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Application of acibenzolar-S-methyl and its resistance mechanism in controlling diseases of Pak-choi
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|摘要:||Acibenzolar-S-methyl (ASM) 係苯并噻二唑 (benzothiadiazole) 的衍生物，其透過植物體內的水楊酸訊號傳遞路徑，誘導植物產生防禦反應而達到防治病害之效，此現象稱為誘導性抗病 (induced resistance)。本研究目的為探討ASM 應用在不結球白菜上是否會造成生理傷害、能否防治不結球白菜炭疽病 (anthracnose) 和苗立枯病 (damping-off)、及ASM誘導抗病之可能作用機制。以葉面噴灑和種子浸泡2種方式處理1、10和100 mg/L ASM，觀察對王冠和三鳳2 號品種的生長影響。結果顯示施用高濃度 (100 mg/L) ASM 時會引起植株鮮重減少、矮化或葉片畸形等藥害 (phytotoxicity)；而種子方面，浸泡高濃度ASM 的時間越久，則越容易引起種子發芽率降低或幼苗生長勢較差等徵狀。在防治炭疽病試驗中，於接種病原菌前預先處理1 和10 mg/L ASM，經統計分析其罹病度後得知，於接種前1天施用1與10 mg/L ASM 之防治效果可達80%以上，而接種前4天內皆為有效防治之施藥時間點，防治效果可超過50%，其中預先處理10 mg/L ASM 之防治效果與百克敏和亞托敏的防治效果相同。於種子浸泡ASM 防治不結球白菜苗立枯病之試驗中，僅有以10 mg/L ASM 處理三鳳2號品種種子18小時以上能防治立枯病，然此種處理條件亦會對幼苗生長造成影響，對植株生長影響甚大，顯示ASM 適合以種子浸泡之方式處理不結球白菜。本試驗分析不結球白菜抗病相關之酵素，結果得知王冠品種之超氧化物歧化酶 (superoxidase dismutase, SOD) 活性於處理1 mg/L ASM 後24小時達最高，而三鳳2號品種之SOD 活性於處理10 mg/L ASM 後24小時達最高；而接種炭疽病菌後，王冠品種的活性皆明顯高於對照組，三鳳2號品種的活性則於12小時後明顯高於對照組0.7倍。然於接種前3小時施用1與10 mg/L ASM 之王冠品種於6小時起SOD 活性開始上升，在24小時後活性增加0.5與0.7 倍；三鳳2 號品種分別預處理1 與10 mg/L ASM 之後的SOD 活性於3和6小時後開始明顯增加，24小時後活性增加0.3與0.4倍。於過氧化氫酶 (catalase, CAT) 之活性測定，顯示處理10 mg/L ASM 的王冠品種於6 小時活性會降低0.6倍，但隨後又回復至與對照組無明顯差異，然三鳳2號品種於施用各處理所測得之CAT活性皆無明顯差異。另王冠品種於接種病原菌後3與18小時，CAT活性明顯降低0.8與0.5倍；而三鳳2號品種則於接種後6小時，活性明顯減少0.8倍。若接種前3 小時預處理10 mg/L ASM 之王冠品種，CAT 活性於6 小時後明顯降低0.8倍，而三鳳2 號品種預處理1mg/L ASM 之活性，於12 小時後明顯降低0.7倍。過氧化酵素 (peroxidase, POD) 活性測定指出，處理1 mg/L ASM 後王冠品種之POD於第5天活性增加達1.4倍，處理10與100 mg/L ASM 後POD於第4天活性達到最高，分別較對照組增加1.9與4.2倍；三鳳2號品種處理1與10 mg/L ASM後之POD活性於1天後開始增加，而100 mg/L ASM 處理之植株於4 天後開始增加，且所有處理組之活性皆可持續至第7 天，活性分別較對照組增加0.5、1.0、0.9
倍。此兩種品種接種病原菌後，王冠品種的POD 活性於1 天後開始上升，於第6天時明顯增加0.9倍；而三鳳2 號品種之活性則於第6天才明顯增加0.3倍。王冠品種預處理1 mg/L ASM 之POD活性於3天後開始顯著增加，第6 天明顯增加達0.7倍；而預處理10 mg/L ASM之活性於3小時內立刻被活化，於第3天明顯增加達0.6倍；另預處理100 mg/L ASM之活性於1天起顯著增加，則於第5 天明顯增加達1.7倍。三鳳2 號品種預處理1 mg/L ASM之POD活性與對照組無明顯差異；而預處理10 mg/L ASM後之活性於第3天明顯增加0.7倍；另預處理100 mg/L ASM植株之POD 活性於第6天後明顯增加0.4倍。於苯丙胺酸氨裂解酵素(phenylalanine ammonia lyase, PAL)之活性測定，結果顯示王冠品種處理1 與10 mg/L ASM後，PAL活性於第3天明顯增加0.1與0.2 倍；100 mg/L ASM 處理之植株體內活性於5 天後明顯增加，於第7天活性增加達0.2倍。而三鳳2號品種施用1、10、100 mg/L ASM後7天，PAL 活性分別明顯增加0.4、0.4、0.6倍。於接種病原菌後，王冠品種於6天後，PAL活性明顯增加0.2倍；而三鳳2號品種的活性無任何改變。此外，王冠品種在接種前預先處理1 mg/L ASM之PAL活性無任何改變，而預處理10與100 mg/L ASM 後，PAL活性於第6天皆明顯增加0.2倍。三鳳2號品種預處理1 mg/L ASM 後PAL 活性無任何改變，而預處理10 mg/L ASM 植株的活性，則於6天後明顯增加0.3倍；另預處理100 mg/L ASM 後的PAL活性於3小時與第4天顯著增加0.4與0.2倍。前人研究指出，植物抗病性的產生除上述與抗病有關酵素外，亦包含病程相關蛋白質 (pathogenesis-related proteins, PR protein) 和總酚化物 (total phenolic compounds) 等，未來將進一步分析其他與抗病性產生有關之機制。|
Acibenzolar-S-methyl (ASM) is one of the derivatives of benzothiadiazole. It can induce plant defense responses to control diseases by salicylic acid signaling pathway. Thus, ASM can be an alternative control agent in protecting crops. In this study, the objectives are 1) estimation of phytotoxicity caused by ASM on Pak-choi (Brassica campestris L. Chinese Group), 2) efficacy of controlling anthracnose and damping-off of Pak-choi, 3) possibility of resistant mechanisms inducing by ASM in Pak-choi. Based on leaf spraying and seed soaking, two Pak-choi cultivars of Wang Guan and San Feng No.2 were treated with 1, 10, and 100 mg/L ASM, respectively. The leaf spraying results indicated that Pak-choi could showe some abnormal symptoms caused by 100 mg/L ASM, including fresh weight reduction, dwarf or deformation of leave shape. For seed treatment, the rate of seed germination were reduced, or even germination the seedling did not show healthy under 10 or 100 mg/L ASM. Comparing the efficacy of two cultivars pre-treated with 1 and 10 mg/L ASM 1 day or 4 day before inoculated 105 conidia/ ml C. higginsianum PA01could induce resistance and reduce more than 80% or 50% disease severity, respectively. Consequently, the two cultivars pre-treated with 10 mg/L ASM showed same efficacy as treated fungicides of pyraclostrobin and azoxystrobin. The other side, only San Feng No.2 cultivar showed good efficacy to againt damping-off caused by Rhizoctonia solani when the seeds soaked with 10 mg/L ASM for 18 hrs. However, the seedings of two cultivars showed abnormal symptoms after treated with 10 mg/L ASM. Thus, seed treatment might not good method for controlling damping-off. For carring out the resistance mechanism of Pak-choi, several enzymes corresponded with crops resistance were analyzed, including superoxidase dismutase (SOD), catalase (CAT), peroxidase (POD) and phenylalanine ammonia lyase (PAL). The activity of SOD in Wang Guan cultivar was highest at 24 hrs after treated with 1 mg/L ASM, however, the activity of SOD in San Feng No.2 cultivar was highest at 24 hrs after treated with 10 mg/L ASM. The activities of SOD in Wang Guan cultivar were 0.6 ~ 0.8 times high; meanwhile, the activity of SOD in San Feng No.2 cultivar was 0.7 times high at 12 hrs after inoculated with C. higginsianum PA01. The activities of SOD in Wang Guan cultivar were 0.7 and 0.5 times high at 24 hrs, and the activities of SOD in San Feng No.2 cultivar were 0.3 and 0.4 high at 24 hrs after pre-treated with 1 and 10 mg/L ASM. In the activity of CAT, the Wang Guan cultivar was reduced 0.6 times high at 6 hrs after treated 10 mg/L ASM and subsequently returned to normal level. Moreover, the activity of CAT in San Feng No.2 cultivar did not show significant difference after treated with ASM. After inoculation, the activities of CAT in Wang Guan cultivar were reduced 0.8 and 0.5 times at 3 and 18 hrs and the activity of CAT in San Feng No.2 cultivar was reduced 0.8 times at 6 hrs. The other side, the activity of CAT in Wang Guan cultivar was reduced 0.8 times at 6 hrs after pre-treated with 10 mg/L ASM, however, the activity of CAT in San Feng No.2 cultivar was reduced 0.7 times at 12 hrs after pre-treated with 1 mg/L ASM. The POD test indicated that the activity of POD in Wang Guan cultivar was increased around 1.4 times high at 5 days after treated 1 mg/L ASM, and the POD activities were 1.9 and 4.2 times high at 4 days after treated 10 and 100 mg/L ASM, respectively. Meanwhile, the activity of POD in San Feng No.2 cultivar was increased at 1 day after treated with 1 and 10 mg/L, and the activity increased at 4 days after with 100 mg/L. In this test, the activities of POD in the two cultivars were induced and the activities could be persisted for 7 days. Comparing the POD activities without inoculation, the POD activities in Wang Guan cultivar and San Feng No.2 cultivar were increased 0.9 and 0.3 times at 6 days after inoculation. Moreover, the POD activities of Wang Guan cultivar were highest at 6 days, 3 days and 5 days after pre-treated with 1, 10, and 100 mg/L ASM, respectively. However, the activity of POD in San Feng No.2 cultivar did not show significant difference after pre-treated with 1 mg/L ASM. The activity of POD in San Feng No.2 cultivar was increased 0.7 times at 3 days after pre-treated with 10 mg/L ASM, and the activity of POD in San Feng No.2 cultivar was increased 0.4 times at 6 days after pre-treated with 100 mg/L ASM. For the activity of PAL in Pak-choi, the Wang Guan cultivar was increased significantly 0.1 and 0.2 times high at 3 days after treated with 1 and 10 mg/L ASM, and the PAL activity was increased significantly at 5 days and increased 0.2 times high at 7 days after treated with 100 mg/L ASM. The other sides, the activities of PAL in San Feng No.2 were increased 0.4, 0.4, and 0.6 times high at 7 days after treated with 1, 10, and 100 mg/L ASM, respectively. The activity of PAL in Wang Guan cultivar was increased 0.2 times at 6 days, however, the activity of PAL in San Feng No.2 cultivar did not show significant difference after inoculating. The activity of PAL in Wang Guan cultivar did not show significant difference after pre-treated with 1 mg/L ASM, however, the activities of PAL in Wang Guan cultivar were both increased 0.2 times at 6 days after pre-treated with 10 and 100 mg/L ASM. The activity of PAL in San Feng No.2 cultivar did not show significant difference after pre-treated with 1 mg/L ASM, however, the activity of PAL in San Feng No.2 cultivar was increased 0.3 times at 6 days after pre-treated with 10 mg/L ASM. Moreover, the activities of PAL in San Feng No.2 were increased 0.4 and 0.2 times at 3 hrs and 4 days after pre-treated with 100 mg/L ASM. In this study, the other defense responses of crops were not examined, including pathogenesis-related (PR) proteins or content of total phenolic compounds. These resistance mechanisms will be carried out in future.
|Appears in Collections:||植物病理學系|
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