以防治油菜菌核病為目標(biāo)的核盤菌乙酰乳酸合酶抑制劑的篩選

溫雪瑋，陳泱泱，武斌，胡東維，梁五生

以防治油菜菌核病為目標(biāo)的核盤菌乙酰乳酸合酶抑制劑的篩選

溫雪瑋，陳泱泱，武斌，胡東維，梁五生*1

（浙江大學(xué)農(nóng)業(yè)與生物技術(shù)學(xué)院生物技術(shù)研究所/農(nóng)業(yè)部作物病蟲分子生物學(xué)重點(diǎn)實(shí)驗(yàn)室，杭州310058）

為開發(fā)新的油菜菌核病防治藥劑，選取7種乙酰乳酸合酶（acetolactate synthase,ALS）潛在抑制劑（氯嘧磺隆、芐嘧磺隆、氯磺隆、甲嘧磺隆、咪唑乙煙酸、滅草喹和磺胺噻唑）作為3類ALS酶抑制劑（磺酰脲類、咪唑啉酮類、磺胺噻唑類）的代表，對(duì)其防治油菜菌核病的性能進(jìn)行測(cè)試。結(jié)果顯示，1.0 mg/L滅草喹、磺胺噻唑、氯嘧磺隆、芐嘧磺隆和氯磺隆對(duì)核盤菌ALS酶活性有較強(qiáng)的抑制作用，而相同劑量的甲嘧磺隆的抑制作用微弱，咪唑乙煙酸無抑制作用。相應(yīng)地，半最大效應(yīng)濃度（half-maximal effective concentration，EC50）值顯示，核盤菌的菌絲生長(zhǎng)對(duì)滅草喹、磺胺噻唑、氯嘧磺隆、芐嘧磺隆和氯磺隆的抑制作用較敏感，而對(duì)甲嘧磺隆和咪唑乙煙酸的敏感性較弱。氯嘧磺隆、芐嘧磺隆、氯磺隆、滅草喹和甲嘧磺隆對(duì)油菜葉片ALS酶活性有強(qiáng)烈的抑制作用，咪唑乙煙酸和磺胺噻唑的抑制作用明顯比上述5種抑制劑弱。氯嘧磺隆、芐嘧磺隆、氯磺隆、滅草喹、甲嘧磺隆和咪唑乙煙酸處理對(duì)油菜幼苗生長(zhǎng)發(fā)育有明顯的負(fù)面影響，主要表現(xiàn)為生長(zhǎng)速率下降，植株明顯比對(duì)照矮小，葉片變小，葉色變黃。磺胺噻唑處理對(duì)油菜幼苗生長(zhǎng)發(fā)育的負(fù)面影響明顯比上述6種抑制劑弱。選擇對(duì)核盤菌ALS酶活性和菌落生長(zhǎng)的抑制作用都較強(qiáng)、而對(duì)油菜ALS酶活性的抑制作用和對(duì)油菜幼苗生長(zhǎng)的負(fù)面影響最弱的磺胺噻唑進(jìn)行抗核盤菌侵染試驗(yàn)。結(jié)果顯示，磺胺噻唑處理顯著地降低了核盤菌對(duì)油菜葉片的侵染程度，對(duì)油菜菌核病產(chǎn)生了較明顯的防治效果。上述研究結(jié)果表明，磺胺噻唑可作為分子結(jié)構(gòu)基礎(chǔ)來進(jìn)一步開發(fā)以核盤菌ALS酶為作用靶標(biāo)的油菜菌核病防治劑。

乙酰乳酸合酶；菌核?。缓吮P菌；油菜；磺胺噻唑

核盤菌（Sclerotinia sclerotiorum）是一種可對(duì)農(nóng)業(yè)產(chǎn)生嚴(yán)重負(fù)面影響的植物病原真菌。該真菌可侵染超過400種植物，其中包括不少重要的農(nóng)作物和蔬菜[1-4]。油菜是我國(guó)主要的油料作物，也是世界范圍內(nèi)重要的油料作物。核盤菌是油菜的主要病害之一，可導(dǎo)致油菜菌核?。╮apeseed Sclerotinia disease）。該病在我國(guó)一般年份的發(fā)病率為10%～30%，導(dǎo)致5%～30%的產(chǎn)量損失；流行年份在一些嚴(yán)重田塊的發(fā)病率可超過80%[5-6]。

由于目前尚無核盤菌高抗油菜品種，油菜種植者仍主要依賴殺菌劑來防治該病害。多年來我國(guó)一直使用以多菌靈和甲基托布津?yàn)橹鞯谋讲⑦溥蝾悮⒕鷦﹣矸乐斡筒司瞬7]。由于長(zhǎng)期、反復(fù)地使用多菌靈，目前已在許多地區(qū)監(jiān)測(cè)到抗多菌靈的核盤菌菌株[7-9]。因此，有必要指導(dǎo)油菜種植者更加科學(xué)地使用已有殺菌劑，另外還需要不斷開發(fā)新的油菜菌核病防治藥劑。

乙酰乳酸合酶（acetolactate synthase,ALS），又稱乙酰羥基酸合酶（acetohydroxyacid synthase），是植物和微生物細(xì)胞中催化支鏈氨基酸生物合成過程中第一步反應(yīng)的酶[10-12]。通過抑制植物細(xì)胞中的ALS酶活性可以破壞支鏈氨基酸生物合成，阻斷蛋白質(zhì)合成，干擾細(xì)胞分裂，結(jié)果影響植物生長(zhǎng)，甚至可能導(dǎo)致植物死亡[12-13]。由于動(dòng)物細(xì)胞內(nèi)不含ALS酶，在田間施用ALS酶抑制劑作為除草劑對(duì)人、畜等動(dòng)物安全，故此酶是除草劑研發(fā)中的一種重要靶標(biāo)[14-15]。近年來，以人體病原細(xì)菌和真菌的ALS酶作為靶標(biāo)開發(fā)新的人用抗生素和殺菌劑的研究報(bào)道逐漸增多[16-21]，但嘗試?yán)肁LS酶抑制劑來防治植物真菌病害的研究目前只見到1篇文獻(xiàn)報(bào)道[22]。

我們從NCBI數(shù)據(jù)庫中搜取核盤菌ALS酶的氨基酸序列（XP_001592310.1）和油菜ALS酶的氨基酸序列（CAA77613.1），通過序列比對(duì)后發(fā)現(xiàn)兩者的同源性只有36.48%（圖1）。如此低的同源性暗示理論上有可能篩選到對(duì)核盤菌ALS酶作用較強(qiáng)、但對(duì)油菜ALS酶作用較弱的抑制劑，用于防治油菜菌核病。本文報(bào)道了我們對(duì)這一可能性進(jìn)行探索的試驗(yàn)結(jié)果。

圖1 核盤菌和油菜的ALS酶氨基酸序列比較Fig.1 Comparison of the amino acid sequences of acetolactate synthases of Sclerotinia sclerotiorum and rapeseed

1 材料與方法

1.1 試驗(yàn)材料

核盤菌分離物（7-3）分離自油菜種植地，之前已用于進(jìn)行其他一些研究[23-25]。油菜品種為浩油11。

核盤菌的培養(yǎng)按照之前報(bào)道的方法進(jìn)行[23-25]。在用固體平板培養(yǎng)時(shí)，將馬鈴薯葡萄糖瓊脂（potato dextrose agar,PDA）培養(yǎng)基倒至直徑為9 cm的培養(yǎng)皿中制成PDA平板，將核盤菌菌核接種于PDA平板表面中心位置，然后置于暗處22℃下培養(yǎng)。繼代培養(yǎng)時(shí)用滅過菌的5 mm內(nèi)徑打孔器從PDA平板上培養(yǎng)的核盤菌菌落外緣打取菌碟接種于新的PDA平板上，使菌絲面與培養(yǎng)基接觸，置于暗處22℃下培養(yǎng)。在用液體培養(yǎng)基培養(yǎng)時(shí)，如上用打孔器從PDA平板上培養(yǎng)的核盤菌菌落外緣打取菌碟接種到用三角瓶盛裝的馬鈴薯葡萄糖（potato dextrose,PD）培養(yǎng)基中，然后置于暗處22℃下振蕩培養(yǎng)。

油菜苗的培養(yǎng)按照常規(guī)方法進(jìn)行。將油菜種子用水沖洗干凈后在蒸餾水中浸泡1 d，然后置于濕潤(rùn)的濾紙上，放入人工氣候箱中光照萌發(fā)（22℃，光/暗=16 h/8 h）。用塑料杯（開口直徑為6.8 cm）分裝適量的營(yíng)養(yǎng)土，將種子萌發(fā)后獲得的油菜幼苗植于營(yíng)養(yǎng)土中，每杯土栽1株苗，將幼苗放入人工氣候箱中光照培養(yǎng)（22 ℃，光/暗=16 h/8 h）。

1.2 核盤菌菌絲ALS酶的提取

用PD培養(yǎng)基培養(yǎng)核盤菌6 d，取菌絲用2層紗布過濾并盡量擠去培養(yǎng)基，稱取適量菌絲，置于用液氮預(yù)冷過的研缽中，依次加入適量石英砂和液氮，將菌絲研磨成粉末，然后按菌絲鮮質(zhì)量∶緩沖液體積＝1∶2的比例加入ALS酶提取緩沖液[0.1 mol/L磷酸鉀緩沖液（pH 7.5），1.0 mmol/L焦磷酸氯化硫胺素，0.1 mmol/L FAD，3.0 mmol/L MgCl2]，充分混勻，冰浴20 min后用2層紗布過濾，將濾液在4℃、1.2萬r/min條件下離心20 min，收集上清液，即核盤菌菌絲ALS粗酶液。按參考文獻(xiàn)[26]的方法測(cè)定提取的粗酶液中蛋白質(zhì)濃度。

1.3 油菜葉片ALS酶的提取

從油菜幼苗上剪取適量葉片，用水沖洗干凈后吸去表面水分，稱鮮質(zhì)量，置于用液氮預(yù)冷過的研缽中，加入適量石英砂，再加液氮研磨為粉末，按葉片鮮質(zhì)量∶緩沖液體積＝1∶5的比例加入ALS酶提取緩沖液（組成同核盤菌菌絲ALS酶的提?。?，充分混勻，冰浴20 min后用2層紗布過濾，將濾液在4℃、1.2萬r/min條件下離心20 min，收集上清液，即油菜葉片ALS粗酶液。按參考文獻(xiàn)[26]的方法測(cè)定提取的粗酶液中蛋白質(zhì)濃度。

1.4 ALS酶潛在抑制劑對(duì)核盤菌和油菜ALS酶的抑制效力測(cè)定

研究表明，不同物種的ALS酶之間可能有差異，抑制劑的抑制效果有物種特異性[27-32]。在試驗(yàn)前因不明確本文測(cè)試的ALS酶抑制劑對(duì)核盤菌ALS酶及后續(xù)油菜ALS酶的抑制效力，所以將它們稱為“核盤菌和油菜ALS酶的潛在抑制劑”。經(jīng)過調(diào)研文獻(xiàn)，選取以下7種ALS酶潛在抑制劑進(jìn)行試驗(yàn)：氯嘧磺隆、芐嘧磺隆、氯磺隆、甲嘧磺隆、咪唑乙煙酸、滅草喹和磺胺噻唑。其中：氯嘧磺隆、芐嘧磺隆、氯磺隆和甲嘧磺隆作為磺酰脲類ALS酶抑制劑的代表，咪唑乙煙酸和滅草喹作為咪唑啉酮類ALS酶抑制劑的代表，磺胺噻唑作為磺胺噻唑類ALS酶抑制劑的代表。以上7種ALS酶潛在抑制劑均購自上海阿拉丁生化科技股份有限公司。

[33]報(bào)道的方法測(cè)定ALS酶的活性并略作改進(jìn)，即用乙酰乳酸脫羧酶取代硫酸進(jìn)行脫羧反應(yīng)。反應(yīng)分3類：1）對(duì)照反應(yīng)管。取試管，加入1 mL前面提取的粗酶液和50 μL 30%硫酸，搖勻，再加入1 mL反應(yīng)緩沖液[50 mmol/L磷酸鉀緩沖液（pH 7.0），200 mmol/L丙酮酸鈉，0.5 mmol/L焦磷酸氯化硫胺素，0.03 mmol/L FAD，2.0 mmol/L MgCl2]，以及10 μL稀釋100倍的乙酰乳酸脫羧酶溶液，搖勻，37℃水浴反應(yīng)1 h。2）正常反應(yīng)管。取試管，加入1 mL粗酶液，1 mL反應(yīng)緩沖液，10 μL稀釋100倍的乙酰乳酸脫羧酶溶液和50 μL蒸餾水，搖勻，37℃水浴反應(yīng)1 h。3）加抑制劑反應(yīng)管。取試管，加入1 mL粗酶液，適量ALS酶潛在抑制劑溶液，1 mL反應(yīng)緩沖液，10 μL稀釋100倍的乙酰乳酸脫羧酶溶液，加適量蒸餾水使反應(yīng)液體積一致，并使抑制劑終質(zhì)量濃度為1 mg/L，搖勻，37℃水浴反應(yīng)1 h。上述3類反應(yīng)管水浴反應(yīng)后每管都依次加入0.5%肌酸500 μL、9% α-萘酚溶液500 μL（用4 mol/L NaOH配制），60℃水浴加熱15 min，室溫下放置15 min，5 000 r/min離心5 min，收取上清液。將從對(duì)照反應(yīng)管得到的上清液用來調(diào)零，測(cè)定從正常反應(yīng)管和加抑制劑反應(yīng)管得到的上清液在530 nm處的吸光度值D（530 nm）。通過D（530 nm）、反應(yīng)液中的蛋白質(zhì)量和反應(yīng)時(shí)間計(jì)算酶活性及抑制劑對(duì)ALS酶活性的抑制率。以U表示酶活性單位，1 U指每毫克蛋白質(zhì)每小時(shí)使反應(yīng)液在530 nm處吸光度的變化值為1.0。抑制劑對(duì)ALS酶活性的抑制率＝（正常反應(yīng)管的酶活性－加抑制劑反應(yīng)管的酶活性）/正常反應(yīng)管的酶活性×100%。測(cè)定試驗(yàn)重復(fù)3次，每次試驗(yàn)中每類反應(yīng)管都至少平行測(cè)定3份。

1.5 核盤菌對(duì)ALS酶潛在抑制劑的敏感性測(cè)定

將抑制劑分別溶解后取適量加入PDA培養(yǎng)基中，倒至9 cm直徑培養(yǎng)皿中，每種ALS酶潛在抑制劑都制成含不同濃度抑制劑的敏感性測(cè)定PDA平板，用滅過菌的5 mm內(nèi)徑打孔器從繼代培養(yǎng)PDA平板上培養(yǎng)2 d的核盤菌菌落外緣打取菌碟，接種于敏感性測(cè)定PDA平板中心，然后置于暗處22℃下培養(yǎng)，觀察菌絲生長(zhǎng)情況。每種濃度至少重復(fù)3份。當(dāng)無ALS酶潛在抑制劑PDA平板（對(duì)照平板）上的菌落快長(zhǎng)滿平板時(shí)，從2個(gè)互相垂直的方向測(cè)量所有PDA平板上的菌落直徑，所得數(shù)據(jù)用于計(jì)算EC50值（抑制50%菌絲生長(zhǎng)的有效濃度）。EC50值的計(jì)算按參考文獻(xiàn)[22-23]的方法進(jìn)行。

1.6 ALS酶潛在抑制劑對(duì)油菜幼苗生長(zhǎng)發(fā)育的影響試驗(yàn)

培養(yǎng)油菜幼苗至適當(dāng)大小（約3～5葉期），用鑷子夾取脫脂棉吸取ALS酶潛在抑制劑溶液（1.0 mg/L），輕柔涂抹油菜幼苗第1片真葉的上表面，將幼苗置于人工氣候箱中，22℃下光照培養(yǎng)。每天觀察記錄幼苗的生長(zhǎng)發(fā)育情況。試驗(yàn)重復(fù)3次，在每次試驗(yàn)中每種處理至少重復(fù)5株。

1.7 ALS酶潛在抑制劑對(duì)核盤菌侵染油菜的防治效果測(cè)定

按參考文獻(xiàn)[1]的方法進(jìn)行核盤菌侵染油菜離體葉片試驗(yàn)。取油菜葉片，先后用自來水和蒸餾水沖洗干凈。取直徑11 cm的玻璃培養(yǎng)皿，鋪上2層粗濾紙，加入適量無菌水使濾紙充分潤(rùn)濕。然后將葉片鋪于濕潤(rùn)的濾紙上，用醫(yī)用無菌脫脂棉吸取無菌水包裹葉柄保濕，在非葉脈位置用針尖刺一小孔，用滅過菌的5 mm內(nèi)徑打孔器從繼代培養(yǎng)PDA平板上培養(yǎng)2 d的核盤菌菌落外緣打取菌碟，接種于油菜離體葉片的小孔上，使菌絲面與葉片接觸。將接種了菌碟的葉片分成2組：一組用1.0 mg/L ALS酶潛在抑制劑溶液進(jìn)行葉面噴霧處理，使葉面均勻分布細(xì)小液滴；另一組用無菌蒸餾水對(duì)葉面進(jìn)行類似的噴霧處理，作為有接菌但無抑制劑處理的對(duì)照。另外取一組葉片作為空白（無菌）接種對(duì)照，即用滅菌的5 mm內(nèi)徑打孔器從PDA無菌平板上打取無菌瓊脂圓塊，接種于油菜離體葉片的小孔上。將上述放有試驗(yàn)葉片的培養(yǎng)皿蓋上玻璃蓋后置于人工氣候箱中，22℃下光照培養(yǎng)，1 d后拍照，并測(cè)量最寬處和最窄處的長(zhǎng)度來計(jì)算病斑幅度[病斑幅度=（最寬處長(zhǎng)度＋最窄處長(zhǎng)度）/2]。試驗(yàn)重復(fù)3次，在每次試驗(yàn)中每組處理或?qū)φ罩辽俳臃N10片油菜葉。

2 結(jié)果與分析

2.1 ALS酶潛在抑制劑對(duì)核盤菌ALS酶活性的抑制作用

檢測(cè)結(jié)果（圖2）表明：當(dāng)抑制劑質(zhì)量濃度為1.0 mg/L時(shí)，氯嘧磺隆、芐嘧磺隆、氯磺隆、甲嘧磺隆、滅草喹和磺胺噻唑這6種抑制劑均對(duì)核盤菌ALS酶活性有一定的抑制作用，且以滅草喹的抑制作用最強(qiáng)，甲嘧磺隆的抑制作用最弱，僅有微弱的抑制作用；而1.0 mg/L咪唑乙煙酸對(duì)核盤菌ALS酶活性無抑制作用。

圖2 7種ALS酶潛在抑制劑對(duì)核盤菌和油菜ALS酶活性的抑制作用Fig.2 Inhibitory effects of seven potential acetolactate synthase(ALS)inhibitors on the activity of ALS from Sclerotinia sclerotiorum and rapeseed

2.2 ALS酶潛在抑制劑對(duì)核盤菌菌落生長(zhǎng)的抑制作用

為了探明本試驗(yàn)7種ALS酶潛在抑制劑對(duì)核盤菌菌絲生長(zhǎng)的影響，利用PDA平板測(cè)試了這些抑制劑在不同質(zhì)量濃度條件下對(duì)核盤菌菌落生長(zhǎng)的影響。結(jié)果表明，滅草喹和磺胺噻唑在0.1 mg/L時(shí)即有明顯的抑制作用，氯嘧磺隆、芐嘧磺隆和氯磺隆自1.0 mg/L起有明顯的抑制作用，而甲嘧磺隆和咪唑乙煙酸在50 mg/L時(shí)的抑制作用仍很弱（圖3）。此外，對(duì)7種ALS酶潛在抑制劑對(duì)核盤菌的EC50值測(cè)算結(jié)果表明：滅草喹、磺胺噻唑、氯嘧磺隆、芐嘧磺隆、氯磺隆對(duì)核盤菌的EC50值小于10 mg/L，表明核盤菌對(duì)這5種抑制劑相對(duì)較敏感；而甲嘧磺隆和咪唑乙煙酸對(duì)核盤菌的EC50值都大于50 mg/L，表明核盤菌對(duì)這2種抑制劑相對(duì)不太敏感（表1）。這與它們對(duì)核盤菌ALS酶活性抑制作用強(qiáng)弱的變化趨勢(shì)一致。說明核盤菌對(duì)這些抑制劑的敏感性強(qiáng)弱與它們抑制核盤菌ALS酶活性的作用強(qiáng)弱相關(guān)。

圖3 7種ALS酶潛在抑制劑對(duì)PDA平板上核盤菌菌落生長(zhǎng)的影響Fig.3 Effects of seven potential acetolactate synthase inhibitors on the growth of Sclerotinia sclerotiorum colony on potato dextrose agar plates

2.3 ALS酶潛在抑制劑對(duì)油菜ALS酶活性的抑制作用

檢測(cè)結(jié)果表明，7種ALS酶潛在抑制劑對(duì)油菜葉片ALS酶活性均有一定的抑制作用，其中氯嘧磺隆、芐嘧磺隆、氯磺隆、滅草喹和甲嘧磺隆這5種抑制劑的抑制作用較強(qiáng)，而咪唑乙煙酸和磺胺噻唑的抑制作用較弱，尤其是磺胺噻唑的抑制作用最弱（圖2）。比較它們對(duì)油菜和核盤菌ALS酶的抑制作用可見，除磺胺噻唑外，其余6種ALS酶潛在抑制劑對(duì)油菜ALS酶的抑制作用都明顯強(qiáng)于對(duì)核盤菌ALS酶的抑制作用。

表1 7種ALS酶潛在抑制劑對(duì)核盤菌的EC50值Table 1 EC50values of seven potential acetolactate synthase inhibitors to Sclerotinia sclerotiorum

2.4 ALS酶潛在抑制劑對(duì)油菜幼苗生長(zhǎng)發(fā)育的影響

試驗(yàn)結(jié)果（圖4）表明：氯嘧磺隆、芐嘧磺隆、氯磺隆、滅草喹、甲嘧磺隆和咪唑乙煙酸這6種ALS酶潛在抑制劑在1.0 mg/L時(shí)即對(duì)油菜幼苗的生長(zhǎng)發(fā)育有明顯的負(fù)面影響，主要表現(xiàn)為生長(zhǎng)速率下降，植株明顯比對(duì)照矮小，葉片變小，葉色變黃；而磺胺噻唑?qū)τ筒擞酌绲纳L(zhǎng)發(fā)育雖然也有一定程度的負(fù)面影響，但其影響程度明顯比上述6種抑制劑弱。比較各抑制劑對(duì)油菜ALS酶活性的抑制作用強(qiáng)弱和對(duì)油菜幼苗生長(zhǎng)發(fā)育的影響程度可以看出，各抑制劑對(duì)油菜幼苗生長(zhǎng)發(fā)育的影響程度與抑制油菜ALS酶活性的作用強(qiáng)弱基本相關(guān)。

圖4 7種ALS酶潛在抑制劑對(duì)油菜幼苗生長(zhǎng)發(fā)育的影響Fig.4 Effects of seven potential acetolactate synthase inhibitors on the growth and development of rapeseed seedlings

2.5 ALS酶潛在抑制劑對(duì)核盤菌侵染油菜葉片的影響

圖5 磺胺噻唑處理對(duì)核盤菌侵染油菜葉片的影響Fig.5 Effect of sulfathiazole treatment on the infection of Sclerotinia sclerotiorum to rapeseed leaves

油菜菌核病防治劑需要對(duì)核盤菌有較好的滅殺或抑制作用，但不能影響油菜的生長(zhǎng)發(fā)育。上述結(jié)果顯示，在7種ALS酶潛在抑制劑中，核盤菌對(duì)滅草喹、磺胺噻唑、氯嘧磺隆、芐嘧磺隆和氯磺隆敏感，其中磺胺噻唑?qū)τ筒薃LS酶活性的抑制作用和對(duì)油菜幼苗生長(zhǎng)發(fā)育的負(fù)面影響最弱，因此，磺胺噻唑用于防治油菜菌核病的潛力最大。為此，我們測(cè)試了磺胺噻唑處理對(duì)核盤菌侵染油菜葉片的影響。結(jié)果（圖5）顯示：核盤菌菌絲侵染油菜葉片的能力非常強(qiáng)，1 d內(nèi)即可誘發(fā)明顯的病斑；而在油菜葉片接種核盤菌后噴施磺胺噻唑（1 mg/L），可顯著降低核盤菌侵染油菜葉片所誘發(fā)的病斑幅度。這表明磺胺噻唑處理對(duì)核盤菌侵染油菜的進(jìn)程有明顯的抑制作用。因此，磺胺噻唑處理可對(duì)油菜菌核病產(chǎn)生較明顯的防治效果。

3 討論與結(jié)論

動(dòng)物細(xì)胞內(nèi)不含ALS酶。迄今關(guān)于ALS酶的研究絕大多數(shù)是以植物或細(xì)菌為研究對(duì)象，另外有極少量以真菌為材料。研究植物ALS酶主要是為了研發(fā)以ALS酶為靶標(biāo)的除草劑[15]。細(xì)菌ALS酶研究主要以大腸桿菌為材料，研究ALS酶的活性調(diào)控機(jī)制[29]。近年來以人體病原細(xì)菌ALS酶為靶標(biāo)開發(fā)人用抗生素逐漸引起關(guān)注。例如結(jié)核分枝桿菌（Mycobacterium tuberculosis）是一種可引發(fā)人體結(jié)核病（如肺結(jié)核）的病原細(xì)菌。JUNG等[21]最近從6 800種化合物中篩選到15種對(duì)結(jié)核分枝桿菌ALS酶具有較強(qiáng)抑制作用的化合物，它們?cè)?0 μmol/L時(shí)對(duì)結(jié)核分枝桿菌ALS酶活性的抑制率即大于80%，其中5種三唑并嘧啶類化合物的抑制效果尤其突出，其半最大抑制效應(yīng)濃度（half-maximal inhibitory concentration,IC50）值為 0.4～1.24 μmol/L，更重要的是它們對(duì)一些從臨床病樣中分離得到的具有廣譜耐藥性的結(jié)核分枝桿菌株系也有顯著的抑制作用，其MIC50值（50%minimal inhibitory concentration，50%最低抑菌質(zhì)量濃度）只有0.2～0.8 mg/L。這一質(zhì)量濃度水平與常用的抗結(jié)核病藥物異煙肼和利福平的MIC50值濃度水平相當(dāng)，因此上述篩選得到的ALS酶抑制劑有望用于開發(fā)以ALS酶為作用靶標(biāo)的抗結(jié)核病藥物。迄今為止，對(duì)真菌ALS酶的研究報(bào)道仍非常少，但與細(xì)菌ALS酶研究發(fā)展趨勢(shì)類似，以人體病原真菌ALS酶為靶標(biāo)開發(fā)人用殺菌劑近年來逐漸引起關(guān)注。比如白色念珠菌（Candida albicans）是一種人體病原真菌，可侵犯人體的皮膚、黏膜而引發(fā)炎癥，甚至可導(dǎo)致內(nèi)臟或全身感染。LEE等[16]研究證實(shí)了幾種磺酰脲類化合物對(duì)白色念珠菌的ALS酶和菌落生長(zhǎng)有抑制作用，其中乙基-2-{[（4-碘-6-甲氧基嘧啶-2-基）氨甲酰]氨磺酰}苯甲酸酯的抑制作用最強(qiáng)，其MIC90值（90%minimal inhibitory concentration，90%最低抑菌質(zhì)量濃度）為0.7 mg/L。

涉及植物病原真菌ALS酶的研究目前只見到2篇文獻(xiàn)報(bào)道[22,34]。DU等[34]報(bào)道了稻瘟病菌（Magnaporthe oryzae）的ALS酶為稻瘟病菌實(shí)現(xiàn)其致病性所必需。葉朝燕等[22]測(cè)試了14種嘧啶氧芐胺類化合物對(duì)鐮刀菌（Fusarium graminearum）ALS酶活性及菌落生長(zhǎng)的抑制效果，這也是涉及利用ALS酶抑制劑來防治植物真菌病害的唯一研究報(bào)道。這一報(bào)道以及上文所述以ALS酶為靶標(biāo)的人用抗生素和殺菌劑的研究進(jìn)展啟示我們利用ALS酶抑制劑來防治油菜菌核病可能具有較大潛力。由于植物細(xì)胞內(nèi)含有ALS酶，因此研發(fā)以ALS酶為靶標(biāo)的植物真菌病害防治劑需要確保不影響植物的ALS酶。目前研究已證實(shí)，ALS酶與其抑制劑之間存在緊密的結(jié)構(gòu)對(duì)應(yīng)關(guān)系[27-32]，ALS酶一個(gè)氨基酸的變化即可能改變其對(duì)某種抑制劑的敏感性。比如LEE等[28]報(bào)道，誘導(dǎo)大麥（Hordeum vulgare L.）ALS酶編碼基因的一個(gè)堿基發(fā)生替換，導(dǎo)致ALS酶的653位絲氨酸突變成天門冬酰胺，結(jié)果大麥從對(duì)咪唑啉酮類除草劑甲氧咪草煙敏感變?yōu)椴幻舾校籅ROSNAN等[31]報(bào)道早熟禾（Poa annua L.）ALS酶編碼基因的2個(gè)堿基發(fā)生替換，導(dǎo)致ALS酶的205位丙氨酸突變成苯丙氨酸，結(jié)果早熟禾產(chǎn)生對(duì)多類除草劑（咪唑啉酮類、磺酰脲類、三唑并嘧啶類、磺酰基氨基-碳酰-三唑啉酮類、嘧啶硫苯甲酸類）的抗性；HUANG等[32]報(bào)道反枝莧（Amaranthus retroflexus L.）ALS酶的376位天冬氨酸突變成谷氨酸，反枝莧對(duì)除草劑咪唑乙煙酸從敏感變?yōu)椴幻舾?。比?duì)核盤菌ALS酶和油菜ALS酶的氨基酸序列后發(fā)現(xiàn)，兩者的同源性只有36.48%。因此，從理論上有可能篩選到對(duì)油菜ALS酶作用較弱、但對(duì)核盤菌ALS酶作用較強(qiáng)的抑制劑，用于防治油菜菌核病。本研究對(duì)7種ALS酶潛在抑制劑（氯嘧磺隆、芐嘧磺隆、氯磺隆、甲嘧磺隆、咪唑乙煙酸、滅草喹和磺胺噻唑）防治油菜菌核病的可能性進(jìn)行了試驗(yàn)。結(jié)果發(fā)現(xiàn)，滅草喹、磺胺噻唑、氯嘧磺隆、芐嘧磺隆和氯磺隆對(duì)核盤菌ALS酶活性和核盤菌菌落生長(zhǎng)都具有較好的抑制作用，而這5種抑制劑中磺胺噻唑?qū)τ筒薃LS酶活性的抑制作用和對(duì)油菜幼苗生長(zhǎng)發(fā)育的影響都最弱?？购吮P菌侵染試驗(yàn)的結(jié)果顯示，噴施磺胺噻唑顯著地降低了核盤菌對(duì)油菜葉片的侵染程度。這些結(jié)果表明，磺胺噻唑用于防治油菜菌核病具有較大的潛力。由于磺胺噻唑目前已被作為醫(yī)用抗菌藥物，用于防治肺炎球菌、腦膜炎雙球菌、淋球菌和溶血性鏈球菌等人體病原菌的感染?；谵r(nóng)用和醫(yī)用抗菌素分開的原則，磺胺噻唑本身不宜作為油菜菌核病的防治劑使用，但本試驗(yàn)結(jié)果表明，磺胺噻唑可作為分子結(jié)構(gòu)基礎(chǔ)來進(jìn)一步開發(fā)以核盤菌ALS酶為作用靶標(biāo)的油菜菌核病防治劑。

在對(duì)核盤菌ALS酶或油菜ALS酶的抑制效力方面，本試驗(yàn)結(jié)果顯示，2種咪唑啉酮類ALS酶潛在抑制劑（咪唑乙煙酸和滅草喹）的抑制效力差異顯著，4種磺酰脲類ALS酶潛在抑制劑（氯嘧磺隆、芐嘧磺隆、氯磺隆和甲嘧磺?。┑囊种菩Яσ簿哂幸欢ú町?。這說明ALS酶抑制劑的側(cè)鏈基團(tuán)對(duì)其抑制效力具有重要影響，也再次證明ALS酶與其抑制劑之間存在緊密的結(jié)構(gòu)對(duì)應(yīng)關(guān)系。目前對(duì)這一結(jié)構(gòu)對(duì)應(yīng)關(guān)系已有一些研究[25-30]，但仍了解不夠，因此，對(duì)其繼續(xù)進(jìn)行系統(tǒng)深入的研究將有助于研發(fā)更多更佳的以ALS酶為靶標(biāo)的油菜菌核病防治藥劑，以及其他植物真菌病害的防治藥劑。

參考文獻(xiàn)(References):

[1] WANG C G,YAO J,DU X Z,et al.The Arabidopsis mediator complex subunit16 is a key component of basal resistance against the necrotrophic fungal pathogen Sclerotinia sclerotiorum.Plant Physiology,2015,169(1):856-872.

[2] BOLAND G J,HALL R.Index of plant hosts of Sclerotinia sclerotiorum.Canadian Journal of Plant Pathology,1994,16(2):93-108.

[3] BARDIN S D,HUANG H C.Research on biology and control of Sclerotinia diseases in Canada.Canadian Journal of Plant Pathology,2001,23(1):88-98.

[4] YOUNG C S,CLARKSON J P,SMITH J A,et al.Environmental conditions influencing Sclerotinia sclerotiorum infection and disease development in lettuce.Plant Pathology,2004,53(4):387-397.

[5] 汪雷,劉瑤,丁一娟,等.油菜菌核病研究進(jìn)展.西北農(nóng)林科技大學(xué)學(xué)報(bào)(自然科學(xué)版),2015,43(10):85-93.WANG L,LIU Y,DING Y J,et al.Advance in Sclerotinia stem rot of rapeseed.Journal of Northwest A&F University(Natural Science Edition),2015,43(10):85-93.(in Chinese with English abstract)

[6] LI G Q,HUANG H C,MIAO H J,et al.Biological control of sclerotinia diseases of rapeseed by aerial applications of the mycoparasite Coniothyrium minitans.European Journal of Plant Pathology,2006,114(4):345-355.

[7] 周鋒,王彥芬,張小磊,等.油菜菌核病病原菌對(duì)多菌靈抗藥性的研究進(jìn)展.湖南農(nóng)業(yè)科學(xué),2012(17):82-84.ZHOU F,WANG Y F,ZHANG X L,et al.Advances in resistance of Sclerotinia sclertiorum to carbendazim.Hunan Agricultural Sciences,2012(17):82-84.(in Chinese with English abstract)

[8] 楊敬輝,潘以樓,朱桂梅,等.油菜菌核病菌對(duì)多菌靈和乙霉威的抗藥性機(jī)理.植物保護(hù)學(xué)報(bào),2004,31(1):74-78.YANG J H,PAN Y L,ZHU G M,et al.Mechanism of resistance of Sclerotinia sclertiorum to carbendazim and diethofencarb.Acta Phytophylacica Sinica,2004,31(1):74-78.(in Chinese with English abstract)

[9] 李偉,李偉,周益軍,等.江蘇省油菜菌核病菌對(duì)多菌靈的敏感性.中國(guó)油料作物學(xué)報(bào),2007,29(1):63-68.LI W,LI W,ZHOU Y J,et al.Sensitivity of Sclerotinia sclerotiorum isolates to carbendazim in Jiangsu Province.Chinese Journal of Oil Crop Sciences,2007,29(1):63-68.(in Chinese with English abstract)

[10]MCCOURT J A,DUGGLEBY R G.Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids.Amino Acids,2006,31(2):173-210.

[11]LIU Y D,LI Y Y,WANG X Y.Acetohydroxyacid synthases:Evolution,structure,and function.Applied Microbiology and Biotechnology,2016,100(20):8633-8649.

[12]GALILI G,AMIR R,FERNIE A R.The regulation of essential amino acid synthesis and accumulation in plants.Annual Review of Plant Biology,2016,67:153-178.

[13]RAY T B.Site of action of chlorsulfuron:Inhibition of valine and isoleucine biosynthesis in plants.Plant Physiology,1984,75(3):827-831.

[14]MCCOURT J A,PANG S S,KING-SCOTT J,et al.Herbicidebinding sites revealed in the structure of plant acetohydroxyacid synthase.Proceedings of the National Academy of Sciences of the USA,2006,103(3):569-573.

[15]POWLES S B,YU Q.Evolution in action:Plants resistant to herbicides.Annual Review of Plant Biology,2010,61:317-347.

[16]LEE Y T,CUI C J,CHOW E W L,et al.Sulfonylureas have antifungal activity and are potent inhibitors of Candida albicans acetohydroxyacid synthase.Journal of Medicinal Chemistry,2013,56(1):210-219.

[17]KREISBERG J F,ONG N T,KRISHNA A,et al.Growth inhibition of pathogenic bacteria by sulfonylurea herbicides.Antimicrobial Agents and Chemotherapy,2013,57(3):1513-1517.

[18]RICHIE D L,THOMPSON K V,STUDER C,et al.Identification and evaluation of novel acetolactate synthase inhibitors as antifungal agents.Antimicrobial Agents and Chemotherapy,2013,57(5):2272-2280.

[19]PUE N,GUDDAT L W.Acetohydroxyacid synthase:A target for antimicrobial drug discovery.Current Pharmaceutical Design,2014,20(5):740-753.

[20]GOKHALE K, TILAK B. Mechanisms of bacterial acetohydroxyacid synthase(AHAS)and specific inhibitors of Mycobacterium tuberculosis AHAS as potential drug candidates against tuberculosis.Current Drug Targets,2015,16(7):689-699.

[21]JUNG I P,HA N R,LEE S C,et al.Development of potent chemical antituberculosis agents targeting Mycobacterium tuberculosis acetohydroxyacid synthase.International Journal of Antimicrobial Agents,2016,48(3):247-258.

[22]葉朝燕,張陽,汪冒君,等.鐮刀菌乙酰乳酸合成酶抑制劑的篩選及抗菌性分析.浙江大學(xué)學(xué)報(bào)(理學(xué)版),2015,42(2):166-171,220.YE C Y,ZHANG Y,WANG M J,et al.Screening and fungistatic activity ofacetolactate synthase inhibitors for Fusarium graminearum.Journal of Zhejiang University(Science Edition),2015,42(2):166-171,220.(in Chinese with English abstract)

[23]XU T,YAO F,LIANG W S,et al.Involvement of alternative oxidase in the regulation of growth,development,and resistance to oxidative stress ofSclerotinia sclerotiorum.Journalof Microbiology,2012,50(4):594-602.

[24]XU T,WANG Y T,LIANG W S,et al.Involvement of alternative oxidase in the regulation of Sclerotinia sclerotiorum sensitivity to fungicides of azoxystrobin and procymidone.Journalof Microbiology,2013,51(3):352-358.

[25]梁五生,徐婷,姚飛,等.交替氧化酶可增強(qiáng)核盤菌對(duì)多菌靈的敏感性.中國(guó)農(nóng)學(xué)通報(bào),2016,32(6):193-197.LIANG W S,XU T,YAO F,et al.Enhancing effect of alternative oxidase on sensitivity of Sclerotinia sclerotiorum to carbendazim.Chinese Agricultural Science Bulletin,2016,32(6):193-197.(in Chinese with English abstract)

[26]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry,1976,72(1/2):248-254.

[27]KALME S,PHAM C N,GEDI V,et al.Inhibitors of Bacillus anthracis acetohydroxyacid synthase.Enzyme and Microbial Technology,2008,43(3):270-275.

[28]LEE H,RUSTGI S,KUMAR N,et al.Single nucleotide mutation in the barley acetohydroxy acid synthase(AHAS)gene confers resistance to imidazolinone herbicides.Proceedings of the National Academy of Sciences of the USA,2011,108(21):8909-8913.

[29]GEDI V,YOON M Y.Bacterial acetohydroxyacid synthase and its inhibitors:A summary of their structure,biological activity and current status.The FEBS Journal,2012,279(6):946-963.

[30]EVANS D L,JOSHI S V.Elucidating modes of activation and herbicideresistancebysequenceassemblyand molecular modelling of the acetolactate synthase complex in sugarcane.Journal of Theoretical Biology,2016,407:184-197.

[31]BROSNAN J T,VARGAS J J,BREEDEN G K,et al.A new amino acid substitution(Ala-205-Phe)in acetolactate synthase(ALS)confers broad spectrum resistance to ALS-inhibiting herbicides.Planta,2016,243(1):149-159.

[32]HUANG Z F,CHEN J Y,ZHANG C X,et al.Target-site basis for resistance to imazethapyr in redroot amaranth(Amaranthus retroflexus L.).Pesticide Biochemistry and Physiology,2016,128:10-15.

[33]SINGH B K,STIDHAM M A,SHANER D L.Assay of acetohydroxyacid synthase.Analytical Biochemistry,1988,171(1):173-179.

[34]DU Y,ZHANG H F,HONG L,et al.Acetolactate synthases MoIlv2 and MoIlv6 are required for infection-related morphogenesis in Magnaporthe oryzae.Molecular Plant Pathology,2013,14(9):870-884.

Screening of acetolactate synthase inhibitors against Sclerotinia sclerotiorum for the purpose of controlling rapeseed Sclerotinia disease.Journal of Zhejiang University(Agric.&Life Sci.),2017,43(5):589-598

WEN Xuewei,CHEN Yangyang,WU Bin,HU Dongwei,LIANG Wusheng*
(Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects/Institute of Biotechnology,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,China)

acetolactate synthase;Sclerotinia disease;Sclerotinia sclerotiorum;rapeseed;sulfathiazole

S 432.44

A

10.3785/j.issn.1008-9209.2017.02.201

Summary Rapeseed is an oil crop with quite large growing areas in China,and also one of the main oil crops in the world.As one of the most serious diseases in rapeseed,Sclerotinia disease,caused by the fungal pathogen Sclerotinia sclerotiorum,can result in significant yield loss.At present,no rape cultivars are available that are resistant to S.sclerotiorum.Using fungicides is the main measure to control rapeseed Sclerotinia disease in China.Carbendazim,as a common fungicide,has been frequently applied to control rapeseed Sclerotinia disease for long.Increasing evidence of fungicide resistance in populations of S.sclerotiorum has been found.Therefore,new fungicides are constantly being requested to control rapeseed Sclerotinia disease.Acetolactate synthase(ALS)is the enzyme that catalyzes the first step in the branched-chain aminoacid biosynthesis pathway in plants and microbes,and is the target of some herbicides.Recently,some studies have reported the development of new antibiotics to control bacterial pathogens or new fungicides to control fungal pathogens of human beings with ALS as the action target.However,there are few studies on developing new fungicides to control fungal pathogens of plants with ALS as the action target.

國(guó)家自然科學(xué)基金（31370279）；浙江省自然科學(xué)基金（LY13C140001）；浙江省教育廳科研計(jì)劃項(xiàng)目（Y201327327）。

梁五生（http://orcid.org/0000-0001-9360-1187），Tel:+86-571-86433024，E-mail:liangws@zju.edu.cn

（First author）：溫雪瑋（http://orcid.org/0000-0002-0886-3930），E-mail:1018559277@qq.com

2017-02-20；接受日期（Accepted）:2017-04-28

In the present study,seven potential ALS inhibitors(chlorimuron-ethyl,bensulfuron-methyl,chlorsulfuron,sulfometuronmethyl,imazethapyr,imazaquin and sulfathiazole)were used to determine the effectiveness of controlling rapeseed Sclerotinia disease.Chlorimuron-ethyl,bensulfuron-methyl,chlorsulfuron and sulfometuron-methyl were chosen as the representatives of sulfonylurea-type ALS inhibitors.Imazethapyr and imazaquin were the representatives of imidazolinone-type ALS inhibitors,and sulfathiazole was the representative of sulfathiazole-type ALS inhibitors.

The results indicated that the activity of ALS in S.sclerotiorum could not been inhibited by 1.0 mg/L imazethapyr.In contrast,the treatment with 1.0 mg/L chlorimuron-ethyl,bensulfuron-methyl,chlorsulfuron,sulfometuron-methyl,imazaquin and sulfathiazole showed more or less inhibitory effects on the ALS activity of S.sclerotiorum.Among them,imazaquin had the strongest inhibitory effect on ALS,but sulfometuron-methyl showed the weakest inhibitory effect.Imazaquin and sulfathiazole could effectively suppress the growth of S.sclerotiorum colony on potato dextrose agar plates at the concentration of 0.1 mg/L.Chlorimuron-ethyl,bensulfuron-methyl and chlorsulfuron had inhibitory effect at the concentration of 1.0 mg/L.However,sulfometuron-methyl and imazethapyr showed weak inhibitory effect on the growth of S.sclerotiorum colony even at the concentration of 50 mg/L.The half-maximal effective concentrations(EC50)of imazaquin,sulfathiazole,chlorimuron-ethyl,bensulfuron-methyl and chlorsulfuron were determined to be less than 10 mg/L,but EC50values of sulfometuron-methyl and imazethapyr were determined to be more than 50 mg/L.All the seven potential ALS inhibitors showed inhibitory effects on the activity of ALS in rapeseed leaves at the concentration of 1.0 mg/L.The inhibitory effects of chlorimuron-ethyl,bensulfuronmethyl,chlorsulfuron,imazaquin and sulfometuron-methyl were much stronger than those of imazethapyr and sulfathiazole.The inhibitory effect of sulfathiazole was the weakest among the seven inhibitors.Significant damaging influences were observed on rapeseed seedlings by using chlorimuron-ethyl,bensulfuron-methyl,chlorsulfuron,imazaquin,sulfometuron-methyl and imazethapyr at the concentration of 1.0 mg/L,such as decreasing growth rate,dwarfing,reducing leaf size and leaf yellowing,but no significant influences were observed by treating rapeseed seedlings with sulfathiazole.The use of sulfathiazole could exert remarkable inhibitory effects against ALS activity and colony growth of S.sclerotiorum without giving rise to harmful influence on rapeseed seedlings.Thus,the effect of sulfathiazole on S.sclerotiorum infection to rapeseed was tested.The result indicated that the treatment with sulfathiazole significantly reduced the infection scale of S.sclerotiorum in rapeseed leaves.

In conclusion,the present research indicated that S.sclerotiorum was sensitive to imazaquin,sulfathiazole,chlorimuron-ethyl,bensulfuron-methyl and chlorsulfuron other than sulfometuron-methyl and imazethapyr.On the other hand,rapeseed was sensitive to all the seven ALS inhibitors but imazethapyr and sulfathiazole.It is suggested that sulfathiazole treatment is an effective strategy to control rapeseed Sclerotinia disease caused by S.sclerotiorum.Therefore,this study concludes that sulfathiazole is a potential molecular structure basis for controlling rapeseed Sclerotinia disease with ALS of S.sclerotiorum as an action target.