4種殺菌劑及其復配劑對番茄灰霉病菌的毒力

劉圣明, 海 飛, 車志平, 田月娥, 劉 欣

(1. 河南科技大學林學院植物保護系,洛陽 471003；2. 河南農業職業學院農業工程系,鄭州 451450)

4種殺菌劑及其復配劑對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. 河南科技大學林學院植物保護系,洛陽 471003；2. 河南農業職業學院農業工程系,鄭州 451450)

采用菌絲生長速率法測定了咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環唑4 種殺菌劑及其兩元復配劑對番茄灰霉病菌的毒力。結果顯示,咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環唑對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。復配劑啶酰菌胺∶苯醚甲環唑1∶5、1∶3和1∶1,咯菌腈∶苯醚甲環唑1∶5增效作用最明顯;復配劑咯菌腈∶苯醚甲環唑1∶3，咯菌腈∶氟啶胺1∶3,啶酰菌胺∶咯菌腈5∶1,啶酰菌胺∶苯醚甲環唑3∶1具有增效作用,SR值范圍為1.5～4.05,其中以復配劑啶酰菌胺∶苯醚甲環唑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唑4 種不同作用機制的殺菌劑可以交替或復配使用,以阻止或延緩灰霉病菌抗藥性的進一步加劇,為灰霉病的綜合防控和抗藥性治理提供理論依據。

灰霉病； 灰葡萄孢； 殺菌劑； 復配劑

由灰葡萄孢BotrytiscinereaPers.exFr.,無性型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、莖和果實的腐爛,影響作物的品質和產量,尤其在溫室和大棚等設施蔬菜上危害尤為嚴重[1-5]。灰霉病在保護地番茄上嚴重發生時,能造成番茄花、葉和果實軟腐,減產50%以上[6-8]。由于缺少高抗灰霉病的番茄品種,從20世紀70年代開始,生產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灰霉病進行化學防治,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,極易對施用的殺菌劑產生抗藥性,導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和施藥量的不斷增加,我國江蘇、河南、山東、北京和遼寧等地相繼出現了單抗、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]研究發現江蘇省南京市和淮陰市灰霉病菌對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%；山東省灰霉病菌對四類殺菌劑的四抗菌株比例為100%。2016年Liu等[10]研究發現河南省灰霉病菌對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產生了較高比例的抗藥性。為了阻止或延緩抗藥性的進一步加劇,延長藥劑的使用壽命,應科學選用合適的殺菌劑防治灰霉病。

采用不同作用機制的殺菌劑交替或復配使用,是阻止或延緩病原菌抗藥性進一步加劇的主要策略。因此,本研究采用菌絲生長速率法,測定了咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環唑4種不同作用機制的殺菌劑單劑及其兩元復配劑對番茄灰霉病菌的毒力,對生產上綜合防控灰霉病和科學合理地用藥具有重要的指導意義。

1 材料與方法

1.1 供試菌株

供試菌株為實驗室保存的經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(中國)有限公司提供；97%氟啶胺原藥,江蘇優士化學有限公司提供；95.4%苯醚甲環唑原藥,先正達(中國)有限公司提供,上述3種藥劑均預溶于甲醇,配制成104μg/mL母液。96.2%啶酰菌胺原藥,巴斯夫(中國)有限公司提供,預溶于丙酮,配制成104μg/mL的母液。

1.3 供試培養基

PDA培養基: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種殺菌劑毒力測定

采用菌絲生長速率法測定咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環唑對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培養基中,制成系列濃度(表1)的含藥PDA平板,以不加藥劑為對照。用直徑為5 mm的打孔器,在25℃黑暗條件下培養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唑系列濃度的PDA平板上。將各平板置于恒溫培養箱內,25℃黑暗條件下培養3 d,測量并記錄菌落直徑。各處理3次重復,試驗重復3次。根據灰霉病菌在不同濃度藥劑平板上的線性生長速率,計算出各濃度藥劑對病原菌的生長抑制率,用DPS統計軟件進行處理,求出各藥劑的EC50。

表1 4種殺菌劑毒力測定濃度

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苯醚甲環唑Difenoconazole0,0.3125,0.625,1.25,2.5,5,10

1.5 復配劑毒力測定

采用菌絲生長速率法測定復配劑對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唑4種藥劑按照表2所示的比例分別進行兩元復配后,分別加入到滅菌熱熔冷卻至50℃左右的PDA培養基中,制成含復配劑系列濃度(表2)的PDA平板。按照1.4測定和計算方法,求出各復配藥劑的EC50。利用以下公式求出SR值[18],當SR值大于等于1.5時為增效作用,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)為實際測量抑制中濃度。

表2 復配劑不同比例濃度設計

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

復配劑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 結果與分析

2.1 4種殺菌劑對番茄灰霉病菌的毒力

采用菌絲生長速率法測定了咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環唑對番茄灰霉病菌的毒力。結果(表3)顯示,4種藥劑對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種藥劑對番茄灰霉病菌的菌絲生長都有較好的抑制作用,4種單劑中咯菌腈對番茄灰霉病菌的毒力最高。

2.2 復配劑對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唑4 種藥劑分別按照一定比例進行兩元復配,采用菌絲生長速率法測定各復配劑對番茄灰霉病菌的毒力。結果(表 4)顯示,不同組合、不同配比的復配劑對番茄灰霉病菌的毒力不同。復配劑啶酰菌胺：苯醚甲環唑1∶5、1∶3和1∶1、咯菌腈∶苯醚甲環唑1∶5增效作用最明顯；復配劑咯菌腈∶氟啶胺1∶3、咯菌腈∶苯醚甲環唑1∶3、啶酰菌胺∶咯菌腈5∶1、啶酰菌胺∶苯醚甲環唑3∶1具有增效作用,SR范圍為1.5～4.05,其中以復配劑啶酰菌胺∶苯醚甲環唑1∶5增效作用最好,其SR為4.05；其余不同配比的組合復配劑具有相加作用,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相關系數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苯醚甲環唑difenoconazoleY=4.8934+0.3360X2.08740.9971

表4 不同復配劑對番茄灰霉病菌的毒力

Table 4 Toxicities of mixtures of fungicides againstBotrytiscinereabased on mycelial growthinvitro

復配劑Mixture藥劑比例Fungicideproportion毒力回歸方程Toxicityregressionequation相關系數Correlationcoefficient實際測量抑制中濃度/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,屬于抗藥性發生風險高的病原菌,極易對防治藥劑產生抗藥性[20]。采用不同作用機制的殺菌劑交替或復配使用,是阻止或延緩病原菌抗藥性進一步加劇的主要策略。咯菌腈屬于苯基吡咯類非內吸性殺菌劑,是滲透信號傳導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,是解偶聯劑,破壞氧化磷酸化[22]；啶酰菌胺屬于新型煙酰胺類內吸性殺菌劑,是呼吸作用抑制劑[23]；苯醚甲環唑屬于三唑類殺菌劑,是甾醇脫甲基化抑制劑[24]。本研究采用菌絲生長速率法測定了咯菌腈、氟啶胺、啶酰菌胺和苯醚甲環唑4種不同作用機制的殺菌劑單劑及其兩元復配劑對番茄灰霉病菌的毒力。

單劑研究結果顯示,4種藥劑對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)和苯醚甲環唑(2.087 4 μg/mL),表明4種藥劑對番茄灰霉病菌菌絲生長均有較好的抑制作用。咯菌腈和氟啶胺單劑對番茄灰霉病菌具有較高的毒力,而且在田間尚沒有發現抗咯菌腈和氟啶胺的番茄灰霉病菌菌株[25-26]。近年來,雖然陸續在田間發現了抗苯醚甲環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]。復配劑研究結果顯示,不同配比的各組合復配劑具有增效或相加作用,沒有拮抗作用,表明4種殺菌劑在兩兩復配使用時各藥劑的作用機理互不影響。兩元復配劑中以啶酰菌胺∶苯醚甲環唑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種不同作用機制的殺菌劑及其兩元復配劑對番茄灰霉病菌均有較高的毒力,建議生產中采用這4種不同作用機制的殺菌劑進行交替或復配使用,以阻止或延緩灰霉菌抗藥性的進一步發展,為灰霉病的綜合防控和抗藥性治理提供理論依據。

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(責任編輯: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

國家自然科學基金青年科學基金(31301688)；公益性行業(農業)科研專項(201303023)；河南省自然科學基金(162300410079)；河南科技大學博士科研啟動基金(09001589)

S 436.412

B

10.3969/j.issn.0529-1542.2017.02.042

* 通信作者 E-mail:liushengmingzb@163.com