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Scaled-up ethyl formate fumigation to replace methyl bromide on traded mushroom to disinfest mushroom fly (Lycoriella mali)

Scaled-up ethyl formate fumigation to replace methyl bromide on traded mushroom to disinfest... Mushroom fly, Lycoriella mali (Diptera: Sciaridae), is the primary pest in imported mushrooms. The amount of Tricho - loma matsutake imported from China increases every fall when it is harvested. When importing T. matsutake, disinfes- tation using methyl bromide (MB) or phosphine (PH ) is performed to prevent the introduction of L. mali. However, MB will be phased out due to ozone-depletion, chronic toxicity to workers, and residual issues. PH fumigation in mushroom disinfestation requires a long exposure time (24 h). In this study, we used ethyl formate (EF), which can replace MB and reduce exposure time. The efficacy of EF, PH3 and EF + PH3 on L. mali was evaluated. Using 4-h EF fumigation at 5 °C, the 3rd and 4th instar was the most tolerant stage in terms of 99% killed lethal concentration × time products (LCt ). When 4-h EF fumigation at 5 °C was applied on all stages of L. mali, the LCt values of EF 99% 99% 3 3 3 were 73.1 g h/m to the 1st and 2nd instar, 112.9 g h/m to the 3rd and 4th instar, 68.9 g h/m to pupae, and 20.1 g h/ m to adult. It was confirmed that combination treatment with EF + PH had a synergistic effect on L. mali. The 3 3 3 LCt of EF + 0.5 g/m of PH to the 3rd and 4th instar was 48.3 g h/m . When only 140 g/m of EF was applied for 99% 3 3 3 4 h at > 5 °C and 35 g/m of EF + 0.5 g/m of PH for 4 h at > 5 °C in commercial trials containing T. matsutake, proven efficacy (100%) on L. mali was confirmed. In the case of EF treatment only, phytotoxic damage occurred due to high Ct products, and there was no phytotoxic damage in combination treatment with EF + PH . This study provides a new guideline for EF + PH combination treatment within a shorter exposure time (4 h) than existing PH treatment (24 h) 3 3 and replacement of MB use. Keywords: Lycoriella mali, Ethyl formate, Phosphine, Alternatives, EF + PH , Synergistic effect Introduction Efficacy of synthetic pesticides such as diflubenzuron, Flies of the family Sciaridae occur almost worldwide in diazinon, methoprene, and phosphate insecticides such many different cultivating and perishable commodities as dimethoate and acephate in L. mali has been reported [1]. Significant loss of cultivating mushrooms caused [2]. by several species such as Lycoriella mali and Lycoriella In the mushroom trade among several countries ingenua of sciarid in the mushroom industry has been including China and Korea, Lycoriella sp. is classified reported from the USA, UK, and South Korea [2–5]. as a quarantine pest in some countries, including South Korea, and must be treated by phytosanitary disinfes- tation at ports. According to KATI [6], South Korea *Correspondence: Byungholee@hotmail.com exported 7584 t of Pleurotus eryngii, the main exported Institute of Life Scienc, Gyeongsang National University, Jinju 52828, mushroom, and imported 145 t of Tricholoma matsutake Republic of Korea from mainly China in 2019. Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Kwon et al. Appl Biol Chem (2021) 64:64 Page 2 of 11 According to the phytosanitary guideline in Korea, Materials and methods imported mushrooms infested with L. mali must be Insects and chemicals chemically treated with methyl bromide (MB). In the Lycoriella mali was collected from a mushroom farm case of exported mushrooms, pest free inspection was in Yeongcheon, Gyeongbuk, South Korea during 2020. the last option to avoid rejecting them in countries where L. mali were transferred and reared in an insect rear- they are imported with the option not to treat with MB ing room at Gyeongsang National University. L. mali because it caused loss of quality. As well as phytotoxic was maintained in the insect rearing room at 24°C and damage to mushrooms treated with MB, its use has been 60–70% relative humidity (RH) with a 16:8 [L:D] h. phasing out because of ozone depletion properties and Pleurotus eryngii was provided as a food source. Female chronic toxicity to human in Korea [7]. MB fumiga- adults of L. mali lay eggs on water agar (2%) in the insect tion on food commodities could be more difficult in the breeding dish (100 mm × 40 mm). Larvae pupated within future because there is a need to update residual bro- 5–6  days, and adults emerged within 25  days; 1st, 2nd, mide ion and MB itself post all type of food commodi- 3rd and 4th instar larvae, pupae, and adults were used ties associated with the new Positive List System (PLS) in this study. EF (Fumate , >  99% purity; Hoengseong, in Korea [8]. In Korea, MB fumigation on treaded mush- Korea) was supplied by Safefume Co. Ltd in Korea. Phos- rooms will be discontinued after 2022 (Personal com- phine was purchased as ECO Fume (2% PH + 98% CO ) 2 3 2 munication with MG Park), because there is currently no from Cytec (Sydney, Australia). consumer safety data supported in Korea. In the case of PH fumigation, as current alternative options, commer- Egg hatching test at low temperature cial adaptation might be difficult because a long expo - Egg hatching studies of L. mali were performed at 5 ± 0.5°C sure time is required in grains (> 5 days) and mushrooms in an incubator. The eggs were collected form rearing cages (> 1  days), which could shorten the shelf life of mush- with 200 mated females on the Pleurotus eryngii over 1 day rooms [9]. and treated immediately. Before fumigation to eggs, 2% Ethyl formate (EF), an alternative to MB, is known agar medium was laid on the bottom of the breeding dish to be safer than other fumigants in the workplace and (50 mm × 15 mm) to maintain moisture and cut pine of P. there is residual free regulation in many countries eryngii was placed on the agar medium. Then 20 eggs of L. because it is globally classified as a food additive. In mali were transferred to each cut pine of P. eryngii. Because practice, EF fumigation has been used in imported com- the egg color is transparent, the cut pine of P. eryngii was modities such as fruits, vegetables, nursery plants, etc. dyed using natural pigments to make observation of the [10, 11]. A new concept of EF application technology eggs easier. Following placcement in a 5°C incubator, the with N (Non-CO ) was developed and used commer- 2 2 egg hatching rate was observed for 5 to 14 days. After 72 h cially in Korea [9]. of treatment, eggs were checked hatching rate. Treatment Although EF was effective fumigant, it has high sorp - was replicated three times, and the control was replicated tion [12] to commodities like perishable fruits and veg- 10 times at room temperature (24 ± 1°C). etables and more less vaporization at low temperature [13]. PH was good at permeability to commodites like timber [14]. Thus the new concept of fumigation has Efficacy to developmetal stages of L. mali with EF been studied that combined EF and PH , which was more in a laboratory experiment effective to insect pests and less phytotoxic damage to Efficacy of EF was evaluated for three different devel - commodities [15, 16]. opmental stages (adult, larvae and pupae) of L mali. In No study evaluating the efficacy of EF on L. mali has the case of EF mixed with PH studies, the 3rd and 4th been reported. Herein, we suggest new disinfestation instars of L mali, which is the most tolerant stage to EF, guidelines for disinfestation of L. mali using EF and was evaluated. Adults of L. mali were transferred from a EF + PH , which are a replacement for MB. breeding dish (50 mm × 15 mm) using collecting equip- We evaluated (1) Efficacy of EF for 4 h-fumigation on L. ment (Fulton, MX-991/U, Georgia) within three days of mali in lab studies, (2) sorption studies on several types developing an adult. Adults of L. mali were fumigated of mushroom and gas penetration under imported con- at 5°C for 4  h with 1.0–9.0  g/m of EF. The 1st and 2nd ditions, (3) Synergestic effect of EF and PH to L. mali instar and the 3rd and 4th instar were classified and and Phytotoxic damage to mushroom with EF + PH tested. From 1 to 7  days after hatching, the 1st and 2nd fumigation. (4) application of liquid EF with N applica- instar and 7 to 14  days were classified into the 3rd and tion on a commercial scale for confirmation on imported 4th instar. The pupae were used within two days after mushrooms. K won et al. Appl Biol Chem (2021) 64:64 Page 3 of 11 pupation. Larvae and pupae stages of L. mali were trans- (FPD) and HP-PLOT/Q (30  m × 530  µm × 40  µm, Agi- ferred from water agar (2%) to a breeding dish (50  mm lent, Santa Clara, CA) operating in split mode (10:1). The × 15 mm). The 1st and 2nd instar of L. mali were fumi - temperature of the injector and the oven was 200℃. The gated at 5°C for 4 h with 1.0–30.0 g/m of EF. The 3rd and temperature of the detector was 250°C. The injection vol - 4th instar were fumigated at 5°C for 4 h with 1.0–45.0 g/ umes and flow rate of EF and PH were 60 µl and 20 and m of EF. The pupae were fumigated at 5°C for 4  h with 1.5 and 5 ml/min, respectively. 1.0–40.0 g/m of EF. The concentration of EF was measured using an Agi - Determination of the synergistic effect of ethyl formate lent portable GC 17A (Shimadzu, Kyoto, Japan) equipped mixed with phosphine against the 3rd and 4th larvae of L. with a flame ionization detector (FID) after separation on mali a DB5-MS Column (30  m × 0.25  mm i.d., 0.25  µm film After efficacy to EF alone fumigation and EF combined thickness; J&W Scientific, Folsom CA). The oven tem -with PH fumigation, we evaluated synergistic effect. perature was 100°C The injector and detector tempera - A synergistic effect was measured by synergistic ratios tures were 250 and 280°C, respectively. Helium was used (SRs). Synergistic ratios are defined by Hewlett and as a carrier gas at the flow rate of 1.5 mL/min. Headspace Plackett [17] and based on equation. 1. EF was calculated by the peak area against external EF L(Ct) of ethyl formate only standards. SR = (1) L(Ct) of ethyl formate + phosphine After completion of 4 h fumigation, treated L. mali was transferred to an insect rearing room at 24°C and 60–70% *SR = 1 describes additive action, relative humidity (RH) with a 16:8 [L:D] h. The mortality SR < 1 describes antagonism, of the treated L. mali larvae and adults were determined SR < 1 describes synergism. by visual inspection of movement using a microscope 1 day after fumigation. The mortality of the treated L. mali Sorption test of T. mastutake for 4 h EF fumigation pupae was determined by checking the number of adults in a laboratory experiment for 5  days after fumigation. Usually, the rate of pupae to Evaluation of sorption of EF on T. matsutake was per- adults in the control group was 50% level. All treatments formed in a 6.8 L desiccator under lab condition. Each T. and controls were replicated three times. matsutake was filled at a 0.5, 1, and 1.5% filling ratio (w/v) The temperature was recorded using data loggers for the sorption test; 140  g/m of EF was applied using (Thermo Recorder TR-72Ui, T&D Corp., Japan). In this a syringe for 4  h at 5°C. Gas sampling was performed at study, a 6.8 L desiccator was used in the experiment as a time intervals (10 min, 1, 2, and 4 h) and measurements fumigation chamber. The desiccators were sealed with a were performed using an Agilent portable GC 17A (Shi- glass stopper equipped with a septum (Alltech Associates madzu, Kyoto, Japan) equipped with a flame ionization Australia, Cat. No. 15419). The exact volume of desicca - detector (FID) after separation on a DB5-MS Column tors was measured using weigh of water. The desiccators (30 m × 0.25 mm i.d., 0.25 µm film thickness; J&W Scien - were tightly sealed with high vacuum grease (Dow Corn- tific, Folsom, CA). ing, USA). A filter paper (Whatman No. 1) was inserted into the glass stopper to make clear evaporation in the desiccator for the injected EF. A magnetic bar to stir the Gas penetration test of EF on packed mushrooms fumigant was located at the bottom of the desiccator. The with logistic consideration (Styrofoam box) dose of fumigant and Ct products was calculated using In the case of import and export Tricholoma matsutake, the method reported by Ren et al. [14]. a styrofoam box with an ice pack was used as packing materials. Evaluation of gas penetration of EF on packed mushrooms (Tricholoma matsutake) was performed in Efficacy to 3rd and 4th larvae of L. mali with EF combined 3 a 0.275  m fumigation chamber at 5°C. Each mushroom with  PH in a laboratory experiment was filled at a 1.0% filling ratio (w/v) for the gas penetra - For EF + PH efficacy studies, the 3rd and 4th instars of L 3 tion test; 35, 70 and  140  g/m of EF was applied using a mali were trasnffered on agar petri dish in desiccator (6.8 special vaporizer (supplied by Safefume Co.) for 4  h at L). The larvae in the desiccators were fumigated at 5°C for 5°C. The two conditions (inside, outside of the Styrofoam 4  h with 4.0–28.0  g/m of EF mixed with 0.5 and 1.0  g/ box) of EF concentration were analyzed. Gas sampling m of PH . Analysis of EF concentration was as shown was performed at time intervals and measurements were above, the concentration of PH was measured using Agi- performed using an Agilent portable GC 17A. lent 7890A equipped with a flame photometric detector Kwon et al. Appl Biol Chem (2021) 64:64 Page 4 of 11 Commercial scaled‑up fumigation Phytotoxic assessment EF alone fumigation on Tricholoma matsutake The phytotoxic damage of EF on mushrooms (Pleuro - Scale-up fumigation was performed using a 25  m tus eryngii, Tricholoma matsutake,) was evaluated in Tarp-fumigation chamber in the Incheon airport ware- scaled-up studies (5  m fumigation chamber). Two types house in South Korea. Tricholoma matsutake were of mushrooms were filled at a 1.5% filling ratio; 140  g/ 3 3 placed in a styrofoam box (1.7% f.r.) and the cover m of EF for Pleurotus eryngii and 140  g/m of EF and 3 3 of the box was opened, which is based on commer- 35  g/m of EF + 0.5  g/m PH for Tricholoma matsutake cial use. Then, 140  g/m of EF was applied for 4  h at were applied using a special vaporizer (supplied by Safe- 5°C. Liquid EF was vaporized with SFM-I (supplied by fume Co.) for 4 h at 5°C. After completion of fumigation, Safefume Co.) with N as the carrier gas and a fan was mushrooms were transferred to storage at 5 ± 0.8°C. The placed at the bottom of the Tarp-chamber for efficient deterioration degrees were classified according to symp - gas circulation. The concentrations of the inside and toms of the water condensation surface of the cap and outside of the styrofoam box were analyzed. Three of changing a soft cap, grill, stem parts (0: Non, 1: water the insect breeding dishes containing > 300 larvae of L. condensation, 2: water condensation following changing mali (total 1079) were then located in the styrofoam a soft cap and grill parts 3: water condensation follow- box. Gas sampling was collected at time intervals (0, ing changing a soft cap, grill and then stem part). A color 1, 2, and 4 h) and EF concentration inside and outside change (hue value) using a color meter (TES 135A, Tai- of the styrofoam box was measured using GC-FID. wan), weght loss (%, weight differences before and after Because measurement with GC-FID directly at the treatment) and market value based on the hardness of field is impossible, the concentration was checked in head parts (the harder the best, the softer the worst) were advance at the field using a gas analyzer (IBRID MX6; evaluated 3-d after fumigation. Industrial Scientific, Pittsburgh, PA, USA). A previous study found no sigfinicant difference in the measured concentration of IBRID and GC-FID. Fumigated larvae Statistical analysis of L. mali were transferred to an insect rearing room. Analysis of the toxicological dose response to EF by L. The mortality of larvae was determined 1-d after fumi- mali was based on a Probit analysis (Finney, 1971). As gation. All treatments and controls were replicated part of the analysis, the slopes of the Probit transfor- three times. mations were determined as well as Chi-square tests of data homogeneity for different treatments. The indices of toxicity measurement derived from this analysis were EF fumigation mixed with  PH on Tricholoma matsutake L(Ct) = median lethal concentration that causes 50% Scale-up fumigation was performed using a 5  m fumi- 50% response (mortality) and L(Ct) = lethal concentration gation chamber in the same APQA site in South Korea. 99% that causes 99% response (mortality) of exposed L. mali Tricholoma matsutake were placed in a styrofoam box determined from a range of at least 10 different Ct prod - (0.5% f.r.) and the cover of the box was opened, which ucts to ensure that the observed data covered mortality is based on commercial use. Then, 35 g/m of EF mixed from 0 to 100% and adequately covered the intermediate with 0.5  g/m of PH was applied for 4  h at 5°C. Liq- range. For analysis of the hatchability of eggs on two tem- uid EF was vaporized using a vaporizer (supplied by peratures and phytotoxic damage assessment of EF fumi- Safefume Co.) with N as the carrier gas and a mini fan gation, on mushrooms, a T-test procedure was used to was placed at the bottom of the chamber for efficient compare the two sample means. The EF alone fumigation gas circulation. The concentration of PH was achieved and EF combined with PH fumigation against phytotox- by injecting 125  ml of 2% PH into a 5  m fumigation 3 icity of mushrooms were compared using Tukey’s test in chamber. The concentrations of the inside and outside scaled-up trials. All statistical analyses were performed of the styrofoam box were analyzed. Three insect breed - using SAS (ver. 9.4; SAS Institute Inc.) [18]. ing dishes containing > 300 larvae of L. mali (total 1,097) were then located in the styrofoam box. Gas sampling was collected at time intervals (0, 1, 2, and 4 h) and EF Results and discussion and PH concentrations inside and outside of the sty- Egg hatchability test at low temperature rofoam box were easured using GC-FID and GC-FPD. This study was conducted to investigate eggs hatching in Fumigated larvae of L. mali were transferred to an low temperature conditioned mushrooms. According to insect rearing room. The mortality of larvae was deter - previous ecological studies on L. mali, the average length mined 1-d after fumigation. All treatments and controls of a generation from egg to adult was 28 d at 21°C [19]; it were replicated three times. K won et al. Appl Biol Chem (2021) 64:64 Page 5 of 11 Table 1 Egg hatchability of Lycoriella mali under two conditions (5 ± 0.5℃, 24 ± 1.0℃) Day after oviposition Temperature (mean ± SE, No. of tested No. of hatched p‑ value Hatchability ℃) (mean ± SE, %) 5 5 ± 0.5 60 0 < 0.0001 0.0 ± 0.0 24 ± 1.0 60 52 88.3 ± 1.7* 6 5 ± 0.5 60 0 < 0.0001 0.0 ± 0.0 24 ± 1.0 60 60 100.0 ± 0.0 7 5 ± 0.5 60 0 < 0.0001 0.0 ± 0.0 24 ± 1.0 60 60 100.0 ± 0.0 8 5 ± 0.5 60 0 – 0.0 ± 0.0 9 5 ± 0.5 60 0 – 0.0 ± 0.0 10 5 ± 0.5 60 0 – 0.0 ± 0.0 11 5 ± 0.5 60 0 – 0.0 ± 0.0 12 5 ± 0.5 60 0 – 0.0 ± 0.0 13 5 ± 0.5 60 0 – 0.0 ± 0.0 14 5 ± 0.5 60 0 – 0.0 ± 0.0 All the unhatched eggs were emergedll at 6-d after oviposition - impossible to check Table 2 LCt (Lethal Concentration × time) value of EF fumigation for 4 h expoure on Lycoriella mali at 5 ± 0.5 ℃ Stage L(Ct) (95% CL) L(Ct) (95% CL) Slope ± SE df χ 50% 99% 1st, 2nd instar 43.6 (37.1–53.3) 73.1 (68.4–100.2) 3.1 ± 0.3 21 28.9 3rd, 4th instar 27.84 (22.53–33.01) 112.9 (90.70–152.25) 3.2 ± 0.3 28 48.47 Pupae 36.8 (28.9–46.1) 68.9 (57.6–97.1) 4.3 ± 0.3 15 40.2 Adults 7.8 (5.8–13.9) 20.1 (16.6–26.2) 2.0 ± 0.4 13 32.1 took five days from eggs to hatching. The egg hatchabil - ity under normal temperature condition (24 ± 1°C) were Table 3 Efficacy of PH3, EF and EF + PH fumigation for 4 h 88.3 ± 1.7% and 100.0 ± 0.0% at the day of the 5th and 6th expoure on Lycoriella mali at 5 ± 0.5℃ day after oviposition (Table  1). However, we confirmed Fumigant dose Ct products (g h/m ) Mortality ± SE (%) that the eggs under low temperature (5.0 ± 0.5°C) did not hatch at all. Based on logistic distribution of imported Control 0.0 0.0 ± 0.0 T. matsutake from China, it takes at least five days from PH 0.5 g/m 1.8 0.0 ± 0.0 harvest to consumers, all logistics is under < 5°C, mean- PH 1.0 g/m 3.5 0.0 ± 0.0 ing that survival of the egg might be diffidult under cold EF 6.0 g/m 14.8 20.0 ± 2.9 temperature conditions if found in imported mushrooms. EF 10.0 g/m 26.4 46.7 ± 1.7 EF 18.0 g/m 44.9 86.7 ± 1.7 Efficacy to developmetal stages of L. mali with EF EF 26.0 g/m 65.5 95.0 ± 0.0 3 3 in a laboratory experiment PH 0.5 g/m + EF 6.0 g/m 1.8 + 15.2 41.7 ± 1.7 The efficacy of 4  h EF fumigation (practical exposure PH 0.5 g/m + EF 10.0 g/ 1.8 + 26.6 40.0 ± 2.9 condtion using EF fumigations in Korea) on larvae, PH 0.5 g/m + EF 18.0 g/ 1.8 + 45.7 100.0 ± 0.0 pupae, and adults stages of L. mali at 5℃ is shown in 3 Table 2. For the 1st and 2nd of L. mali larvae, the L(Ct) 50% PH 0.5 g/m + EF 26.0 g/ 1.8 + 65.7 100.0 ± 0.0 and L(Ct) values of EF were 43.6, 73.1 g h/m . For the 99% 3rd and 4th of L. mali larvae, the L(Ct) and L(Ct) 50% 99% PH 1.0 g/m + EF 18.0 g/ 3.6 + 47.0 100.0 ± 0.0 values of EF were 25.0 and 112.9  g  h/m . The L(Ct) 50% and L(Ct) values of EF on L. mali pupae were 36.8 and PH 1.0 g/m + EF 26.0 g/ 3.5 + 65.6 100.0 ± 0.0 99% 3 3 3 68.9  g  h/m andadults were 7.8 and 20.1  g  h/m at 5°C (Table  2), respectively. Thus, the order of susceptively Kwon et al. Appl Biol Chem (2021) 64:64 Page 6 of 11 Table 4 LCt (Lethal concentration x time) value of EF mixed with 0.5 g/m PH for 4 h exposure on Lycoriella mali Temp (℃) Stage L(Ct) (95% CL) L(Ct) (95% CL) Slope ± SE df χ 50% 99% 5 3rd, 4th instar 34.4 (33.3–35.7) 48.3 (44.9–53.9) 15.8 ± 1.8 16 77.59 Table 5 Syngeristic efficacy of EF mixed with 0.5 g/m PH on 18.0  g/m for 4  h fumigation, Otherwise, EF combined 3rd–4th larvae stage of Lycoriella mali with PH of 0.5  g/m fumigation for 4  h was controlled a b 100% against 3rd and 4th instar of L. mali (Table 3). The Temp (℃) Stage Synergistic ratios Synergistic ratios L(Ct) L(Ct) L(Ct) and L(Ct) values of EF + 0.5 g/m PH against 50% 99% 50% 99% 3 the 3rd and 4th of L. mali were 34.4 and 48.3  g  h/m at 5 3rd–4th larvae 0.81 2.34 5  °C, respectively (Table  4). According to the results of Synergistic ratios (SRs): L(Ct) of ethyl formate only/ L(Ct) of ethyl 50% 50% a preliminary experiment at 5°C, the 3rd and 4th instar formate + 0.5 g/m phosphine were the most tolerant stages of L. mali. It was confirmed Synergistic ratios (SRs): L(Ct) of ethyl formate only/ L(Ct) of ethyl 99% 99% that application of EF + 0.5  g/m PH to L. mali had a formate + 0.5 g/m phosphine synergistic effect when treated L(Ct) at 5°C (Table  5). 99% The fumigant, PH was required more longer exposure of L. mali life stages to EF based on L(Ct) values was 99% times to kill insect pests than EF [21] and EF was needed adults > pupae > 1st and 2nd instar > 3rd and 4th instar to high concentration to control insect pests at low tem- larvae. Drosophila suzukii (Diptera: Drosophilidae) of the perature [22]. But mixed usage of these fumigants would other flies was also most tolerant larval stage except for be better to control insect pests than alone [15, 16]. eggs [20]. Sorption test of T. mastutake for 4 h EF fumigation in a laboratory experiment Efficacy to 3rd and 4th larvae of L. mali with EF combined Based on the efficacy studies resulting in L(Ct) val- 99% with  PH in a laboratory experiment ues of EF on L. mali larvae, which is the most tolerant to The efficacy of EF + 0.5  g/m PH fumigation for 4  h on EF fumigation in this study, estimated applicable sched- the 3rd and 4th of L. mali at 5°C is shown in Table 3. The ules (140  g/m EF for 4  h exposure) were performed mortality of L. mali was no effect with PH of 0.5 and on T. matsutake. The losses of EF inside the fumigated 1.0  g/m for 4  h fumigation, and was 86.7% with EF of desiccators and their adsorption are shown in Fig.  1. 0.9 0.50% 1% 1.50% 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Fumiga on me (h) Fig. 1 Concentration loss of EF fumigation (140 g/m at 5℃ for 4 h) on Tricholoma matsutake C/C0 K won et al. Appl Biol Chem (2021) 64:64 Page 7 of 11 The concentration of EF decreased during 4  h of expo - 100  g/m of EF might be applicable. However, > 100  g/ sure on mushrooms. Loss rate of EF on T.matsutake was m of EF could be obtained with proven efficacy (> 99%) relatively low, approximately 30% at f.r. 1.5%. But EF was under the worst circumstance of commercials. EF con- required high concentration due to their high sorption to centration inside the styrofoam box containing T. mat- commodities like fruits and vegetable, which can cause to sutake was lower than outside. The difference of EF phytoxic damage to commodities [23–25]. concentration inside and outside the box might be dif- ferent depending on fanning conditions (running time, Gas penetration test of EF on packed mushroom numbers of fans, locations of fan, electrical capacity, with styrofoam box etc.) during the EF fumigition. Based on unpubuished A gas penetration test was performed using a 0.275  m data, we suggested using a fan for more than 2 h at low- fumigation chamber with different filling ratios of T. middle capactity for even distribution of gas. matsutake. As a result of EF gas penetration, there was a difference between the inside and outside of Commercial sized EF fumigation the styrofoam box (Fig.  2). When EF 35, 70, 140  g/ EF alone fumigation on Tricholoma matsutake m was applied, Ct products inside the styrofoam box The scaled-up EF fumigation to confirm disinfestation 3 3 were 60.2 ± 2.4, 95.0 ± 2.9 and 167.1 ± 6.1  g  h/m at on L. mali was performed using a 25 m Tarp-fumigation f.r. 1.0%, respectively. However, EF Ct products out- chamber filled with T. matsutake (1.7% f.r. w/v). When side the styrofoam box were 89.4 ± 2.2, 136.3 ± 2.3 140  g/m was applied for 4  h at 5°C, L. mali achieved and 219.7 ± 4.1  g  h/m at f.r. 1.0%, respectively, shown 100% mortality (total 1079 of fumigated L. mali lar- in Fig.  2. Regarding the initial concentration, the con- vae used). The acculatumed Ct products of EF were 3 3 centration of inside was 15–40% of the concentration 159.0 ± 3.1, 147.2 ± 2.8  g  h/m inside, 195.3 ± 4.3  g  h/m of outside. After 2  h of treatment, the inside and out- outside the styrofoam box (Fig. 3, Table 6). We confirmed side concentrations were similar. The calculation of Ct more than achievable L(Ct) values and the loss rates of 99% products in the lab might be different when applied EF were approximately 70–80% for 4  h exposure, which in actual scaled-up trials because it is denpendent on was similar to previous lab condition studies. When con- sealing conditions, temperature, and condition of com- ducting this test, we tried to confirm the mortality for L. modities (water content, logistic packing, etc.). Accord- mali naturally infested in T. matsutake, however collec- ing to our data when determing EF concentration to tion of a large amount of T. matsutake was impossible achieve target Ct products (112.9  g  h/m , Probit esti- and the number of infested L. mali per mushroom was mation for 99% mortality on L. mali) with 4 h exposure, too small. For a small number of L. mali, 100% mortality 100 35 g/m3 O 70 g/m3 O 140 g/m3 O 35 g/m3 I 70 g/m3 I 140 g/m3 I 0123 4 Fumiga on me (h) Fig. 2 Gas concentration, when fumigated with 35, 70, and 140 g/m EF for 4 h inside (I) and outside (O) of styrofoam box containing 1.0% f.r. of Tricholoma matsutake (temp. 5.1 ± 0.3℃) EF Concentra on (g/m ) Kwon et al. Appl Biol Chem (2021) 64:64 Page 8 of 11 S. B. O S. B. I#1 S. B. I#2 0123 4 Fumigaon me (h) 3 3 Fig. 3 Gas concentration when fumigated with 140 g/m EF for 4 h on Tricholoma matsutake (f.r. 1.7%) in scaled-up trials (25 m Tarp-fumigation chamber, temp.: 7.3 ± 0.4 ℃). *Gas concentration inside (S. B. I) and outside (S. B. O) the styrofoam box Table 6 Scaled-up trials of liquid EF-fumigaion and EF + PH3 fumigation on Tricholoma matsutake 3 3 2 Type and size of fumigation Appl. Dose (g/m ) Packing and Ct products (g h/m ) Mort. (%) (dead/total) Deterioration Market value gas sampling degree Fumigation chamber – Untreated – 0 (0/159) 0 a B1 B2 C (25 m ) # EF 140.0 S.B.(I 1) 159.0 ± 3.1 100 (331/331) 3 b D D D S.B.(I 2) 147.2 ± 2.8 100 (387/387) 3 b D D D S.B.(O) 195.3 ± 4.3 100 (361/361) 3 b D D D Fumigation chamber – Untreated – 0 (0/159) 0 a B1 B2 C (5 m ) # EF 35.0 + PH 0.5 S.B.(I 1) 50.6 ± 0.6 100 (397/397) 0 a B1 B2 C S.B.(I 2) 50.2 ± 1.1 100 (356/356) 0 a B1 B2 C S.B.(O) 66.4 ± 0.1 100 (344/344) 0 a B1 B2 C p-value < 0.0001 < 0.0001 S.B: Styrofoam box, Inside (I), Outside (O) 1. Condition: f.r. 1.7%, 7.33 ± 0.39 ℃ 2. Condition: f.r. 0.5%, 4.94 ± 0.21 ℃ The deterioration degrees. 0: Non, 1: water condensation, 2: water condensation following changing soft a cap and grill parts 3: water condensation following changing soft a cap, grill and then stem part Market value based on commercial grades. A: button, B1: young mushroom, B2: mature mushroom, C: overmature mushroom, D: not for sale was achieved. Regarding weight loss, in assessment of 3 d matsutake (0.5% f.r. w/v). When 35  g/m of EF + 0.5  g/ after fumigation, T. matsutake was not significantly dif - m of PH was applied for 4 h at 5 °C, L. mali achieved ferent (df: 16, p-value: 0.8062) (Fig.  4). Likewise, regard- 100% mortality (total 1,097 of fumigated L. mali lar- ing color change, T. matsutake was not significantly vae used). The acculatumed Ct products of EF were 3 3 different (df: 16, p-value: 0.8198) (Fig. 5.). 50.6 ± 0.6, 50.2 ± 1.1  g  h/m inside, 66.4 ± 0.1  g  h/m outside the styrofoam box (Fig.  6, Table  3). We con- EF fumigation mixed with PH3 on Tricholoma matsutake firmed more than achievable L(Ct) values and the 99% We proposed EF + PH combination fumigation. It loss rates of EF were approximately 60–70% for 4  h was performed using a 5  m container filled with T. exposure, which was similar to previous lab condition EF concentraon (g/m ) K won et al. Appl Biol Chem (2021) 64:64 Page 9 of 11 3 3 3 Fig. 4 Weight loss after 3-d fumigation on mushrooms fumigated with 140 g/m EF only and 35 g/m EF mixed with 0.5 g/m PH (4 h exposure). NS: Not significant 3 3 3 Fig. 5 Color change after 3-d fumigation on mushrooms fumigated with 140 g/m EF only and 35 g/m EF mixed with 0.5 g/m PH (4 h exposure). NS: Not significant Kwon et al. Appl Biol Chem (2021) 64:64 Page 10 of 11 S. B. I#1 S. B. I#2 S. B. O#1 0123 4 Fumigation time (h) 3 3 3 Fig. 6 Gas concentration when fumigated with 35 g/m EF + 0.5 g/m PH for 4 h on Tricholoma matsutake (f.r. 0.5%) in scale-up trials. (5 m fumigation chamber, Temp.: 5.0 ± 0.2 ℃) *Gas concentration inside (S. B. I) and outside (S. B. O) the styrofoam box Author details studies. Regarding weight loss, in assessment of 3-d Division of Applied Life Science (BK21 + Program), Gyeongsang National after fumigation, T. matsutake was not significantly University, Jinju 52828, Republic of Korea. Institute of Life Scienc, Gyeongsang different (df: 18, p -value: 0.4423) (Fig.  4). Regarding National University, Jinju 52828, Republic of Korea. Animal and Plant Quaran- tine Agency, Gimcheon 39660, Republic of Korea. USDA-ARS, US Pacific Basin color change, T. matsutake was not significantly differ - Agricultural Research Center, Hilo, HI 96720, USA. ent (df: 16, p-value: 0.3473) (Fig. 5). Thus, EF combined with PH fumigation was no damage to mushrooms 3 Received: 18 May 2021 Accepted: 18 August 2021 unlike EF alone fumigation. There were previous stud - ies reported that EF + CO [26], PH + CO for reduc- 2 3 2 ing LT (Lethal time) values [27] and PH + O [28] for 3 2 increasing efficacy and decreasing phytotoxic damage References 1. Lewandowski M, Sznyk A, Bednarek A (2004) Biology and morphometry to commodities. of Lycoriella ingenua (Diptera: Sciaridae). Biol Lett 41:41–50 2. Cantelo WW (1979) Lycoriella mali: control in mushroom compost by Acknowledgements incorporation of insecticide into compost. J Econ Enotomol 71:703–705 This research funded byAnimal and Plant Quarantine Agency in Republic 3. White PF (1985) Pests and pesticides. In: Flegg PB, Spencer DM, Wood of Korea. Also we thank to Korean Mushroom Association (KMA) to support DA (eds) The biology and technology of the cultivated mushroom. Wiley, commercial trials. Chichester, pp 279–293 4. Kim KJ, Hwang CY (1996) An investigation of insect pest on the mush- Authors’ contributions room (Lentinus edodes, Pleurotus ostreatus) in south region of Korea. B.H.L, K.W.K and M.G.P designed the experiments. T.H.K and D.B.K conducted Korean J Appl Entomol 35:45–51 experiments, results analysis and interpretation. G.H.R and T.H.K wrote the 5. Shamshad A, Clift AD, Mansfield S (2008) Toxicity of six commercially manuscript. All authors read and approved the final manuscript. formulated insecticides as biopesticides to third instar of mushroom sciarid, Lycoriella ingenua Dofour (Diptera: Sciaridae), in New South Wales, Funding Australia. Aust J Entomol 47:256–260 This study was supported by APQA(Animal and Plant Quarntine Agency) 6. Kati (2020) https:// www. kati. net/ stati stics/ perio dPerf orman ce. do, last project (P.N. PQ20203B012). modified 20 Nov 2020. Accessed 21 Feb2021 7. Choi J, Lim E, Park MG, Cha W (2020) Assessing the retest reliability of pre- Availability of data and materials frontal EEG markers of brain rhythm slowing in the eyes-closed resting The datasets used and/or analyzed during the current study are avaliable from state. Clin EEG Neurosci 51(5):1–9 the corresponding author on reasonable request. 8. Bae EY, Lee EK (2009) Pharmacoeconomic guidelines and their implementation in the positive list system in South Korea. Value Health Declarations 12:36–41 9. Yang JO, Park YR, Hyun IH, Kim GH, Kim BS, Lee BH, Ren YL (2016) A Competing interests combination treatment using ethyl formate and phosphine to control The authors declare that they have no competing interests. EF concentration (gh/m ) K won et al. Appl Biol Chem (2021) 64:64 Page 11 of 11 planococcus citri (Hemiptera: Pseudococcidae) on pineapples. J Econ for controlling Pseudococcus longispinus (Hemiptera: Pseudococcidae) Entomol 109(6):2355–2363 and Pseudococcus orchidicola on imported foliage nursery plants. J Econ 10. Simpson T, Bikoba V, Mitcham EJ (2004) Eec ff ts of ethyl formate on fruit Entomol 112(5):2149–2156 quality and target pest mortality for harvested strawberries. Postharvest 22. Agarwal M, Ren YL, Newman J, Learmonth S (2015) Ethyl formate: a Biol Technol 34:313–319 potential disinfestation treatment for eucalyptus weevil (Gonipterus 11. Simpson T, Bikoba V, Tipping C, Mitcham EJ (2007) Ethyl formate as a platensis) (Coloptrea: Curculionidae) in apples. J Econ Entomol postharvest fumigant for selected pests of table grapes. J Econ Entomol 108(6):2566–2571 100:1084–1090 23. Kim BS, Shin EM, Park YJ, Yang JO (2020) Susceptibility of the cigarette 12. Smit R, Jooste MM, Addison MF, Johnson SA (2020) Etyhl formate fumiga- bettle Lasioderma serricorne (Fabricius) to phosphine, ethyl formate and tion: its effect on stone and pome fruit quality, and grain chinch bug their combination, and the sorption and desorption of fumigants on (Macchiademus diplopterus) mortality. Sci Hortic 261:1–9 cured tobacco leaves. Insects 11(9):599 13. Lee JS, Kim HK, Kyung YJ, Park GH, Lee BH, Yang JO, Koo HN, Kim GH 24. Lee BH, Kim BS, Yang JO, Park CK, Ren YL (2014) Evaluation of synergistic (2018) Fumigation activity of ethyl formate and phosphine against Tetra- effect between ethyl formate and phosphine for control of three species nychus urticae (Acari: Tetranychidae) on imported sweet pumpkin. J Econ aphids in perishable commodity. 11th International working conference Entomol 111(4):1625–1632 on Stored Product Protection, 24–28 November. Chiang Mai, Thailand, pp 14. Ren YL, Lee BH, Padovan B (2011) Penetration of methyl bromide, sulfuryl 979–984 fluoride, ethanedinitrile and phosphine into timber blocks and the sorp - 25. Kyung YJ, Kim HK, Lee JS, Kim BS, Yang JO, Lee BH, Koo HN, Kim GH (2018) tion rate of the fumigants. J Stored Prod Res 47:63–68 Efficacy and phytotoxicity of phosphine as fumigants for Frankliniella 15. Lee BH, Kim HM, Kim BS, Yang JO, Moon YM, Ren YL (2016) Evaluation of occidentalis ( Thysanoptera: Thripidae) on asparagus. J Econ Entomol the synergistic effect between ethyl formate and phosphine for control 111(6):2644–2651 of Aphis gossypii (Homoptera: Aphididae). J Econ Entomol 109(1):143–147 26. Haritos VS, Damcevski KA, Dojchinov G (2006) Improved efficacy of ethyl 16. Cho SW, Kim HK, Kim BS, Yang JO, Kim GH (2020) Combinatory effect of formate against stored grain insects by combination with carbon dioxide ethyl formate and phosphine fumigation Pseducoccus longispinus and P. in a ‘dynamic’ application. Pest Manag Sci 62:325–333 orchidicola (Hemiptera: Pseudococcidae) mortality and phytotoxicity to 27. Meenatchi R, Alice RPS, Paulin PP (2018) Synergistic effect of phosphine 13 foliage nursery plants. J Asia Pac Entomol 23(1):152–158 and carbon dioxide on the mortality of Tribolium castaneum (Coleoptera: 17. Hewlett PS, Plackett RL (1969) A unfied theory for quantal responses to Tenebrionidae) in paddy. J Agric Sci 10:503–510 mixtures of drugs: non-interactive action. Biometrics 15:591–610 28. Liu SS, Liu YB, Simmons GS (2015) Oxygenated phosphine fumigation 18. Institute SAS (2009) SAS/STAT 9.2 user’s guide, 2nd edn. SAS Institute Inc, for control of light brown apple moth (Lepidoptera: Tortricidae) eggs on Cary cut-flower. J Econ Entomol 108:1630–1636 19. O’connor L, Keli CB, (2005) Mushroom host influence on Lycoriella mali (Diptera: sciaridae) life cycle. J Econ Entomol 98(2):342–349 Publisher’s Note 20. Kwon TH, Park CG, Lee BH, Zarders DR, Roh GH, Kendra PE, Cha DH (2021) Springer Nature remains neutral with regard to jurisdictional claims in pub- Etyhl formate fumigation and ethyl formate plus cold treatment combi- lished maps and institutional affiliations. nation as potential phytosanitary quarantine treatments of Drosophila suzukii in blueberries. J Asia Pac Entomol 24:129–135 21. Kyung YJ, Kim HK, Cho SW, Kim BS, Yang JO, Koo HN, Kim GH (2019) Com- parison of the efficacy and phytotoxicity of phosphine and ethyl formate http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Biological Chemistry Springer Journals

Scaled-up ethyl formate fumigation to replace methyl bromide on traded mushroom to disinfest mushroom fly (Lycoriella mali)

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Abstract

Mushroom fly, Lycoriella mali (Diptera: Sciaridae), is the primary pest in imported mushrooms. The amount of Tricho - loma matsutake imported from China increases every fall when it is harvested. When importing T. matsutake, disinfes- tation using methyl bromide (MB) or phosphine (PH ) is performed to prevent the introduction of L. mali. However, MB will be phased out due to ozone-depletion, chronic toxicity to workers, and residual issues. PH fumigation in mushroom disinfestation requires a long exposure time (24 h). In this study, we used ethyl formate (EF), which can replace MB and reduce exposure time. The efficacy of EF, PH3 and EF + PH3 on L. mali was evaluated. Using 4-h EF fumigation at 5 °C, the 3rd and 4th instar was the most tolerant stage in terms of 99% killed lethal concentration × time products (LCt ). When 4-h EF fumigation at 5 °C was applied on all stages of L. mali, the LCt values of EF 99% 99% 3 3 3 were 73.1 g h/m to the 1st and 2nd instar, 112.9 g h/m to the 3rd and 4th instar, 68.9 g h/m to pupae, and 20.1 g h/ m to adult. It was confirmed that combination treatment with EF + PH had a synergistic effect on L. mali. The 3 3 3 LCt of EF + 0.5 g/m of PH to the 3rd and 4th instar was 48.3 g h/m . When only 140 g/m of EF was applied for 99% 3 3 3 4 h at > 5 °C and 35 g/m of EF + 0.5 g/m of PH for 4 h at > 5 °C in commercial trials containing T. matsutake, proven efficacy (100%) on L. mali was confirmed. In the case of EF treatment only, phytotoxic damage occurred due to high Ct products, and there was no phytotoxic damage in combination treatment with EF + PH . This study provides a new guideline for EF + PH combination treatment within a shorter exposure time (4 h) than existing PH treatment (24 h) 3 3 and replacement of MB use. Keywords: Lycoriella mali, Ethyl formate, Phosphine, Alternatives, EF + PH , Synergistic effect Introduction Efficacy of synthetic pesticides such as diflubenzuron, Flies of the family Sciaridae occur almost worldwide in diazinon, methoprene, and phosphate insecticides such many different cultivating and perishable commodities as dimethoate and acephate in L. mali has been reported [1]. Significant loss of cultivating mushrooms caused [2]. by several species such as Lycoriella mali and Lycoriella In the mushroom trade among several countries ingenua of sciarid in the mushroom industry has been including China and Korea, Lycoriella sp. is classified reported from the USA, UK, and South Korea [2–5]. as a quarantine pest in some countries, including South Korea, and must be treated by phytosanitary disinfes- tation at ports. According to KATI [6], South Korea *Correspondence: Byungholee@hotmail.com exported 7584 t of Pleurotus eryngii, the main exported Institute of Life Scienc, Gyeongsang National University, Jinju 52828, mushroom, and imported 145 t of Tricholoma matsutake Republic of Korea from mainly China in 2019. Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Kwon et al. Appl Biol Chem (2021) 64:64 Page 2 of 11 According to the phytosanitary guideline in Korea, Materials and methods imported mushrooms infested with L. mali must be Insects and chemicals chemically treated with methyl bromide (MB). In the Lycoriella mali was collected from a mushroom farm case of exported mushrooms, pest free inspection was in Yeongcheon, Gyeongbuk, South Korea during 2020. the last option to avoid rejecting them in countries where L. mali were transferred and reared in an insect rear- they are imported with the option not to treat with MB ing room at Gyeongsang National University. L. mali because it caused loss of quality. As well as phytotoxic was maintained in the insect rearing room at 24°C and damage to mushrooms treated with MB, its use has been 60–70% relative humidity (RH) with a 16:8 [L:D] h. phasing out because of ozone depletion properties and Pleurotus eryngii was provided as a food source. Female chronic toxicity to human in Korea [7]. MB fumiga- adults of L. mali lay eggs on water agar (2%) in the insect tion on food commodities could be more difficult in the breeding dish (100 mm × 40 mm). Larvae pupated within future because there is a need to update residual bro- 5–6  days, and adults emerged within 25  days; 1st, 2nd, mide ion and MB itself post all type of food commodi- 3rd and 4th instar larvae, pupae, and adults were used ties associated with the new Positive List System (PLS) in this study. EF (Fumate , >  99% purity; Hoengseong, in Korea [8]. In Korea, MB fumigation on treaded mush- Korea) was supplied by Safefume Co. Ltd in Korea. Phos- rooms will be discontinued after 2022 (Personal com- phine was purchased as ECO Fume (2% PH + 98% CO ) 2 3 2 munication with MG Park), because there is currently no from Cytec (Sydney, Australia). consumer safety data supported in Korea. In the case of PH fumigation, as current alternative options, commer- Egg hatching test at low temperature cial adaptation might be difficult because a long expo - Egg hatching studies of L. mali were performed at 5 ± 0.5°C sure time is required in grains (> 5 days) and mushrooms in an incubator. The eggs were collected form rearing cages (> 1  days), which could shorten the shelf life of mush- with 200 mated females on the Pleurotus eryngii over 1 day rooms [9]. and treated immediately. Before fumigation to eggs, 2% Ethyl formate (EF), an alternative to MB, is known agar medium was laid on the bottom of the breeding dish to be safer than other fumigants in the workplace and (50 mm × 15 mm) to maintain moisture and cut pine of P. there is residual free regulation in many countries eryngii was placed on the agar medium. Then 20 eggs of L. because it is globally classified as a food additive. In mali were transferred to each cut pine of P. eryngii. Because practice, EF fumigation has been used in imported com- the egg color is transparent, the cut pine of P. eryngii was modities such as fruits, vegetables, nursery plants, etc. dyed using natural pigments to make observation of the [10, 11]. A new concept of EF application technology eggs easier. Following placcement in a 5°C incubator, the with N (Non-CO ) was developed and used commer- 2 2 egg hatching rate was observed for 5 to 14 days. After 72 h cially in Korea [9]. of treatment, eggs were checked hatching rate. Treatment Although EF was effective fumigant, it has high sorp - was replicated three times, and the control was replicated tion [12] to commodities like perishable fruits and veg- 10 times at room temperature (24 ± 1°C). etables and more less vaporization at low temperature [13]. PH was good at permeability to commodites like timber [14]. Thus the new concept of fumigation has Efficacy to developmetal stages of L. mali with EF been studied that combined EF and PH , which was more in a laboratory experiment effective to insect pests and less phytotoxic damage to Efficacy of EF was evaluated for three different devel - commodities [15, 16]. opmental stages (adult, larvae and pupae) of L mali. In No study evaluating the efficacy of EF on L. mali has the case of EF mixed with PH studies, the 3rd and 4th been reported. Herein, we suggest new disinfestation instars of L mali, which is the most tolerant stage to EF, guidelines for disinfestation of L. mali using EF and was evaluated. Adults of L. mali were transferred from a EF + PH , which are a replacement for MB. breeding dish (50 mm × 15 mm) using collecting equip- We evaluated (1) Efficacy of EF for 4 h-fumigation on L. ment (Fulton, MX-991/U, Georgia) within three days of mali in lab studies, (2) sorption studies on several types developing an adult. Adults of L. mali were fumigated of mushroom and gas penetration under imported con- at 5°C for 4  h with 1.0–9.0  g/m of EF. The 1st and 2nd ditions, (3) Synergestic effect of EF and PH to L. mali instar and the 3rd and 4th instar were classified and and Phytotoxic damage to mushroom with EF + PH tested. From 1 to 7  days after hatching, the 1st and 2nd fumigation. (4) application of liquid EF with N applica- instar and 7 to 14  days were classified into the 3rd and tion on a commercial scale for confirmation on imported 4th instar. The pupae were used within two days after mushrooms. K won et al. Appl Biol Chem (2021) 64:64 Page 3 of 11 pupation. Larvae and pupae stages of L. mali were trans- (FPD) and HP-PLOT/Q (30  m × 530  µm × 40  µm, Agi- ferred from water agar (2%) to a breeding dish (50  mm lent, Santa Clara, CA) operating in split mode (10:1). The × 15 mm). The 1st and 2nd instar of L. mali were fumi - temperature of the injector and the oven was 200℃. The gated at 5°C for 4 h with 1.0–30.0 g/m of EF. The 3rd and temperature of the detector was 250°C. The injection vol - 4th instar were fumigated at 5°C for 4 h with 1.0–45.0 g/ umes and flow rate of EF and PH were 60 µl and 20 and m of EF. The pupae were fumigated at 5°C for 4  h with 1.5 and 5 ml/min, respectively. 1.0–40.0 g/m of EF. The concentration of EF was measured using an Agi - Determination of the synergistic effect of ethyl formate lent portable GC 17A (Shimadzu, Kyoto, Japan) equipped mixed with phosphine against the 3rd and 4th larvae of L. with a flame ionization detector (FID) after separation on mali a DB5-MS Column (30  m × 0.25  mm i.d., 0.25  µm film After efficacy to EF alone fumigation and EF combined thickness; J&W Scientific, Folsom CA). The oven tem -with PH fumigation, we evaluated synergistic effect. perature was 100°C The injector and detector tempera - A synergistic effect was measured by synergistic ratios tures were 250 and 280°C, respectively. Helium was used (SRs). Synergistic ratios are defined by Hewlett and as a carrier gas at the flow rate of 1.5 mL/min. Headspace Plackett [17] and based on equation. 1. EF was calculated by the peak area against external EF L(Ct) of ethyl formate only standards. SR = (1) L(Ct) of ethyl formate + phosphine After completion of 4 h fumigation, treated L. mali was transferred to an insect rearing room at 24°C and 60–70% *SR = 1 describes additive action, relative humidity (RH) with a 16:8 [L:D] h. The mortality SR < 1 describes antagonism, of the treated L. mali larvae and adults were determined SR < 1 describes synergism. by visual inspection of movement using a microscope 1 day after fumigation. The mortality of the treated L. mali Sorption test of T. mastutake for 4 h EF fumigation pupae was determined by checking the number of adults in a laboratory experiment for 5  days after fumigation. Usually, the rate of pupae to Evaluation of sorption of EF on T. matsutake was per- adults in the control group was 50% level. All treatments formed in a 6.8 L desiccator under lab condition. Each T. and controls were replicated three times. matsutake was filled at a 0.5, 1, and 1.5% filling ratio (w/v) The temperature was recorded using data loggers for the sorption test; 140  g/m of EF was applied using (Thermo Recorder TR-72Ui, T&D Corp., Japan). In this a syringe for 4  h at 5°C. Gas sampling was performed at study, a 6.8 L desiccator was used in the experiment as a time intervals (10 min, 1, 2, and 4 h) and measurements fumigation chamber. The desiccators were sealed with a were performed using an Agilent portable GC 17A (Shi- glass stopper equipped with a septum (Alltech Associates madzu, Kyoto, Japan) equipped with a flame ionization Australia, Cat. No. 15419). The exact volume of desicca - detector (FID) after separation on a DB5-MS Column tors was measured using weigh of water. The desiccators (30 m × 0.25 mm i.d., 0.25 µm film thickness; J&W Scien - were tightly sealed with high vacuum grease (Dow Corn- tific, Folsom, CA). ing, USA). A filter paper (Whatman No. 1) was inserted into the glass stopper to make clear evaporation in the desiccator for the injected EF. A magnetic bar to stir the Gas penetration test of EF on packed mushrooms fumigant was located at the bottom of the desiccator. The with logistic consideration (Styrofoam box) dose of fumigant and Ct products was calculated using In the case of import and export Tricholoma matsutake, the method reported by Ren et al. [14]. a styrofoam box with an ice pack was used as packing materials. Evaluation of gas penetration of EF on packed mushrooms (Tricholoma matsutake) was performed in Efficacy to 3rd and 4th larvae of L. mali with EF combined 3 a 0.275  m fumigation chamber at 5°C. Each mushroom with  PH in a laboratory experiment was filled at a 1.0% filling ratio (w/v) for the gas penetra - For EF + PH efficacy studies, the 3rd and 4th instars of L 3 tion test; 35, 70 and  140  g/m of EF was applied using a mali were trasnffered on agar petri dish in desiccator (6.8 special vaporizer (supplied by Safefume Co.) for 4  h at L). The larvae in the desiccators were fumigated at 5°C for 5°C. The two conditions (inside, outside of the Styrofoam 4  h with 4.0–28.0  g/m of EF mixed with 0.5 and 1.0  g/ box) of EF concentration were analyzed. Gas sampling m of PH . Analysis of EF concentration was as shown was performed at time intervals and measurements were above, the concentration of PH was measured using Agi- performed using an Agilent portable GC 17A. lent 7890A equipped with a flame photometric detector Kwon et al. Appl Biol Chem (2021) 64:64 Page 4 of 11 Commercial scaled‑up fumigation Phytotoxic assessment EF alone fumigation on Tricholoma matsutake The phytotoxic damage of EF on mushrooms (Pleuro - Scale-up fumigation was performed using a 25  m tus eryngii, Tricholoma matsutake,) was evaluated in Tarp-fumigation chamber in the Incheon airport ware- scaled-up studies (5  m fumigation chamber). Two types house in South Korea. Tricholoma matsutake were of mushrooms were filled at a 1.5% filling ratio; 140  g/ 3 3 placed in a styrofoam box (1.7% f.r.) and the cover m of EF for Pleurotus eryngii and 140  g/m of EF and 3 3 of the box was opened, which is based on commer- 35  g/m of EF + 0.5  g/m PH for Tricholoma matsutake cial use. Then, 140  g/m of EF was applied for 4  h at were applied using a special vaporizer (supplied by Safe- 5°C. Liquid EF was vaporized with SFM-I (supplied by fume Co.) for 4 h at 5°C. After completion of fumigation, Safefume Co.) with N as the carrier gas and a fan was mushrooms were transferred to storage at 5 ± 0.8°C. The placed at the bottom of the Tarp-chamber for efficient deterioration degrees were classified according to symp - gas circulation. The concentrations of the inside and toms of the water condensation surface of the cap and outside of the styrofoam box were analyzed. Three of changing a soft cap, grill, stem parts (0: Non, 1: water the insect breeding dishes containing > 300 larvae of L. condensation, 2: water condensation following changing mali (total 1079) were then located in the styrofoam a soft cap and grill parts 3: water condensation follow- box. Gas sampling was collected at time intervals (0, ing changing a soft cap, grill and then stem part). A color 1, 2, and 4 h) and EF concentration inside and outside change (hue value) using a color meter (TES 135A, Tai- of the styrofoam box was measured using GC-FID. wan), weght loss (%, weight differences before and after Because measurement with GC-FID directly at the treatment) and market value based on the hardness of field is impossible, the concentration was checked in head parts (the harder the best, the softer the worst) were advance at the field using a gas analyzer (IBRID MX6; evaluated 3-d after fumigation. Industrial Scientific, Pittsburgh, PA, USA). A previous study found no sigfinicant difference in the measured concentration of IBRID and GC-FID. Fumigated larvae Statistical analysis of L. mali were transferred to an insect rearing room. Analysis of the toxicological dose response to EF by L. The mortality of larvae was determined 1-d after fumi- mali was based on a Probit analysis (Finney, 1971). As gation. All treatments and controls were replicated part of the analysis, the slopes of the Probit transfor- three times. mations were determined as well as Chi-square tests of data homogeneity for different treatments. The indices of toxicity measurement derived from this analysis were EF fumigation mixed with  PH on Tricholoma matsutake L(Ct) = median lethal concentration that causes 50% Scale-up fumigation was performed using a 5  m fumi- 50% response (mortality) and L(Ct) = lethal concentration gation chamber in the same APQA site in South Korea. 99% that causes 99% response (mortality) of exposed L. mali Tricholoma matsutake were placed in a styrofoam box determined from a range of at least 10 different Ct prod - (0.5% f.r.) and the cover of the box was opened, which ucts to ensure that the observed data covered mortality is based on commercial use. Then, 35 g/m of EF mixed from 0 to 100% and adequately covered the intermediate with 0.5  g/m of PH was applied for 4  h at 5°C. Liq- range. For analysis of the hatchability of eggs on two tem- uid EF was vaporized using a vaporizer (supplied by peratures and phytotoxic damage assessment of EF fumi- Safefume Co.) with N as the carrier gas and a mini fan gation, on mushrooms, a T-test procedure was used to was placed at the bottom of the chamber for efficient compare the two sample means. The EF alone fumigation gas circulation. The concentration of PH was achieved and EF combined with PH fumigation against phytotox- by injecting 125  ml of 2% PH into a 5  m fumigation 3 icity of mushrooms were compared using Tukey’s test in chamber. The concentrations of the inside and outside scaled-up trials. All statistical analyses were performed of the styrofoam box were analyzed. Three insect breed - using SAS (ver. 9.4; SAS Institute Inc.) [18]. ing dishes containing > 300 larvae of L. mali (total 1,097) were then located in the styrofoam box. Gas sampling was collected at time intervals (0, 1, 2, and 4 h) and EF Results and discussion and PH concentrations inside and outside of the sty- Egg hatchability test at low temperature rofoam box were easured using GC-FID and GC-FPD. This study was conducted to investigate eggs hatching in Fumigated larvae of L. mali were transferred to an low temperature conditioned mushrooms. According to insect rearing room. The mortality of larvae was deter - previous ecological studies on L. mali, the average length mined 1-d after fumigation. All treatments and controls of a generation from egg to adult was 28 d at 21°C [19]; it were replicated three times. K won et al. Appl Biol Chem (2021) 64:64 Page 5 of 11 Table 1 Egg hatchability of Lycoriella mali under two conditions (5 ± 0.5℃, 24 ± 1.0℃) Day after oviposition Temperature (mean ± SE, No. of tested No. of hatched p‑ value Hatchability ℃) (mean ± SE, %) 5 5 ± 0.5 60 0 < 0.0001 0.0 ± 0.0 24 ± 1.0 60 52 88.3 ± 1.7* 6 5 ± 0.5 60 0 < 0.0001 0.0 ± 0.0 24 ± 1.0 60 60 100.0 ± 0.0 7 5 ± 0.5 60 0 < 0.0001 0.0 ± 0.0 24 ± 1.0 60 60 100.0 ± 0.0 8 5 ± 0.5 60 0 – 0.0 ± 0.0 9 5 ± 0.5 60 0 – 0.0 ± 0.0 10 5 ± 0.5 60 0 – 0.0 ± 0.0 11 5 ± 0.5 60 0 – 0.0 ± 0.0 12 5 ± 0.5 60 0 – 0.0 ± 0.0 13 5 ± 0.5 60 0 – 0.0 ± 0.0 14 5 ± 0.5 60 0 – 0.0 ± 0.0 All the unhatched eggs were emergedll at 6-d after oviposition - impossible to check Table 2 LCt (Lethal Concentration × time) value of EF fumigation for 4 h expoure on Lycoriella mali at 5 ± 0.5 ℃ Stage L(Ct) (95% CL) L(Ct) (95% CL) Slope ± SE df χ 50% 99% 1st, 2nd instar 43.6 (37.1–53.3) 73.1 (68.4–100.2) 3.1 ± 0.3 21 28.9 3rd, 4th instar 27.84 (22.53–33.01) 112.9 (90.70–152.25) 3.2 ± 0.3 28 48.47 Pupae 36.8 (28.9–46.1) 68.9 (57.6–97.1) 4.3 ± 0.3 15 40.2 Adults 7.8 (5.8–13.9) 20.1 (16.6–26.2) 2.0 ± 0.4 13 32.1 took five days from eggs to hatching. The egg hatchabil - ity under normal temperature condition (24 ± 1°C) were Table 3 Efficacy of PH3, EF and EF + PH fumigation for 4 h 88.3 ± 1.7% and 100.0 ± 0.0% at the day of the 5th and 6th expoure on Lycoriella mali at 5 ± 0.5℃ day after oviposition (Table  1). However, we confirmed Fumigant dose Ct products (g h/m ) Mortality ± SE (%) that the eggs under low temperature (5.0 ± 0.5°C) did not hatch at all. Based on logistic distribution of imported Control 0.0 0.0 ± 0.0 T. matsutake from China, it takes at least five days from PH 0.5 g/m 1.8 0.0 ± 0.0 harvest to consumers, all logistics is under < 5°C, mean- PH 1.0 g/m 3.5 0.0 ± 0.0 ing that survival of the egg might be diffidult under cold EF 6.0 g/m 14.8 20.0 ± 2.9 temperature conditions if found in imported mushrooms. EF 10.0 g/m 26.4 46.7 ± 1.7 EF 18.0 g/m 44.9 86.7 ± 1.7 Efficacy to developmetal stages of L. mali with EF EF 26.0 g/m 65.5 95.0 ± 0.0 3 3 in a laboratory experiment PH 0.5 g/m + EF 6.0 g/m 1.8 + 15.2 41.7 ± 1.7 The efficacy of 4  h EF fumigation (practical exposure PH 0.5 g/m + EF 10.0 g/ 1.8 + 26.6 40.0 ± 2.9 condtion using EF fumigations in Korea) on larvae, PH 0.5 g/m + EF 18.0 g/ 1.8 + 45.7 100.0 ± 0.0 pupae, and adults stages of L. mali at 5℃ is shown in 3 Table 2. For the 1st and 2nd of L. mali larvae, the L(Ct) 50% PH 0.5 g/m + EF 26.0 g/ 1.8 + 65.7 100.0 ± 0.0 and L(Ct) values of EF were 43.6, 73.1 g h/m . For the 99% 3rd and 4th of L. mali larvae, the L(Ct) and L(Ct) 50% 99% PH 1.0 g/m + EF 18.0 g/ 3.6 + 47.0 100.0 ± 0.0 values of EF were 25.0 and 112.9  g  h/m . The L(Ct) 50% and L(Ct) values of EF on L. mali pupae were 36.8 and PH 1.0 g/m + EF 26.0 g/ 3.5 + 65.6 100.0 ± 0.0 99% 3 3 3 68.9  g  h/m andadults were 7.8 and 20.1  g  h/m at 5°C (Table  2), respectively. Thus, the order of susceptively Kwon et al. Appl Biol Chem (2021) 64:64 Page 6 of 11 Table 4 LCt (Lethal concentration x time) value of EF mixed with 0.5 g/m PH for 4 h exposure on Lycoriella mali Temp (℃) Stage L(Ct) (95% CL) L(Ct) (95% CL) Slope ± SE df χ 50% 99% 5 3rd, 4th instar 34.4 (33.3–35.7) 48.3 (44.9–53.9) 15.8 ± 1.8 16 77.59 Table 5 Syngeristic efficacy of EF mixed with 0.5 g/m PH on 18.0  g/m for 4  h fumigation, Otherwise, EF combined 3rd–4th larvae stage of Lycoriella mali with PH of 0.5  g/m fumigation for 4  h was controlled a b 100% against 3rd and 4th instar of L. mali (Table 3). The Temp (℃) Stage Synergistic ratios Synergistic ratios L(Ct) L(Ct) L(Ct) and L(Ct) values of EF + 0.5 g/m PH against 50% 99% 50% 99% 3 the 3rd and 4th of L. mali were 34.4 and 48.3  g  h/m at 5 3rd–4th larvae 0.81 2.34 5  °C, respectively (Table  4). According to the results of Synergistic ratios (SRs): L(Ct) of ethyl formate only/ L(Ct) of ethyl 50% 50% a preliminary experiment at 5°C, the 3rd and 4th instar formate + 0.5 g/m phosphine were the most tolerant stages of L. mali. It was confirmed Synergistic ratios (SRs): L(Ct) of ethyl formate only/ L(Ct) of ethyl 99% 99% that application of EF + 0.5  g/m PH to L. mali had a formate + 0.5 g/m phosphine synergistic effect when treated L(Ct) at 5°C (Table  5). 99% The fumigant, PH was required more longer exposure of L. mali life stages to EF based on L(Ct) values was 99% times to kill insect pests than EF [21] and EF was needed adults > pupae > 1st and 2nd instar > 3rd and 4th instar to high concentration to control insect pests at low tem- larvae. Drosophila suzukii (Diptera: Drosophilidae) of the perature [22]. But mixed usage of these fumigants would other flies was also most tolerant larval stage except for be better to control insect pests than alone [15, 16]. eggs [20]. Sorption test of T. mastutake for 4 h EF fumigation in a laboratory experiment Efficacy to 3rd and 4th larvae of L. mali with EF combined Based on the efficacy studies resulting in L(Ct) val- 99% with  PH in a laboratory experiment ues of EF on L. mali larvae, which is the most tolerant to The efficacy of EF + 0.5  g/m PH fumigation for 4  h on EF fumigation in this study, estimated applicable sched- the 3rd and 4th of L. mali at 5°C is shown in Table 3. The ules (140  g/m EF for 4  h exposure) were performed mortality of L. mali was no effect with PH of 0.5 and on T. matsutake. The losses of EF inside the fumigated 1.0  g/m for 4  h fumigation, and was 86.7% with EF of desiccators and their adsorption are shown in Fig.  1. 0.9 0.50% 1% 1.50% 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Fumiga on me (h) Fig. 1 Concentration loss of EF fumigation (140 g/m at 5℃ for 4 h) on Tricholoma matsutake C/C0 K won et al. Appl Biol Chem (2021) 64:64 Page 7 of 11 The concentration of EF decreased during 4  h of expo - 100  g/m of EF might be applicable. However, > 100  g/ sure on mushrooms. Loss rate of EF on T.matsutake was m of EF could be obtained with proven efficacy (> 99%) relatively low, approximately 30% at f.r. 1.5%. But EF was under the worst circumstance of commercials. EF con- required high concentration due to their high sorption to centration inside the styrofoam box containing T. mat- commodities like fruits and vegetable, which can cause to sutake was lower than outside. The difference of EF phytoxic damage to commodities [23–25]. concentration inside and outside the box might be dif- ferent depending on fanning conditions (running time, Gas penetration test of EF on packed mushroom numbers of fans, locations of fan, electrical capacity, with styrofoam box etc.) during the EF fumigition. Based on unpubuished A gas penetration test was performed using a 0.275  m data, we suggested using a fan for more than 2 h at low- fumigation chamber with different filling ratios of T. middle capactity for even distribution of gas. matsutake. As a result of EF gas penetration, there was a difference between the inside and outside of Commercial sized EF fumigation the styrofoam box (Fig.  2). When EF 35, 70, 140  g/ EF alone fumigation on Tricholoma matsutake m was applied, Ct products inside the styrofoam box The scaled-up EF fumigation to confirm disinfestation 3 3 were 60.2 ± 2.4, 95.0 ± 2.9 and 167.1 ± 6.1  g  h/m at on L. mali was performed using a 25 m Tarp-fumigation f.r. 1.0%, respectively. However, EF Ct products out- chamber filled with T. matsutake (1.7% f.r. w/v). When side the styrofoam box were 89.4 ± 2.2, 136.3 ± 2.3 140  g/m was applied for 4  h at 5°C, L. mali achieved and 219.7 ± 4.1  g  h/m at f.r. 1.0%, respectively, shown 100% mortality (total 1079 of fumigated L. mali lar- in Fig.  2. Regarding the initial concentration, the con- vae used). The acculatumed Ct products of EF were 3 3 centration of inside was 15–40% of the concentration 159.0 ± 3.1, 147.2 ± 2.8  g  h/m inside, 195.3 ± 4.3  g  h/m of outside. After 2  h of treatment, the inside and out- outside the styrofoam box (Fig. 3, Table 6). We confirmed side concentrations were similar. The calculation of Ct more than achievable L(Ct) values and the loss rates of 99% products in the lab might be different when applied EF were approximately 70–80% for 4  h exposure, which in actual scaled-up trials because it is denpendent on was similar to previous lab condition studies. When con- sealing conditions, temperature, and condition of com- ducting this test, we tried to confirm the mortality for L. modities (water content, logistic packing, etc.). Accord- mali naturally infested in T. matsutake, however collec- ing to our data when determing EF concentration to tion of a large amount of T. matsutake was impossible achieve target Ct products (112.9  g  h/m , Probit esti- and the number of infested L. mali per mushroom was mation for 99% mortality on L. mali) with 4 h exposure, too small. For a small number of L. mali, 100% mortality 100 35 g/m3 O 70 g/m3 O 140 g/m3 O 35 g/m3 I 70 g/m3 I 140 g/m3 I 0123 4 Fumiga on me (h) Fig. 2 Gas concentration, when fumigated with 35, 70, and 140 g/m EF for 4 h inside (I) and outside (O) of styrofoam box containing 1.0% f.r. of Tricholoma matsutake (temp. 5.1 ± 0.3℃) EF Concentra on (g/m ) Kwon et al. Appl Biol Chem (2021) 64:64 Page 8 of 11 S. B. O S. B. I#1 S. B. I#2 0123 4 Fumigaon me (h) 3 3 Fig. 3 Gas concentration when fumigated with 140 g/m EF for 4 h on Tricholoma matsutake (f.r. 1.7%) in scaled-up trials (25 m Tarp-fumigation chamber, temp.: 7.3 ± 0.4 ℃). *Gas concentration inside (S. B. I) and outside (S. B. O) the styrofoam box Table 6 Scaled-up trials of liquid EF-fumigaion and EF + PH3 fumigation on Tricholoma matsutake 3 3 2 Type and size of fumigation Appl. Dose (g/m ) Packing and Ct products (g h/m ) Mort. (%) (dead/total) Deterioration Market value gas sampling degree Fumigation chamber – Untreated – 0 (0/159) 0 a B1 B2 C (25 m ) # EF 140.0 S.B.(I 1) 159.0 ± 3.1 100 (331/331) 3 b D D D S.B.(I 2) 147.2 ± 2.8 100 (387/387) 3 b D D D S.B.(O) 195.3 ± 4.3 100 (361/361) 3 b D D D Fumigation chamber – Untreated – 0 (0/159) 0 a B1 B2 C (5 m ) # EF 35.0 + PH 0.5 S.B.(I 1) 50.6 ± 0.6 100 (397/397) 0 a B1 B2 C S.B.(I 2) 50.2 ± 1.1 100 (356/356) 0 a B1 B2 C S.B.(O) 66.4 ± 0.1 100 (344/344) 0 a B1 B2 C p-value < 0.0001 < 0.0001 S.B: Styrofoam box, Inside (I), Outside (O) 1. Condition: f.r. 1.7%, 7.33 ± 0.39 ℃ 2. Condition: f.r. 0.5%, 4.94 ± 0.21 ℃ The deterioration degrees. 0: Non, 1: water condensation, 2: water condensation following changing soft a cap and grill parts 3: water condensation following changing soft a cap, grill and then stem part Market value based on commercial grades. A: button, B1: young mushroom, B2: mature mushroom, C: overmature mushroom, D: not for sale was achieved. Regarding weight loss, in assessment of 3 d matsutake (0.5% f.r. w/v). When 35  g/m of EF + 0.5  g/ after fumigation, T. matsutake was not significantly dif - m of PH was applied for 4 h at 5 °C, L. mali achieved ferent (df: 16, p-value: 0.8062) (Fig.  4). Likewise, regard- 100% mortality (total 1,097 of fumigated L. mali lar- ing color change, T. matsutake was not significantly vae used). The acculatumed Ct products of EF were 3 3 different (df: 16, p-value: 0.8198) (Fig. 5.). 50.6 ± 0.6, 50.2 ± 1.1  g  h/m inside, 66.4 ± 0.1  g  h/m outside the styrofoam box (Fig.  6, Table  3). We con- EF fumigation mixed with PH3 on Tricholoma matsutake firmed more than achievable L(Ct) values and the 99% We proposed EF + PH combination fumigation. It loss rates of EF were approximately 60–70% for 4  h was performed using a 5  m container filled with T. exposure, which was similar to previous lab condition EF concentraon (g/m ) K won et al. Appl Biol Chem (2021) 64:64 Page 9 of 11 3 3 3 Fig. 4 Weight loss after 3-d fumigation on mushrooms fumigated with 140 g/m EF only and 35 g/m EF mixed with 0.5 g/m PH (4 h exposure). NS: Not significant 3 3 3 Fig. 5 Color change after 3-d fumigation on mushrooms fumigated with 140 g/m EF only and 35 g/m EF mixed with 0.5 g/m PH (4 h exposure). NS: Not significant Kwon et al. Appl Biol Chem (2021) 64:64 Page 10 of 11 S. B. I#1 S. B. I#2 S. B. O#1 0123 4 Fumigation time (h) 3 3 3 Fig. 6 Gas concentration when fumigated with 35 g/m EF + 0.5 g/m PH for 4 h on Tricholoma matsutake (f.r. 0.5%) in scale-up trials. (5 m fumigation chamber, Temp.: 5.0 ± 0.2 ℃) *Gas concentration inside (S. B. I) and outside (S. B. O) the styrofoam box Author details studies. Regarding weight loss, in assessment of 3-d Division of Applied Life Science (BK21 + Program), Gyeongsang National after fumigation, T. matsutake was not significantly University, Jinju 52828, Republic of Korea. Institute of Life Scienc, Gyeongsang different (df: 18, p -value: 0.4423) (Fig.  4). Regarding National University, Jinju 52828, Republic of Korea. Animal and Plant Quaran- tine Agency, Gimcheon 39660, Republic of Korea. USDA-ARS, US Pacific Basin color change, T. matsutake was not significantly differ - Agricultural Research Center, Hilo, HI 96720, USA. ent (df: 16, p-value: 0.3473) (Fig. 5). Thus, EF combined with PH fumigation was no damage to mushrooms 3 Received: 18 May 2021 Accepted: 18 August 2021 unlike EF alone fumigation. There were previous stud - ies reported that EF + CO [26], PH + CO for reduc- 2 3 2 ing LT (Lethal time) values [27] and PH + O [28] for 3 2 increasing efficacy and decreasing phytotoxic damage References 1. Lewandowski M, Sznyk A, Bednarek A (2004) Biology and morphometry to commodities. of Lycoriella ingenua (Diptera: Sciaridae). Biol Lett 41:41–50 2. 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Cho SW, Kim HK, Kim BS, Yang JO, Kim GH (2020) Combinatory effect of formate against stored grain insects by combination with carbon dioxide ethyl formate and phosphine fumigation Pseducoccus longispinus and P. in a ‘dynamic’ application. Pest Manag Sci 62:325–333 orchidicola (Hemiptera: Pseudococcidae) mortality and phytotoxicity to 27. Meenatchi R, Alice RPS, Paulin PP (2018) Synergistic effect of phosphine 13 foliage nursery plants. J Asia Pac Entomol 23(1):152–158 and carbon dioxide on the mortality of Tribolium castaneum (Coleoptera: 17. Hewlett PS, Plackett RL (1969) A unfied theory for quantal responses to Tenebrionidae) in paddy. J Agric Sci 10:503–510 mixtures of drugs: non-interactive action. Biometrics 15:591–610 28. Liu SS, Liu YB, Simmons GS (2015) Oxygenated phosphine fumigation 18. Institute SAS (2009) SAS/STAT 9.2 user’s guide, 2nd edn. SAS Institute Inc, for control of light brown apple moth (Lepidoptera: Tortricidae) eggs on Cary cut-flower. J Econ Entomol 108:1630–1636 19. O’connor L, Keli CB, (2005) Mushroom host influence on Lycoriella mali (Diptera: sciaridae) life cycle. J Econ Entomol 98(2):342–349 Publisher’s Note 20. Kwon TH, Park CG, Lee BH, Zarders DR, Roh GH, Kendra PE, Cha DH (2021) Springer Nature remains neutral with regard to jurisdictional claims in pub- Etyhl formate fumigation and ethyl formate plus cold treatment combi- lished maps and institutional affiliations. nation as potential phytosanitary quarantine treatments of Drosophila suzukii in blueberries. J Asia Pac Entomol 24:129–135 21. Kyung YJ, Kim HK, Cho SW, Kim BS, Yang JO, Koo HN, Kim GH (2019) Com- parison of the efficacy and phytotoxicity of phosphine and ethyl formate

Journal

Applied Biological ChemistrySpringer Journals

Published: Dec 1, 2021

Keywords: Lycoriella mali; Ethyl formate; Phosphine; Alternatives; EF + PH3; Synergistic effect

References