- 學位論文
光桿菌與蘇力菌於防治秋行軍蟲上的協力殺蟲效應
Synergistic insecticidal effect of Photorhabdus luminescens and Bacillus thuringiensis against fall armyworm (Spodoptera frugiperda)
摘要
混用兩種以上不同作用機制的微生物製劑可發揮協力殺蟲效應並且延緩抗藥性的發生。蘇力菌可分泌晶體毒蛋白使昆蟲中腸穿孔，可能能替代為異小桿線蟲，幫助光桿菌順利進入昆蟲血體腔內引發敗血症；光桿菌則可抑制昆蟲先天免疫反應，可能減少蘇力菌毒殺過程中受到免疫作用的拮抗。本研究分別將光桿菌商品化美國菌株Photorhabdus luminescens, Pl ATCC 29,999以及本土分離之菌株Pl 2103-UV與商品化蘇力菌 Bacillus thuringiensis subsp. kurstaki, Bt ABTS-351進行混和處理，分別以 1) 口服毒性試驗、2) 血淋巴試驗、和3) 中腸組織病理切片等方式確認殺蟲效果、協力殺蟲效應，深入探討可行性高，同時應用Pl與Bt管理秋行軍蟲Spodoptera frugiperda的操作方法。結果顯示隨著混和液當中Bt的比例提升，亦提高了對秋行軍蟲三齡幼蟲的殺蟲活性，尤其1:5處理下Pl ATCC 29,999協力比率 (synergistic ratio) 達到1.98，Pl 2103-UV更高達5.29，表示提升Bt的比率，不僅Bt的殺蟲活性增加，亦有效的提升了協力殺蟲的效應。欲進一步驗證Bt強化了Pl ATCC 29,999於秋行軍蟲血體腔內的感染，以微針 (0.3 mm) 抽取處理後的秋行軍蟲血淋巴液塗盤，結果顯示殺蟲效果最好的1:5 Pl: Bt混和處理於血體腔內的Pl菌量亦為最高 (2.12 × 105 CFU/mL, 96小時)，說明Bt比例提高時，能協助更多Pl入侵秋行軍蟲血體腔。病理切片結果顯示，1:5 Pl: Bt混和處理中，Pl ATCC 29,999特有病徵─腸腔大量囊泡及空泡化─顯著地增加，表示增加Bt比例，同時加劇了Pl ATCC 29,999於腸壁的病理作用；然而1:5混和處理中，Pl 2103-UV的囊泡數卻沒有顯著增加，可以確認兩株光桿菌與Bt協力機制並不相同。透過全基因定序分析兩種光桿菌，發現Pl 2103-UV能夠產生更多種類型的孔蛋白 (Porin)，Porin能參與革蘭氏陰性菌的感染，影響病原細菌的定殖；甚至抑制宿主的免疫，因此抑制了秋行軍蟲對蘇力菌的免疫，因而雖然腸壁沒有顯著的病徵，Pl 2013-UV仍測得最高的殺蟲活性與協力比。鎖定Pl 2103-UV獨有的磷孔蛋白 (Phosphoporin, PhoE)，分析PhoE於秋行軍蟲免疫反應中扮演的角色，能夠深入地探討協力機制的細節。本研究揭示Pl與Bt 1:5混和比例下，Bt協助Pl進入秋行軍蟲血體腔的感染效果，提高協力殺蟲效應。可作為應用Pl為協力角色，搭配其他微生物農藥管理大型鱗翅目害蟲的參考案例，朝向永續田間管理前進。
並列摘要
Combining microbial products with different mechanisms of action can produce synergistic insecticidal effects and slow down resistance development. Bacillus thuringiensis (Bt) can secrete crystalline protein toxins that cause perforation of the insect midgut, potentially replacing Heterorhabditis sp., helping Photorhabdus luminescens (Pl) successfully enter the insect hemocoel and cause septicemia. Pl can suppress the innate immune response of insects, which may reduce antagonism from the immune system during the toxic killing process by Bt. This study separately combined the commercial strain Pl ATCC 29,999 and the local isolate Pl 2103-UV with the commercial Bt subsp. kurstaki ABTS-351 for co-treatment. The insecticidal effect and synergistic activity were evaluated through 1) oral toxicity assays, 2) hemolymph assays, and 3) midgut histopathological sections to investigate the feasibility of applying Pl and Bt for managing Spodoptera frugiperda. The results showed that as the proportion of Bt increased in the mixture, the insecticidal activity against third-instar S. frugiperda larvae also increased. Particularly at the 1:5 ratio, the synergistic ratio reached 1.98 for Pl ATCC 29,999 and 5.29 for Pl 2103-UV, indicating that increasing the Bt ratio enhanced Bt insecticidal activity and effectively improved the synergistic killing effect. To further verify whether Bt enhanced the infection of Pl ATCC 29,999 in the S. frugiperda hemocoel, hemolymph was extracted from treated S. frugiperda using a microinjector (0.3 mm) and plated. The results showed that the 1:5 Pl:Bt mixture had the highest Pl counts in the hemocoel (2.12 × 105 CFU/mL at 96 hours), indicating that a higher Bt proportion assisted more Pl in invading the S. frugiperda hemocoel. Histopathological sections revealed that for the 1:5 Pl:Bt mixture with Pl ATCC 29,999, the characteristic symptoms of extensive vacuolization and vesiculation in the gut lumen significantly increased, indicating that increasing the Bt proportion also exacerbated the pathological effect of Pl ATCC 29,999 on the gut wall. However, for the 1:5 mixture with Pl 2103-UV, there was no significant increase in the number of vesicles, suggesting that the synergistic mechanisms of the two Pl strains with Bt were different. Whole-genome sequencing analysis revealed that Pl 2103-UV could produce more types of porins, which participate in the infection process of Gram-negative bacteria, affecting bacterial colonization and even suppressing host immunity. This may have inhibited the immune response of S. frugiperda against Bt, allowing Pl 2103-UV to achieve the highest insecticidal activity and synergistic ratio despite no significant changes in gut wall pathology. The synergistic mechanism can be further explored in depth by targeting the unique phosphoporin (PhoE) of Pl 2103-UV and analyzing its role in the S. frugiperda immune response. This study revealed that at the 1:5 Pl:Bt ratio, Bt assisted Pl in infecting the S. frugiperda hemocoel, enhancing the synergistic insecticidal effect. This can serve as a reference case for applying Pl as a synergistic agent with other microbial insecticides to manage significant lepidopteran pests, paving the way toward sustainable field management.