NY, USA). The LCT50 and LCT99 values were obtained using the Probit analysis [25] program.

3. Results

3.1. Evaluation of the Susceptibility of P. citri to Liquid EF Treatment

The fumigant efficacy was evaluated when liquid EF treatment was performed on the adults, nymphs, and eggs of P. citri. P. citri adults showed a 100% insecticidal effect when treated with 30.0 mg/L liquid EF for 4 h. At this time, the CT product (concentration × time) was 100.68 mg h/L (Table 1). Nymphs also showed a 100% insecticidal effect against the CT value, similar to adult insects. However, to control the eggs, a 100% insecticidal effect was shown when 70.0 mg/L liquid EF was added for 4 h, and at this time, the CT was 193.67 mg h/L (Table 1).
Statistical analysis was performed using lethal CT on the results of fumigant efficacy evaluation for all forms of P. citri (Table 2). As a result of comparing the lethal dose LCT50 for each fetus, the order of egg (43.946) > adult (39.244) > nymph (19.921) was found. Additionally, the LCT90 and LCT99 values showed a similar tendency to the LCT50 value, which were in the order of eggs (80.339, 131.388) > adults (72.369, 119.193) > nymphs (53.170, 118.383).

3.2. Evaluation of Susceptibility to P. citris according to the N2 Concentration during Simultaneous Treatment with Liquid EF and N2

The test was conducted at the LCT50 concentration of liquid EF (adult; 39.244, nymph; 19.921, egg; 43.946) and at six N2 concentrations of 80, 83, 85, 90, 93, and 95%. The insecticidal effect toward P. citri adults was significant and increased as the N2 concentration increased; the insecticidal effect reached 100% when the N2 concentration was 95%. There was no significant difference in the insecticidal effect between the eggs and nymphs with a change in N2 concentration (Table 3).

3.3. Evaluation of the Susceptibility of P. citris to Liquid EF and PH3 Treatment

The fumigant efficacy was evaluated when liquid EF and PH3 were combined with the adults, nymphs, and eggs of P. citri. P. citri adults showed a 100% insecticidal effect when treated with 8.0 mg/L liquid EF and 0.1 mg/L PH3 for 4 h (Table 4). When PH3 was applied at 0.1 mg/L, the LCT50 and LCT99 values of EF were 2.475 and 12.216 mg h/L, respectively (Table 5). Nymphs showed a 100% insecticidal effect when treated with 10.0 mg/L liquid ethyl formate and 0.1 mg/L phosphine for 4 h (Table 4). When phosphine was treated at 0.1 mg/L, the ethyl formate LCT50 and LCT99 values were 0.060 and 29.660 mg h/L, respectively (Table 5). However, eggs did not show a 100% insecticidal effect when treated with 50 mg/L ethyl formate based on 0.1 mg/L phosphine for 4 h. Treatment with 30.0 mg/L liquid ethyl formate and 0.5 mg/L phosphine showed 100% insecticidal effect when treated for 4 h (Table 6). When phosphine was applied at 0.5 mg/L, the LCT50 and LCT99 values of ethyl formate were 20.063 and 48.824 mg h/L, respectively (Table 5). A 100% insecticidal effect was shown when the phosphine concentration was 1.0 mg/L and the ethyl formate concentration was 30 mg/L with 4 h of treatment (Table 6). When phosphine was applied at 1.0 mg/L, the ethyl formate LCT50 and LCT99 values were 2.280 and 40.474 mg h/L, respectively (Table 5).

3.4. Evaluation of the Phytotoxicity of Imported Bananas when Treated with Liquid Ethyl Formate and Nitrogen

After treating imported bananas with liquid ethyl formate and nitrogen in parallel, phytotoxicity to bananas was evaluated at 1, 7, and 14 days (Table 7). In the case of external chromaticity, there was no significant difference between the treatment group and the untreated group until 7 days after treatment, but a slight change was observed in the EF treatment group compared to the untreated group after 14 days. In the case of internal chromaticity, there was no significant difference between the untreated and treated groups. At 1, 7, and 14 days after treatment, there was no significant difference in the sugar content and fruit weight loss between the treated and untreated groups.

4. Discussion

As an alternative to methyl bromide, Korea mainly uses two fumigants and applies them as alternatives at quarantine sites. These fumigants are an ethyl formate fumigant (EF 16.7%, CO2 83.3%) and a phosphine fumigant (PH3 2%, CO2 98%). The combined fumigant was produced in the form of a mixture of CO2 to suppress the flammability of EF or the explosiveness of PH3. In this form, the cylinder is relatively thick to hold the high pressure of CO2, and the weight of the cylinder increase when the thickness increases. The weight of the basic ball cylinder reaches 50–70 kg. The technology for safely manufacturing cylinders increases the price of this fumigant, making it less economical. In addition, heavy cylinders limit portability in the field. The use of a large amount of CO2 creates the problem of CO2 emission, which negates the purpose of replacing MB as an ozone-depleting substance [26]. Vaporizing liquid EF by using N2 is a method of separating liquid EF and N2 and using it for disinfection treatment. Liquid EF is filled into a CO2 cylinder that is currently in use. This is a method that improves movement safety compared to the method of using a mobile device [17,18,27,28,29]. In this study, EF was injected into a vaporizer in a liquid form rather than a gas form, vaporized, and then injected into the fumigation chamber using N2 gas. In addition to reducing CO2 emissions, this method is reported to be able to process chemicals in a short time and disinfect relatively inexpensively compared to the conventional method [30].
As a result of the test, liquid EF treatment showed that it was more difficult to control P. citri eggs than nymphs and adults. When mixing EF and N2, the control effect on adults increased as the N2 concentration increased, but the control effect on the nymphs and eggs was not increased. Generally, pest eggs are known to be difficult to control with fumigants due to their low respiratory rate [31]. In the case of P. citri, it is also known that egg control is difficult [32]. Accordingly, it has been determined that all scale insects are controlled only when the concentration of eggs is completely controlled. On the other hand, the reason why the control effect of adult embryos increased with increasing N2 treatment concentration seems to be that adult embryos are more affected by oxygen concentration than other embryos [33,34]. In addition, Jamieson et al. [35] reported that the insecticidal effect significantly increased when EF was applied under ultra-low oxygen conditions. An increase in insecticidal effect has also been reported for other gases, and there is a report confirming that the insecticidal effect of rice pests increases as the CO2 concentration increases (5, 10, 20%) when mixing EF and CO2 [13]. However, since there is no enhanced insecticidal effect on eggs with the highest resistance, it seems difficult to reduce the dose compared to existing cylinder products during N2 mixing treatment. As a result of this study, it was determined that N2 can be used as a mixed-treatment gas because the control effect did not decrease with addition, and damage did not occur as the N2 concentration increased during liquid EF treatment.
In the case of PH3 treatment in parallel to enhance the efficacy of liquid EF, the control effect was greatly increased in all embryos, even when a small amount (0.1 g/m3) was applied, and the control effect was especially increased for eggs. Yang et al. [17,18] confirmed the increase in the control effect of scale insects in pineapple when treated with EF and PH3 in a cylinder formulation, and Cho et al. [36] also reported the enhancement of the control effect when treated in parallel against two species of mealybugs of the genus Pseudococcus that attack seedlings. In the results of this study, a synergistic effect can be confirmed, so it seems that liquid EF can enhance the control effect when mixed with EF + PH3 under the same cylinder formulation conditions. For field application of the EF and N2 mixture developed in this study, an evaluation of imported bananas was carried out. Other quality indicators, such as color, did not show a significant difference compared to untreated plants, so it was determined that combined liquid EF and N2 treatment can be used at plant quarantine disinfection treatment sites. It has been reported that liquid EF fumigant can be applied to imported bananas [29]. Additionally, Yang et al. [17,18,27,28] reported that the combined treatment of EF and N2 was effective for reproduction. These results suggest that it can be used as a substitute for MB in disinfection and quarantine sites.

5. Conclusions

The results indicate that concurrent treatment with EF and N2 can be used to control mealybugs in banana fruits, and combined treatment with EF and PH3 can also enhance efficacy against mealybugs.

Author Contributions

Conceptualization, J.-O.Y.; methodology, B.K. and J.-O.Y.; formal analysis, B.K., M.-G.P. and J.-O.Y.; investigation, B.K. and M.-G.P.; resources, G.-H.K. and J.-O.Y.; data curation, B.K., G.-H.K. and J.-O.Y.; writing—original draft preparation, B.K.; writing—review and editing, G.-H.K. and J.-O.Y.; supervision, J.-O.Y.; project administration, J.-O.Y.; funding acquisition, J.-O.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Research Fund of Animal and Plant Quarantine Agency in 2021–2023 (F-1543086-2021-23-02).

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Table 1. Mortality of P. citri (adults, nymphs, and eggs) exposed to liquid EF fumigant for 4 h at 13 °C in a 55 L fumigation chamber.
Table 1. Mortality of P. citri (adults, nymphs, and eggs) exposed to liquid EF fumigant for 4 h at 13 °C in a 55 L fumigation chamber.
Liquid EF
Concentration
(mg/L)		CT
(mg h/L)		AdultsNymphsEggs
nMortality ± SE a (%)nMortality ± SE a
(%)		nMortality ± SE a
(%)
Control0.001400.00 ± 0.00 a c3190.00 ± 0.00 a781.33 ± 1.33 a
1.56.30- b-1738.28 ± 1.91 a--
6.019.381558.95 ± 3.07 a13146.93 ± 12.52 b--
10.022.85----13410.40 ± 4.84 a
12.031.99----8423.70 ± 2.60 b
15.051.0914363.67 ± 7.16 b16285.09 ± 3.91 c--
30.0100.68154100.00 ± 0.00 c185100.00 ± 0.00 c--
40.0112.65----13697.78 ± 2.22 c
60.0192.18148100.00 ± 0.00 c217100.00 ± 0.00 c--
70.0193.67----133100.00 ± 0.00 c
a SE is the standard error. b This concentration was not determined. c Mean values within each column followed by the same letter are not significantly different (p < 0.05).
Table 2. Lethal concentration time (LCT) of citrus P. citri (adult, nymph, and egg) exposed to liquid EF fumigant at 13 °C in a 55 L fumigation chamber.
Table 2. Lethal concentration time (LCT) of citrus P. citri (adult, nymph, and egg) exposed to liquid EF fumigant at 13 °C in a 55 L fumigation chamber.
StagenLCT50 (h)
(95% CL a)		LCT90 (h)
(95% CL)		LCT99 (h)
(95% CL)		Slope (±SE)
Adult74039.244
(15.992–55.618)		72.369
(49.901–116.490)		119.193
(82.976–323.723)		4.823 ± 0.662
Nymph118719.921
(13.260–26.405)		53.170
(41.801–68.909)		118.383
(88.478–181.474)		3.006 ± 0.247
Egg56543.946
(31.501–54.623)		80.339
(65.457–103.177)		131.388
(102.448–201.013)		4.892 ± 0.390
a CL denotes the confidence limit.
Table 3. Mortality of P. citri (adults, nymphs, and eggs) exposed to liquid EF fumigant + nitrogen (N2) for 4 h in a 13 °C fumigation chamber.
Table 3. Mortality of P. citri (adults, nymphs, and eggs) exposed to liquid EF fumigant + nitrogen (N2) for 4 h in a 13 °C fumigation chamber.
N2 Ratio
(%)		EF Concentration (mg/L)
14716
AdultsNymphsEggs
nMortality ± SE a (%)nMortality ± SE a (%)nMortality ± SE a (%)
798914.64 ± 5.92 a b28383.90 ± 2.00 a17884.06 ± 4.87 a
808848.65 ± 9.01 b20586.23 ± 10.62 a15189.09 ± 6.62 a
838750.62 ± 7.72 b25590.06 ± 3.43 a10788.83 ± 7.34 a
858459.38 ± 3.77 b36098.82 ± 0.64 a10779.21 ± 12.54 a
909063.33 ± 7.70 b34197.41 ± 1.41 a16387.09 ± 3.00 a
939070.00 ± 6.94 bc194100.00 ± 0.00 a14595.43 ± 3.16 a
9590100.00 ± 0.00 c18386.89 ± 9.96 a16089.14 ± 6.53 a
a SE is the standard error. b Mean values within each column followed by the same letter are not significantly different (p < 0.05).
Table 4. Comparison of the mortality of P. citri to EF over a range of concentrations and gas mixtures of EF + PH3 at 0.1 mg/L for the adults, nymphs, and eggs of the citrus P. citri under 4 h of exposure at 13 °C.
Table 4. Comparison of the mortality of P. citri to EF over a range of concentrations and gas mixtures of EF + PH3 at 0.1 mg/L for the adults, nymphs, and eggs of the citrus P. citri under 4 h of exposure at 13 °C.
Liquid EF
Concentration (mg/L)		AdultsNymphs
Mortality ± SE a (%)Mortality ± SE a (%)
0.000.00 ± 0.00 a b0.00 ± 0.00 a
0.05- c82.56 ± 6.65 b
0.10-89.56 ± 3.24 bc
1.0045.56 ± 4.01 b89.73 ± 1.71 bc
2.00-94.98 ± 2.00 bc
3.0090.00 ± 8.39 c98.17 ± 0.96 c
8.00100.00 ± 0.00 c-
10.00100.00 ± 0.00 c100.00 ± 0.00 c
a SE is the standard error. b Mean values within each column followed by the same letter are not significantly different (p < 0.05). c This concentration was not performed.
Table 5. Lethal concentration time (LCT) of citrus P. citri (adults, nymphs, and eggs) exposed to liquid EF fumigant at 13 °C in a 55 L fumigation chamber.
Table 5. Lethal concentration time (LCT) of citrus P. citri (adults, nymphs, and eggs) exposed to liquid EF fumigant at 13 °C in a 55 L fumigation chamber.
StagePH3 Conc. (mg/L)nLCT50 (h)
(95% CL a)		LCT99 (h)
(95% CL)		Slope (±SE)
Adults0.14422.475 (0.662–3.979)12.216 (8.705–24.441)3.356 ± 0.408
Nymphs0.16720.060 (0.000–0.305)29.660 (12.485–384.726)0.864 ± 0.173
Eggs0.1206817.113 (9.925–23.743)161.332 (100.773–401.264)2.388 ± 0.332
0.5165720.063 (17.482–22.385)48.824 (40.344–67.509)6.024 ± 0.625
1.011272.280 (0.051–6.252)40.474 (29.003–73.546)1.863 ± 0.345
a CL denotes the confidence limit.
Table 6. Comparison of the mortality of P. citri to EF over a range of concentrations and gas mixtures of EF + PH3 at 0.1 mg/L, EF + PH3 at 0.5 mg/L and EF + PH3 at 1.0 mg/L for eggs of the citrus P. citri under 4 h of exposure at 13 °C.
Table 6. Comparison of the mortality of P. citri to EF over a range of concentrations and gas mixtures of EF + PH3 at 0.1 mg/L, EF + PH3 at 0.5 mg/L and EF + PH3 at 1.0 mg/L for eggs of the citrus P. citri under 4 h of exposure at 13 °C.
Phosphine Conc. (mg/L)
Liquid EF
Concentration (mg/L)		0.10.51.0
Mortality ± SE a (%)Mortality ± SE a (%)Mortality ± SE a (%)
0.013.87 ± 3.58 a b4.63 ± 4.63 a8.65 ± 7.38 a
1.029.16 ± 8.55 a5.60 ± 1.30 a88.86 ± 4.33 b
3.035.26 ± 2.47 a10.29 ± 4.13 a-
5.0- c29.86 ± 14.02 ab93.70 ± 3.43 b
8.079.37 ± 7.58 b66.18 ± 14.77 bc-
10.087.87 ± 1.80 b93.26 ± 4.48 c97.65 ± 2.35 b
20.088.55 ± 2.47 b--
30.097.88 ± 1.28 b100.00 ± 0.00 c100.00 ± 0.00 b
50.099.66 ± 0.34 b-100.00 ± 0.00 b
a SE is the standard error. b Mean values within each column followed by the same letter are not significantly different (p < 0.05). c This concentration was not performed.
Table 7. Characteristics of bananas measured 1, 7, and 14 days after fumigation with EF + N2 fumigant at 13 °C in 0.5 m3.
Table 7. Characteristics of bananas measured 1, 7, and 14 days after fumigation with EF + N2 fumigant at 13 °C in 0.5 m3.
Days aTreatmentExternal Color b (Mean ± SE c)Internal Color (Mean ± SE)Sugar Content
(Brix %)		Weight Loss Rate (%)
L*a*b*L*a*b*
1 daysControl66.23 ± 2.81−6.39 ± 1.1237.53 ± 1.3265.24 ± 2.411.06 ± 0.7923.74 ± 1.7712.10 ± 1.070.00 ± 0.00
EF + N267.16 ± 2.33−7.16 ± 1.6237.12 ± 1.3870.42 ± 3.212.71 ± 3.4626.39 ± 2.5912.52 ± 1.880.00 ± 0.00
7 daysControl66.07 ± 2.360.61 ± 1.2741.92 ± 6.6256.28 ± 2.890.51 ± 0.4525.10 ± 2.0120.80 ± 1.150.42 ± 0.01
EF + N267.61 ± 3.251.61 ± 1.0241.95 ± 5.9661.30 ± 5.21−0.05 ± 3.9320.61 ± 3.7920.62 ± 1.350.35 ± 0.42
14 daysControl60.97 ± 6.034.06 ± 1.4338.50 ± 6.0955.31 ± 7.270.42 ± 1.4715.06 ± 4.0318.16 ± 2.900.77 ± 0.01
EF + N254.77 ± 1.884.52 ± 1.6528.90 ± 2.4356.02 ± 5.260.12 ± 1.6014.31 ± 2.3421.26 ± 1.130.70 ± 0.39
a Days after treatment. b Parameters for the color were based on CIE 1976 (L*, a*, b*) color space. L*: lightness, a*: green/magenta, b*: blue/yellow. c SE is the standard error.
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Kim, B.; Park, M.-G.; Kim, G.-H.; Yang, J.-O. Development of a Concurrent Treatment Technique of Ethyl Formate and Mixtures (Nitrogen, Phosphine) to Control Citrus Mealybug (Planococcus citri). Insects 2023, 14, 720. https://doi.org/10.3390/insects14090720

AMA Style

Kim B, Park M-G, Kim G-H, Yang J-O. Development of a Concurrent Treatment Technique of Ethyl Formate and Mixtures (Nitrogen, Phosphine) to Control Citrus Mealybug (Planococcus citri). Insects. 2023; 14(9):720. https://doi.org/10.3390/insects14090720

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Kim, Bongsu, Min-Goo Park, Gil-Hah Kim, and Jeong-Oh Yang. 2023. "Development of a Concurrent Treatment Technique of Ethyl Formate and Mixtures (Nitrogen, Phosphine) to Control Citrus Mealybug (Planococcus citri)" Insects 14, no. 9: 720. https://doi.org/10.3390/insects14090720

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