劉圣明, 海 飛, 車志平, 田月娥, 劉 欣

(1. 河南科技大學(xué)林學(xué)院植物保護(hù)系,洛陽(yáng) 471003；2. 河南農(nóng)業(yè)職業(yè)學(xué)院農(nóng)業(yè)工程系,鄭州 451450)

4種殺菌劑及其復(fù)配劑對(duì)番茄灰霉病菌的毒力

劉圣明1*, 海 飛2, 車志平1, 田月娥1, 劉 欣1

(1. 河南科技大學(xué)林學(xué)院植物保護(hù)系,洛陽(yáng) 471003；2. 河南農(nóng)業(yè)職業(yè)學(xué)院農(nóng)業(yè)工程系,鄭州 451450)

采用菌絲生長(zhǎng)速率法測(cè)定了咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環(huán)唑4 種殺菌劑及其兩元復(fù)配劑對(duì)番茄灰霉病菌的毒力。結(jié)果顯示,咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環(huán)唑?qū)Ψ鸦颐共【挠行б种浦袧舛?EC50)分別為:0.018 0、0.018 1、1.896 8和2.087 4 μg/mL。復(fù)配劑啶酰菌胺∶苯醚甲環(huán)唑1∶5、1∶3和1∶1,咯菌腈∶苯醚甲環(huán)唑1∶5增效作用最明顯;復(fù)配劑咯菌腈∶苯醚甲環(huán)唑1∶3，咯菌腈∶氟啶胺1∶3,啶酰菌胺∶咯菌腈5∶1,啶酰菌胺∶苯醚甲環(huán)唑3∶1具有增效作用,SR值范圍為1.5～4.05,其中以復(fù)配劑啶酰菌胺∶苯醚甲環(huán)唑1∶5增效作用最好,其SR值為4.05；其余不同配比的各組合復(fù)配劑具有相加作用,其SR值范圍為0.5～1.46。表明咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環(huán)唑4 種不同作用機(jī)制的殺菌劑可以交替或復(fù)配使用,以阻止或延緩灰霉病菌抗藥性的進(jìn)一步加劇,為灰霉病的綜合防控和抗藥性治理提供理論依據(jù)。

灰霉?。?灰葡萄孢； 殺菌劑； 復(fù)配劑

由灰葡萄孢BotrytiscinereaPers.exFr.,無(wú)性型Botryotiniafuckeliana(de Bary) Whetz引起的灰霉病是200多種作物，包括番茄、黃瓜和茄子等蔬菜以及草莓和葡萄等水果上的重要病害,能夠造成花、葉、莖和果實(shí)的腐爛,影響作物的品質(zhì)和產(chǎn)量,尤其在溫室和大棚等設(shè)施蔬菜上危害尤為嚴(yán)重[1-5]?；颐共≡诒Ｗo(hù)地番茄上嚴(yán)重發(fā)生時(shí),能造成番茄花、葉和果實(shí)軟腐,減產(chǎn)50%以上[6-8]。由于缺少高抗灰霉病的番茄品種,從20世紀(jì)70年代開(kāi)始,生產(chǎn)上主要使用苯并咪唑類、二甲酰亞胺類、氨基甲酸酯類和苯胺基嘧啶類殺菌劑對(duì)灰霉病進(jìn)行化學(xué)防治,但由于灰霉病菌具有較快的繁殖速率、較大的遺傳變異性和較高的田間適合度等特點(diǎn),極易對(duì)施用的殺菌劑產(chǎn)生抗藥性,導(dǎo)致田間防治效果下降或喪失[6-13]。隨著苯并咪唑類、二甲酰亞胺類、氨基甲酸酯類和苯胺基嘧啶類殺菌劑施藥次數(shù)和施藥量的不斷增加,我國(guó)江蘇、河南、山東、北京和遼寧等地相繼出現(xiàn)了單抗、雙抗、甚至多抗類型的灰霉病菌菌株[6-7, 10-12, 15-17]。2010年Sun等[12]研究發(fā)現(xiàn)江蘇省南京市和淮陰市灰霉病菌對(duì)常用防治藥劑苯并咪唑類殺菌劑多菌靈、二甲酰亞胺類殺菌劑腐霉利、氨基甲酸酯類殺菌劑乙霉威和苯胺基嘧啶類殺菌劑嘧霉胺的四抗菌株比例分別為51.6%和52.8%；山東省灰霉病菌對(duì)四類殺菌劑的四抗菌株比例為100%。2016年Liu等[10]研究發(fā)現(xiàn)河南省灰霉病菌對(duì)四類殺菌劑的四抗菌株比例為68.1%。先前的研究表明灰霉病菌對(duì)常用的苯并咪唑類、二甲酰亞胺類、氨基甲酸酯類和苯胺基嘧啶類防治藥劑已經(jīng)產(chǎn)生了較高比例的抗藥性。為了阻止或延緩抗藥性的進(jìn)一步加劇,延長(zhǎng)藥劑的使用壽命,應(yīng)科學(xué)選用合適的殺菌劑防治灰霉病。

采用不同作用機(jī)制的殺菌劑交替或復(fù)配使用,是阻止或延緩病原菌抗藥性進(jìn)一步加劇的主要策略。因此,本研究采用菌絲生長(zhǎng)速率法,測(cè)定了咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環(huán)唑4種不同作用機(jī)制的殺菌劑單劑及其兩元復(fù)配劑對(duì)番茄灰霉病菌的毒力,對(duì)生產(chǎn)上綜合防控灰霉病和科學(xué)合理地用藥具有重要的指導(dǎo)意義。

1 材料與方法

1.1 供試菌株

供試菌株為實(shí)驗(yàn)室保存的經(jīng)單孢分離的番茄灰霉病菌。

1.2 供試藥劑

97.9%咯菌腈原藥,先正達(dá)(中國(guó))有限公司提供；97%氟啶胺原藥,江蘇優(yōu)士化學(xué)有限公司提供；95.4%苯醚甲環(huán)唑原藥,先正達(dá)(中國(guó))有限公司提供,上述3種藥劑均預(yù)溶于甲醇,配制成104μg/mL母液。96.2%啶酰菌胺原藥,巴斯夫(中國(guó))有限公司提供,預(yù)溶于丙酮,配制成104μg/mL的母液。

1.3 供試培養(yǎng)基

PDA培養(yǎng)基:馬鈴薯200 g,葡萄糖20 g,瓊脂15～20 g,加蒸餾水至1 000 mL。

1.4 4種殺菌劑毒力測(cè)定

采用菌絲生長(zhǎng)速率法測(cè)定咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環(huán)唑?qū)Ψ鸦颐共【碾x體毒力。將咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環(huán)唑母液稀釋為系列濃度,按照一定的比例分別加入到滅菌熱熔冷卻至50℃左右的PDA培養(yǎng)基中,制成系列濃度(表1)的含藥PDA平板,以不加藥劑為對(duì)照。用直徑為5 mm的打孔器,在25℃黑暗條件下培養(yǎng)3 d的灰霉病菌菌落邊緣打孔制備菌餅,然后用接種針挑取菌餅分別接種于含咯菌腈、氟啶胺、啶酰菌胺或苯醚甲環(huán)唑系列濃度的PDA平板上。將各平板置于恒溫培養(yǎng)箱內(nèi),25℃黑暗條件下培養(yǎng)3 d,測(cè)量并記錄菌落直徑。各處理3次重復(fù),試驗(yàn)重復(fù)3次。根據(jù)灰霉病菌在不同濃度藥劑平板上的線性生長(zhǎng)速率,計(jì)算出各濃度藥劑對(duì)病原菌的生長(zhǎng)抑制率,用DPS統(tǒng)計(jì)軟件進(jìn)行處理,求出各藥劑的EC50。

表1 4種殺菌劑毒力測(cè)定濃度

Table 1 Concentrations of four fungicides used for determination of their toxicity toBotrytiscinerea

殺菌劑Fungicide藥劑濃度/μg·mL-1Fungicideconcentration咯菌腈Fludioxonil0,0.003125,0.00625,0.0125,0.025,0.1,0.4氟啶胺Fluazinam0,0.03125,0.0625,0.125,0.25,0.5,1啶酰菌胺Boscalid0,0.1,0.2,0.4,0.8,1.6,3.2苯醚甲環(huán)唑Difenoconazole0,0.3125,0.625,1.25,2.5,5,10

1.5 復(fù)配劑毒力測(cè)定

采用菌絲生長(zhǎng)速率法測(cè)定復(fù)配劑對(duì)番茄灰霉病菌的離體毒力。將咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環(huán)唑4種藥劑按照表2所示的比例分別進(jìn)行兩元復(fù)配后,分別加入到滅菌熱熔冷卻至50℃左右的PDA培養(yǎng)基中,制成含復(fù)配劑系列濃度(表2)的PDA平板。按照1.4測(cè)定和計(jì)算方法,求出各復(fù)配藥劑的EC50。利用以下公式求出SR值[18],當(dāng)SR值大于等于1.5時(shí)為增效作用,在0.5和1.5之間為相加作用,小于0.5為拮抗作用[19]。

A,B為復(fù)配的藥劑；a,b為藥劑在配方中所占的比例；EC50(Exp)為理論抑制中濃度,EC50(Obs)為實(shí)際測(cè)量抑制中濃度。

表2 復(fù)配劑不同比例濃度設(shè)計(jì)

Table 2 Concentrations of fungicides mixtures used for determination of their toxicity toBotrytiscinerea

復(fù)配劑Mixture藥劑比例Fungicideproportion藥劑濃度/μg·mL-1Fungicideconcentration咯菌腈∶氟啶胺fludioxonil∶fluazinam5∶10,0.003125,0.00625,0.0125,0.025,0.05,0.13∶10,0.003125,0.00625,0.0125,0.025,0.05,0.11∶10,0.003125,0.00625,0.0125,0.025,0.05,0.11∶30,0.003125,0.00625,0.0125,0.025,0.05,0.11∶50,0.003125,0.00625,0.0125,0.025,0.05,0.1咯菌腈∶苯醚甲環(huán)唑fludioxonil∶difenoconazole5∶10,0.003125,0.00625,0.0125,0.025,0.05,0.13∶10,0.003125,0.00625,0.0125,0.025,0.05,0.11∶10,0.005,0.01,0.02,0.04,0.08,0.161∶30,0.0125,0.025,0.05,0.1,0.2,0.41∶50,0.0125,0.025,0.05,0.1,0.2,0.4氟啶胺∶苯醚甲環(huán)唑fluazinam∶difenoconazole5∶10,0.003125,0.00625,0.0125,0.025,0.05,0.13∶10,0.003125,0.00625,0.0125,0.025,0.05,0.11∶10,0.005,0.01,0.02,0.04,0.08,0.161∶30,0.0125,0.025,0.05,0.1,0.2,0.41∶50,0.0125,0.025,0.05,0.1,0.2,0.4啶酰菌胺∶苯醚甲環(huán)唑boscalid∶difenoconazole5∶10,0.1,0.2,0.4,0.8,1.6,3.23∶10,0.1,0.2,0.4,0.8,1.6,3.21∶10,0.15625,0.3125,0.625,1.25,2.5,51∶30,0.3125,0.625,1.25,2.5,5,101∶50,0.3125,0.625,1.25,2.5,5,10啶酰菌胺∶咯菌腈boscalid∶fludioxonil5∶10,0.1,0.2,0.4,0.8,1.6,-3∶10,0.05,0.1,0.2,0.4,0.8,-1∶10,0.03125,0.0625,0.125,0.25,0.5,-1∶30,0.003125,0.00625,0.0125,0.025,0.05,0.11∶50,0.003125,0.00625,0.0125,0.025,0.05,0.1啶酰菌胺∶氟啶胺boscalid∶fluazinam5∶10,0.05,0.1,0.2,0.4,0.8,-3∶10,0.05,0.1,0.2,0.4,0.8,-1∶10,0.05,0.1,0.2,0.4,0.8,-1∶30,0.003125,0.00625,0.0125,0.025,0.05,0.11∶50,0.003125,0.00625,0.0125,0.025,0.05,0.1

2 結(jié)果與分析

2.1 4種殺菌劑對(duì)番茄灰霉病菌的毒力

采用菌絲生長(zhǎng)速率法測(cè)定了咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環(huán)唑?qū)Ψ鸦颐共【亩玖Α＝Y(jié)果(表3)顯示,4種藥劑對(duì)番茄灰霉病菌的有效抑制中濃度(EC50)分別為:0.018 0、0.018 1、1.896 8和2.087 4 μg/mL。表明4種藥劑對(duì)番茄灰霉病菌的菌絲生長(zhǎng)都有較好的抑制作用,4種單劑中咯菌腈對(duì)番茄灰霉病菌的毒力最高。

2.2 復(fù)配劑對(duì)番茄灰霉病菌的毒力

將咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環(huán)唑4 種藥劑分別按照一定比例進(jìn)行兩元復(fù)配,采用菌絲生長(zhǎng)速率法測(cè)定各復(fù)配劑對(duì)番茄灰霉病菌的毒力。結(jié)果(表 4)顯示,不同組合、不同配比的復(fù)配劑對(duì)番茄灰霉病菌的毒力不同。復(fù)配劑啶酰菌胺：苯醚甲環(huán)唑1∶5、1∶3和1∶1、咯菌腈∶苯醚甲環(huán)唑1∶5增效作用最明顯；復(fù)配劑咯菌腈∶氟啶胺1∶3、咯菌腈∶苯醚甲環(huán)唑1∶3、啶酰菌胺∶咯菌腈5∶1、啶酰菌胺∶苯醚甲環(huán)唑3∶1具有增效作用,SR范圍為1.5～4.05,其中以復(fù)配劑啶酰菌胺∶苯醚甲環(huán)唑1∶5增效作用最好,其SR為4.05；其余不同配比的組合復(fù)配劑具有相加作用,其SR范圍為0.5～1.46。

表3 4種藥劑單劑對(duì)番茄灰霉病菌的毒力

Table 3 Toxicities of four fungicides againstBotrytiscinereabased on mycelial growthinvitro

殺菌劑Fungicide毒力回歸方程Toxicityregressionequation有效抑制中濃度/μg·mL-1EC50相關(guān)系數(shù)Correlationcoefficient咯菌腈fludioxonilY=7.4353+1.3965X0.01800.9752氟啶胺fluazinamY=6.8220+1.4530X0.01810.9806啶酰菌胺boscalidY=4.8522+0.5315X1.89680.9798苯醚甲環(huán)唑difenoconazoleY=4.8934+0.3360X2.08740.9971

表4 不同復(fù)配劑對(duì)番茄灰霉病菌的毒力

Table 4 Toxicities of mixtures of fungicides againstBotrytiscinereabased on mycelial growthinvitro

復(fù)配劑Mixture藥劑比例Fungicideproportion毒力回歸方程Toxicityregressionequation相關(guān)系數(shù)Correlationcoefficient實(shí)際測(cè)量抑制中濃度/μg·mL-1EC50(Obs)理論抑制中濃度/μg·mL-1EC50(Exp)SR咯菌腈∶氟啶胺fludioxonil∶fluazinam5∶1Y=7.6503+1.5066X0.96550.01740.01801.033∶1Y=7.6248+1.6215X0.97060.02410.01800.751∶1Y=8.1505+1.8481X0.98680.01970.01800.911∶3Y=7.6422+1.5005X0.97350.01730.03612.091∶5Y=7.9043+1.6027X0.98450.01540.01811.18咯菌腈∶苯醚甲環(huán)唑fludioxonil∶difenoconazole5∶1Y=7.5692+1.8486X0.94280.04280.02160.503∶1Y=7.6786+1.7559X0.95470.02980.02390.801∶1Y=6.8376+1.1391X0.99300.02440.03571.461∶3Y=6.4722+1.0531X0.98780.04000.07021.761∶5Y=5.9144+0.6591X0.95430.04100.10352.52氟啶胺∶苯醚甲環(huán)唑fluazinam∶difenoconazole5∶1Y=7.7887+1.7507X0.97800.02550.02170.853∶1Y=7.4768+1.5073X0.98660.02270.02411.061∶1Y=7.4991+1.6771X0.99300.03230.03591.111∶3Y=6.7693+1.5729X0.98780.07500.07060.941∶5Y=6.5163+1.6174X0.97690.11540.10410.90啶酰菌胺∶苯醚甲環(huán)唑boscalid∶difenoconazole5∶1Y=4.7719+0.6431X0.99282.26291.92610.853∶1Y=4.8478+0.6397X0.99601.29001.94111.501∶1Y=5.1864+0.7542X0.99770.56601.98503.511∶3Y=5.1724+0.6333X0.98430.53432.03623.811∶5Y=5.2068+0.7003X0.95960.50672.05084.05啶酰菌胺∶咯菌腈boscalid∶fludioxonil5∶1Y=6.4202+1.2061X0.99440.06640.10311.553∶1Y=6.5441+1.2267X0.97250.05410.07001.291∶1Y=6.8031+1.1793X0.90890.02960.03571.211∶3Y=6.8539+1.1238X0.98840.04400.02390.541∶5Y=6.9233+1.1434X0.98500.02080.02161.04啶酰菌胺∶氟啶胺boscalid∶fluazinam5∶1Y=6.2188+1.6341X0.98690.17950.10370.583∶1Y=6.6995+1.9924X0.98590.14030.07040.501∶1Y=6.7114+1.1958X0.99130.03700.03590.971∶3Y=7.5472+1.6145X0.99010.02640.02410.911∶5Y=8.1893+1.9301X0.95840.02230.02170.97

3 結(jié)論與討論

灰霉病菌具有繁殖快、遺傳變異性大和田間適合度高等特點(diǎn),屬于抗藥性發(fā)生風(fēng)險(xiǎn)高的病原菌,極易對(duì)防治藥劑產(chǎn)生抗藥性[20]。采用不同作用機(jī)制的殺菌劑交替或復(fù)配使用,是阻止或延緩病原菌抗藥性進(jìn)一步加劇的主要策略?？┚鎸儆诒交量╊惙莾?nèi)吸性殺菌劑,是滲透信號(hào)傳導(dǎo)的分裂蛋白活化激酶/組氨酸激酶抑制劑[21]；氟啶胺屬于苯胺吡啶類殺菌劑,是解偶聯(lián)劑,破壞氧化磷酸化[22]；啶酰菌胺屬于新型煙酰胺類內(nèi)吸性殺菌劑,是呼吸作用抑制劑[23]；苯醚甲環(huán)唑?qū)儆谌蝾悮⒕鷦?是甾醇脫甲基化抑制劑[24]。本研究采用菌絲生長(zhǎng)速率法測(cè)定了咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環(huán)唑4種不同作用機(jī)制的殺菌劑單劑及其兩元復(fù)配劑對(duì)番茄灰霉病菌的毒力。

單劑研究結(jié)果顯示,4種藥劑對(duì)番茄灰霉病菌的有效抑制中濃度由低到高依次為:咯菌腈(0.018 0 μg/mL)、氟啶胺(0.018 1 μg/mL)、啶酰菌胺(1.896 8 μg/mL)和苯醚甲環(huán)唑(2.087 4 μg/mL),表明4種藥劑對(duì)番茄灰霉病菌菌絲生長(zhǎng)均有較好的抑制作用。咯菌腈和氟啶胺單劑對(duì)番茄灰霉病菌具有較高的毒力,而且在田間尚沒(méi)有發(fā)現(xiàn)抗咯菌腈和氟啶胺的番茄灰霉病菌菌株[25-26]。近年來(lái),雖然陸續(xù)在田間發(fā)現(xiàn)了抗苯醚甲環(huán)唑和啶酰菌胺的番茄灰霉病菌菌株,但其抗藥性水平和頻率還處于較低水平[23, 27]。復(fù)配劑研究結(jié)果顯示,不同配比的各組合復(fù)配劑具有增效或相加作用,沒(méi)有拮抗作用,表明4種殺菌劑在兩兩復(fù)配使用時(shí)各藥劑的作用機(jī)理互不影響。兩元復(fù)配劑中以啶酰菌胺∶苯醚甲環(huán)唑1∶5增效作用最好,主要是由于啶酰菌胺是呼吸作用抑制劑,能夠抑制病原菌能量的生成,苯醚甲環(huán)唑是甾醇脫甲基化抑制劑,能夠抑制病原菌的生物合成,兩者相輔相成。綜上所述,4種不同作用機(jī)制的殺菌劑及其兩元復(fù)配劑對(duì)番茄灰霉病菌均有較高的毒力,建議生產(chǎn)中采用這4種不同作用機(jī)制的殺菌劑進(jìn)行交替或復(fù)配使用,以阻止或延緩灰霉菌抗藥性的進(jìn)一步發(fā)展,為灰霉病的綜合防控和抗藥性治理提供理論依據(jù)。

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(責(zé)任編輯:楊明麗)

Toxicity of four fungicides and their mixtures toBotrytiscinereafrom tomato

Liu Shengming1, Hai Fei2, Che Zhiping1, Tian Yue’e1, Liu Xin1

(1.DepartmentofPlantProtection,CollegeofForestry,HenanUniversityofScienceandTechnology,Luoyang471003,China; 2.DepartmentofAgriculturalEngineering,HenanVocationalCollegeofAgriculture,Zhengzhou451450,China)

Toxicities of fludioxonil, fluazinam, boscalid and difenoconazole, and their mixtures toBotrytiscinereafrom tomato were detected by the method of mycelial growth assayinvitro. The results showed that the EC50values for fludioxonil, fluazinam, boscalid and difenoconazole were 0.018 0 μg/mL, 0.018 1 μg/mL, 1.896 8 μg/mL, and 2.087 4 μg/mL, respectively. The mixtures of the fungicides boscalid and difenoconazole with the ratio of 1∶5, 1∶3, 1∶1 and 3∶1, fludioxonil and difenoconazole with the ratio of 1∶5 and 1∶3, fludioxonil and fluazinam with the ratio of 1∶3, and boscalid and fludioxonil with the ratio of 5∶1 demonstrated synergistic inhibition effect, with the synergy ratio ranged from 1.5 to 4.05. Among them, the mixture of boscalid and difenoconazole (1∶5) had the strongest inhibition againstB.cinereawith the synergy ratio of 4.05, indicating synergistic inhibition, while the synergy ratio of other mixtures was 0.5-1.46, indicating additive inhibition. The above results indicated that fludioxonil, fluazinam, boscalid and difenoconazole, and their mixtures can be used alternately in controlling the gray mold disease caused byB.cinerea.

grey mould disease;Botrytiscinerea; fungicide; mixture

2016-04-12

2016-04-25

國(guó)家自然科學(xué)基金青年科學(xué)基金(31301688)；公益性行業(yè)(農(nóng)業(yè))科研專項(xiàng)(201303023)；河南省自然科學(xué)基金(162300410079)；河南科技大學(xué)博士科研啟動(dòng)基金(09001589)

S 436.412

B

10.3969/j.issn.0529-1542.2017.02.042

* 通信作者 E-mail:liushengmingzb@163.com