Risk Assessment on Resistance to Pyraclostrobin of Colletotrichum gloeosporioides
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摘要:
目的 探明福建省大豆胶孢炭疽菌群体对吡唑醚菌酯的敏感程度及其与其他杀菌剂的交互抗性。 方法 采用菌丝生长速率法对2023年分离自福建省5个地区的112株大豆胶孢炭疽菌进行敏感性分析,以药剂驯化法获得抗性突变体,测定抗性突变体的遗传稳定性、适合度及对4种不同杀菌剂之间的交互抗性。 结果 吡唑醚菌酯对112个菌株的EC50值介于 0.0682 ~1.0309 µg·mL−1,变异系数为15.12,敏感性频率分布为连续性单峰曲线,符合偏正态分布,EC50平均值0.2036 ±0.1215 µg·mL−1,可作为大豆胶孢炭疽菌对吡唑醚菌酯的敏感性基线;从2株大豆胶孢炭疽菌野生菌株中诱导获得4株中抗吡唑醚菌酯的突变体,1株低抗吡唑醚菌酯的突变体,突变频率为8.3×10−4,其抗药性性状能稳定遗传,抗性突变体与其亲本菌株相比,对温度的敏感性存在差异,抗性突变体菌丝的生长速率、产孢能力及致病力,通常略低于其亲本菌株的生存适应度,抗性突变体在田间可能存在一定适合度代价,但抗性突变体具有一定的自然竞争优势;吡唑醚菌酯与不同类杀菌剂多菌灵、苯醚甲环唑、咪鲜胺之间不存在交互抗性,与同属一类的杀菌剂啶氧菌酯存在明显的交互抗性。结论 福建省大豆胶孢炭疽菌对吡唑醚菌酯具有较高的敏感性,但存在中等抗性风险,生产中可将吡唑醚菌酯与不同类杀菌剂多菌灵、苯醚甲环唑、咪鲜胺混合或交替使用,以延缓大豆胶孢炭疽菌抗药性的产生。 Abstract:Objective Drug sensitivity to and cross-resistance of pyraclostrobin with other fungicides of Colletotrichum gloeosporioides that caused anthracnose in soybeans in Fujian Province were investigated. Methods Sensitivity to pyraclostrobin of 112 strains of C. gloeosporioides isolated from 5 regions in the province in 2023 was determined using the mycelial production rate method. Resistant mutants of the pathogen were obtained by fungicide domestication to determine their genetic stability, fitness, and possible cross-resistance to 4 fungicides. Results The EC50 of pyraclostrobin for the 112 C. gloeosporioides strains ranged 0.0682 µg·mL−1 to 1.0309 µg·mL−1 with a coefficient of variation of 15.12 and averaged 0.203 6±0.121 5 µg·mL−1 as the sensitivity baseline. The distribution of the sensitivity frequency was in a continuous unimodal function consistent with the continuous skewed normal distribution. Induced from two wild C. gloeosporioides, 4 moderate resistant strains and one low resistant strain were secured at a frequency of 8.3×10−4. The mutants stably inherited the drug resistance traits but differed from their parents in temperature sensitivity and were slightly lower on mycelial growth rate, sporulation ability, and pathogenicity. In the field, they might sacrifice other natural competitive advantages as a fitness cost in exchange for the drug resistance. Pyraclostrobin showed no cross-resistance with carbendazim, difenoconazole, and prochloraz but did with the same type of fungicide, picoxystrobin. Conclusion Although the C. gloeosporioides isolated in Fujian were relatively high in sensitivity to pyraclostrobin, they represented merely a moderate drug resistance risk. Nonetheless, in practice, it was plausible to blend pyraclostrobin or apply alternatively with fungicides such as carbendazim, difenoconazole, and prochloraz to delay such potential drawback. -
表 1 福建不同地区大豆胶孢炭疽菌对吡唑醚菌酯的敏感性
Table 1. Sensitivity to pyraclostrobin of C. gloeosporioides isolated from regions in Fujian
采集地区
Sampling region菌株数
Number of isolates吡唑醚菌酯EC50值
EC50 of Pyraclostrobin/(µg·mL−1)菌株间变异系数
Variation factor范围 Range 平均值 Mean 闽东地区 Mindong region 24 0.0682 ~0.3777 0.1662 ±0.0633 a5.54 闽南地区 Minnan region 31 0.0763 ~0.3506 0.1845 ±0.0751 a4.60 闽西地区 Minxi region 27 0.0801 ~0.6428 0.2451 ±0.1359 a8.02 闽北地区 Minbei region 15 0.1284 ~1.0309 0.2387 ±0.2269 a8.03 闽中地区 Minzhong region 15 0.0725 ~0.3007 0.1933 ±0.0670 a4.15 总计 Total 112 0.0682 ~1.0309 0.2036 ±0.1215 a15.12 表中数据为平均数±标准差。同列数据后不同小写字母分别表示经Duncan氏新复极差法检验差异显著(P<0.05)。下同。
Data are mean±SD; those with different lowercase letters on same column indicate significant difference at P<0.05 in Duncan's new multiple range test.表 2 吡唑醚菌酯抗性突变体连续转代培养后EC50和抗性倍数的变化
Table 2. Changes on EC50 and resistance multiple of pyraclostrobin-resistant C. gloeosporioides mutants after continuous generational transference
菌株
Strain第一代
1st generation第三代
3rd generation第五代
5th generation第七代
7th generationEC50 /(µg·mL−1) RM EC50/(µg·mL−1) RM EC50/(µg·mL−1) RM EC50/(µg·mL−1) RM CG23017 0.1349 / 0.1396 / 0.1369 / 0.1344 / CG23017-1 1.7673 13.10 1.7823 12.77 1.7121 12.5 1.6661 12.39 CG23017-2 0.6674 4.95 0.5275 3.78 0.4891 3.57 0.4376 3.26 CG23017-3 1.9268 14.29 1.9084 13.67 1.8966 13.85 1.8612 13.85 CG23052 0.1611 / 0.1647 / 0.1623 / 0.1580 / CG23052-1 3.0151 18.71 3.0198 18.34 2.9742 18.33 2.9574 18.72 CG23052-2 2.7140 16.84 2.7191 16.51 2.7200 16.76 2.6425 16.73 RM为抗性倍数。
RM: Resistance multiple.表 3 抗性突变体及其亲本菌株对温度的敏感性测定
Table 3. Mycelial growth of resistant mutants and parents under different temperatures
菌株 Strain 菌落直径 Colony diameter/mm 16 ℃ 20 ℃ 25 ℃ 28 ℃ 35 ℃ CG23017 31.33±0.58 cd 43.33±1.53 c 59.33±2.31 c 63.67±1.53 b 20.67±1.53 a CG23017-1 28.67±0.58 e 41.33±1.15 cd 58.33±1.15 c 60.67±1.53 b 18.67±3.06 ab CG23017-2 25.33±1.15 f 37.67±1.15 e 56.67±1.53 c 60.33±2.31 b 19.33±1.53 ab CG23017-3 29.67±1.53 de 39.33±1.53 de 57.33±1.53 c 60.67±2.31 b 19.67±1.15 ab CG23052 35.67±2.08 a 48.33±1.15 a 72.67±0.58 a 74.67±1.53 a 17.67±1.53 ab CG23052-1 34.33±1.53 ab 45.67±1.15 b 69.67±1.15 b 71.67±1.53 a 18.33±0.58 ab CG23052-2 32.33±1.53 bc 46.33±0.58 ab 68.33±1.53 b 71.33±2.52 a 16.67±2.52 b 表 4 抗性突变体及其亲本菌株菌丝生长速率、产孢能力、致病力测定
Table 4. Mycelial growth rate, sporulation per area, and disease index of resistant mutants and parents
菌株
Strain菌丝生长速率
Mycelium growth rate/(mm·d−1)单位面积产孢量
Sporulation per area/(×103个·mm−2)病情指数
Disease indexCG23017 9.1±0.22 b 4.58±0.27 ab 47.52±2.34 a CG23017-1 8.67±0.22 b 4.55±0.19 ab 40.37±4.56 a CG23017-2 8.62±0.33 b 4.32±0.08 b 27.08±4.83 b CG23017-3 8.67±0.33 b 4.50±0.26 ab 42.48±10.06 a CG23052 10.67±0.22 a 4.77±0.25 a 41.48±8.82 a CG23052-1 10.24±0.22 a 4.74±0.23 a 26.63±6.32 b CG23052-2 10.19±0.36 a 4.61±0.2 ab 37.43±0.89 ab -
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