臺灣秋行軍蟲 (Spodoptera frugiperda) 對二醯胺類殺蟲劑抗藥性機制探討

Abstract

秋行軍蟲 (Spodoptera frugiperda (J.E. Smith)) 為原產於美洲的植食性害蟲，危害的作物種類眾多，對農業生產造成極大的威脅。秋行軍蟲自 2019 年入侵臺灣至今已接近五年，極有可能對目前所使用的防治藥劑產生抗藥性。因此，本論文於 2022 年自彰化縣、雲林縣、嘉義縣、臺南市和花蓮縣這五個臺灣主要的玉米產區採集田間秋行軍蟲，以飼料混拌法測試三齡幼蟲對硫敵克 (thiodicarb)、賜諾特 (spinetoram)、諾伐隆 (novaluron)、氟大滅 (flubendiamide) 和剋安勃 (chlorantraniliprole) 這五種緊急防治用藥的感受性。結果發現採自田間秋行軍蟲對賜諾特的敏感性最高，而對氟大滅的敏感性最低，其中彰化、嘉義、臺南地區的族群對氟大滅之百分之九十致死濃度 (90% lethal concentration, LC90) 超過田間推薦施用濃度。另外，各地區的秋行軍蟲對剋安勃的半致死濃度 (medium lethal concentration, LC50) 與感性蟲相比皆出現較大的抗性比，顯示目前田間秋行軍蟲對氟大滅及剋安勃這兩種二醯胺類 (diamide) 藥劑出現抗藥性。進一步選擇敏感性相對較低之臺南地區秋行軍蟲，比較三、四和五齡期秋行軍蟲對上述五種藥劑的感受性，發現除了氟大滅以外，五齡蟲對其餘緊急防治藥劑的 LC90 皆未超過田間推薦藥劑濃度。為了進一步瞭解秋行軍蟲對氟大滅及剋安勃產生抗藥性的成因，分別將採自彰化和臺南地區的田間族群以氟大滅和剋安勃篩選，建立抗性品系。經過 11 代及 10 代的篩選後，秋行軍蟲抗性蟲對氟大滅和剋安勃的抗性分別增加了 18.0 倍和 25.8 倍。接著針對氟大滅及剋安勃的作用標的魚尼丁受體上已知及可能的鱗翅目突變位點 K4642R、Y4643F、I4734C、S4864L、V4890M 和 G4891V 進行點突變偵測，發現抗性蟲在 4643 及 4890 這兩個位點有核酸序列的突變，但並未發現有任何的胺基酸位點產生突變。之後分別以胡椒基丁醚 (piperonyl butoxide, PBO)、1-氟-2,4-二硝基苯 (1-fluoro-2 4-dinitrobenzene, DNFB) 和磷酸三苯酯 (triphenyl phosphate, TPP) 這三種協力劑測試對氟大滅與剋安勃抗性蟲的協力效果，結果發現 PBO、DNFB 及 TPP 對氟大滅抗性蟲的協力比分別為 1.75、1.18 和 1.23，其中 PBO 具有顯著的協力效果，而 DNFB 與 TPP 則沒有顯著的協力效果；對剋安勃抗性蟲的協力效果分別為 3.03、5.88 及 28.8，且 PBO、DNFB 和 TPP 皆具有顯著的協力效果。進一步分析感性蟲與抗性蟲的酵素活性，以 ethoxycoumarin-O-deethylase (ECOD) 法測量細胞色素 P450 活性，發現氟大滅抗性蟲的活性為感性蟲的 1.24 倍並具有顯著差異 (p = 0.0012)，而剋安勃抗性蟲活性為感性蟲的 0.96 倍且沒有顯著差異 (p = 0.1971)；使用 1-Chloro-2,4-dinitrobenzene (CDNB) 與 3,4-dichloronitrobenzene (DCNB) 這兩種受質測試穀胱甘肽 S-轉移酶活性，以 CDNB 為受質發現氟大滅與剋安勃抗性蟲的活性分別為感性蟲的 1.27 及 1.33 倍，亦具有顯著差異 (p = 0.001)，但以DCNB 為受質則感性蟲與抗性蟲則無顯著的差異 (p = 0.126)；使用 α-naphthyl acetate (α-NA) 和 β-naphthyl acetate (β-NA) 這兩種受質測試酯酶活性，結果發現以 α-NA 為受質時，氟大滅與剋安勃抗性蟲的酯酶活性分別為感性蟲的1.24及1.52 倍且與感性蟲有顯著差異 (p < 0.0001)。以 β-NA 作為受質，氟大滅與剋安勃抗性蟲的酯酶活性分別為感性蟲的1.86和1.94 倍，亦與感性蟲有顯著差異 (p < 0.0001)。最後在交互抗性的測試中，發現對氟大滅與剋安勃產生抗性之抗性蟲間，會對二藥劑有交互抗性產生，但與硫敵克、賜諾特、諾伐隆則沒有交互抗性產生。本論文結果除了可作為田間防治的參考外，亦能作為國內秋行軍蟲對二醯胺類藥劑抗藥性研究的基礎。

The fall armyworm (Spodoptera frugiperda (J.E. Smith)) is an herbivorous pest originating from the Americas which causes damage to many crops and poses a great threat to agricultural production. It initially invaded Taiwan in 2019 and may have begun developing resistance to currently used emergency insecticides in the ensuing five years. This study collected field fall armyworms from five major corn-producing areas in Changhua, Yunlin, Chiayi, Tainan, and Hualien in 2022, and used the diet-incorporation method to test the susceptibility of third instar larvae to five emergency insecticides: thiodicarb, spinetoram, novaluron, flubendiamide, and chlorantraniliprole. The results showed that fall armyworms collected from field displayed the highest susceptibility to spinetoram and the lowest susceptibility to flubendiamide. Additionally, the 90% lethal concentration (LC90) of Changhua, Chiayi, and Tainan strains to flubendiamide exceeded recommended concentrations. Furthermore, the medium lethal concentration (LC50) of field fall armyworms showed a greater resistance ratio to chlorantraniliprole compared with the susceptible strain, indicating that field fall armyworms may be resistant to the two diamides of flubendiamide and chlorantraniliprole. We further chose Tainan strain, which had the lowest susceptibility, testing the susceptibilities to 3rd, 4th & 5th instar larvae of five pesticides showed that. Except for flubendiamide, the LC90 of the fifth instar to other emergency insecticides did not exceed recommended concentrations. In order to understand the mechanism of fall armyworm resistance to flubendiamide and chlorantraniliprole, field populations from Changhua and Tainan were selected with flubendiamide and chlorantraniliprole to establish resistant strains, respectively. After 11 and 10 generations of selection, resistant strain of fall armyworm to flubendiamide and chlorantraniliprole increased 18.0 and 25.8 times, respectively. After detecting point mutations at the known and possible mutation sites of K4642R、Y4643F、I4734C、S4864L、V4890M and G4891V on target ryanodine receptors, nucleic acid sequence mutations at the two sites 4643 and 4890 were found in resistant strains, but no mutations were found at any amino acid sites. Three synergists, piperonyl butoxide (PBO), 1-fluoro-2 4-dinitrobenzene (DNFB), and triphenyl phosphate (TPP), were used to test the synergistic effects to resistant strains. The results showed that the synergistic ratios of PBO, DNFB and TPP against flubendiamide-resistant fall armyworms were 1.75, 1.18 and 1.23 respectively. PBO had a significant synergistic effect, while DNFB and TPP had no significant synergistic effect; and the synergistic ratios on chlorantraniliprole-resistant fall armyworms were 3.03, 5.88 and 28.8 respectively. PBO, DNFB and TPP all had significant synergistic effects against chlorantraniliprole-resistant fall armyworms. We conducted further analysis of the enzyme activities in susceptible and resistant strains. Using ethoxycoumarin-O-deethylase (ECOD) to measure cytochrome P450 activity, and found that the activities of the flubendiamide-resistant strain was significantly 1.24 times higher than that of the susceptible strain (p = 0.0012), while the activities of chlorantraniliprole-resistant strain was 0.96 times that of the susceptible strain, showing no significant difference (p = 0.1971). The two substrates 1-Chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB) were used to test glutathione S-transferase activity. For CDNB, the activities of the flubendiamide-resistant and chlorantraniliprole-resistant strains were both significantly (p = 0.001) higher than the susceptible strain at 1.27 and 1.33 times, respectively. DCNB showed no significant differences in the activities between the susceptible and resistant strains (p = 0.126). Esterase activity was tested using α-naphthyl acetate (α-NA) and β-naphthyl acetate (β-NA) as substrates. The results showed that when α-NA was used as substrate, the esterase activities of flubendiamide-resistant and chlorantraniliprole-resistant strains were both significantly (p < 0.0001) higher than the susceptible strain at 1.24 and 1.52 times, respectively. When β-NA was used as the substrate, the esterase activities of flubendiamide-resistant and chlorantraniliprole-resistant strains were 1.86 and 1.94 times that of the susceptible strain, respectively, and were also significantly different from the susceptible strain (p < 0.0001). Finally, cross-resistance tests showed that resistant strains displayed cross-resistance to flubendiamide and chlorantraniliprole, but not to thiodicarb, spinetoram, and novaluron. The results of this study can be used as a reference for field control, and can also serve as the basis for research on the resistance of fall armyworms to diamide pesticides.