致病疫霉对缬菌胺敏感基线的建立及抗性风险评估

杨坡; 吴杰; 路粉; 赵建江; 毕秋艳; 韩秀英; 李洋; 王文桥

doi:10.16801/j.issn.1008-7303.2021.0171

致病疫霉对缬菌胺敏感基线的建立及抗性风险评估

doi: 10.16801/j.issn.1008-7303.2021.0171

杨坡^1,,
吴杰²,
路粉²,
赵建江²,
毕秋艳²,
韩秀英²,
李洋²,
王文桥^2, ,

1.
河北农业大学植物保护学院，河北保定 071000
2.
河北省农林科学院植物保护研究所，河北保定 071000

基金项目: 国家重点研发计划项目 (2016YFD0201000)；河北省重点研发计划项目 (21326510D)；国家重点研发计划 (2016YFD0200503-6)

详细信息

作者简介:
杨坡，Yangpo0326@163.com

通讯作者:
王文桥，wenqiaow@163.com

中图分类号: S481.4
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- 被引次数: 0
出版历程
- 收稿日期: 2021-09-10
- 录用日期: 2021-10-24
- 网络出版日期: 2021-11-11
- 刊出日期: 2022-06-10

Establishment of sensitivity baseline of Phytophthora infestans to valifenalate and risk assessment of resistance

1.
College of Plant Protection, Agricultural University of Hebei, Baoding 071000, Hebei Province, China
2.
Institute of Plant Protection, Hebei Academy of Agricultural&Forestry Sciences, Baoding 071000, Hebei Province, China

Funds: National Key R&D Program of China (2016YFD0201000), Key R&D Program in Hebei Province (21326510D), National Key R&D Program of China (2016YFD0200503-6)

摘要

摘要: 为建立致病疫霉Phytophthora infestans (Mont.) de Bary对缬菌胺的敏感基线，采用菌丝生长速率法测定了从河北省、黑龙江省、内蒙古自治区、贵州省和四川省未使用过缬菌胺的地区采集分离的105个致病疫霉菌株对缬菌胺的敏感性；为明确致病疫霉对缬菌胺产生抗性突变体的难易程度，进行了紫外诱导和药剂驯化试验；为明确缬菌胺与常用药剂之间的交互抗性，测定了8个抗缬菌胺突变体及其6个亲本敏感菌株对6种常用杀菌剂的敏感性。结果表明：105株致病疫霉对缬菌胺的EC₅₀值范围为0.0594~0.159 mg/L，平均EC₅₀值为(0.102 ± 0.024) mg/L，不同敏感性菌株的频率呈连续单峰曲线分布，未发现敏感性下降的亚群体，因此可将缬菌胺对105株致病疫霉的平均EC₅₀值作为致病疫霉对缬菌胺的敏感基线；通过紫外诱变敏感菌株菌丝体获得了4个抗缬菌胺的突变体，其抗性水平介于 3.1~14.9倍之间，突变频率为0.54%，通过紫外照射敏感菌株孢子囊悬浮液获得了2个抗性水平分别为8.1倍和8.2倍的抗性突变体，突变频率为1.33 × 10⁻⁷；通过在含缬菌胺的黑麦蔗糖琼脂培养基上继代培养敏感菌株11代，获得2个抗性水平分别为3.1倍和9.4倍的抗性突变体。缬菌胺与烯酰吗啉和双炔酰菌胺存在交互抗性，与氟吡菌胺、嘧菌酯、甲霜灵和霜脲氰不存在交互抗性。初步推测致病疫霉对缬菌胺具有低到中等抗性风险，建议在生产上将缬菌胺与其他类型杀菌剂交替或混合使用，以延缓致病疫霉对缬菌胺抗性的产生。
- 致病疫霉 /
- 缬菌胺 /
- 敏感基线 /
- 抗性突变体 /
- 交互抗药性 /
- 抗性风险
Abstract: In order to establish the baseline sensitivity of Phytophthora infestans (Mont.) de Bary to valifenalate, the sensitivities to valifenalate of 105 P.infestans isolates collected from Hebei, Heilongjiang, Guizhou, Sichuan and Inner Mongolia Autonomous Region, where valifenalate had never been used,were determined by mycelial growth rate method. To clarify the difficulty degree of acquisition of valifenalate-resistant mutants in P. infestans, UV mutagensis and fungicide adaptation test were conducted. In order to clarify the patterns of cross-resistance between valifenalate and fungicides regularly used for late blight control, the sensitivities to valifenalate and six fungicides of eight resistant mutants and their six parent sensitive strains were tested. The results showed that the EC₅₀ values ranged from 0.0594 mg/L to 0.159 mg/L and the mean EC₅₀ value of 105 isolates was (0.102±0.024) mg/L. The frequency of sensitivity to valifenalate distributed as a unimodal curve, and there was no subgroup with sensitivity declined, so the mean EC₅₀ value could be regarded as the sensitivity baseline to valifenalate in P. infestans. Four mutants resistant to valifenalate with the resistance factors ranging from 3.2 to 14.9 were obtained through UV-irradiating mycelia of strains sensitive to valifenalate and the resistance mutagenesis frequency was 0.54%. Two mutants resistant with the resistance factors of 8.1 and 8.2 were obtained through UV-irradiating sporangia of strains sensitive to valifenalate and the resistance mutagenesis frequency was 1.33 × 10⁻⁷. Two mutants resistant to valifenalate with the resistance factors from 3.1 and 9.4 were obtained through 11 generation subcultures. No cross resistance relationship existed between valifenalate and azoxystrobin or fluopicolide or metalaxyl or cymoxanil, but cross resistance existed between valifenalate and mandipropamid or dimethomorph. It was suggested that P. infestans could have a low to moderate resistance risk to valifenalate. Therefore, valifenalate should be used for late blight in mixtures or rotation with other fungicides of different action mechanisms in production to avoid or delay the buildup of valifenalate resistance in P. infestans.
- Phytophthora infestans /
- valifenalate /
- sensitivity baseline /
- resistant mutants /
- cross-resistance /
- risk assessment

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图 1 105株致病疫霉菌株对缬菌胺的敏感性分布

Figure 1. Senisitivity distribution of 105 isolates of Phytophthora infestans to valifenalate

下载: 全尺寸图片幻灯片

图 2 致病疫霉对缬菌胺的敏感基线

Figure 2. Sensitivity baseline of Phytophthora infestans to valifenalate

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图 3 致病疫霉对缬菌胺及其他6种杀菌剂交互抗性模式

Figure 3. Cross-resistance patterns of P. infestans to valifenalate and the other 6 fungicides

下载: 全尺寸图片幻灯片

表 1 供试菌株采集时间及地点

Table 1. Collection time and location of the tested strains

地区　　　 Region	菌株数 Number of strains	采集时间 (年-月) Collection date (Year-Month)	采集地点　　　 Collection locality
内蒙古自治区 Inner Mongolia Autonomous Region	20	2018-08	锡林郭勒盟多伦县；赤峰市喀喇沁旗 Duolun County, Xilinguole League; Kalaqin Banner, Chifeng City
四川省 Sichuan Province	20	2018-08	宜宾市宜宾县；眉山市青神；乐山市犍为县Yibin County, Yibin City; Qingshen County, Meishan City; Qianwei County, Leshan City
贵州省 Guizhou Province	15	2018-08	贵阳市花溪区；贵阳市息烽县Huaxi district, Guiyang City; Xifeng County, Guiyang City
河北省 Hebei Province	25	2018-08	围场满族蒙古族自治县；张家口市沽源县Weichang Manchu and Mongolian Autonomous County; Guyuan County, Zhangjiakou City
黑龙江省 Heilongjiang Province	25	2018-08	齐齐哈尔讷河市；绥化市北林区；大兴安岭加格达奇区Nehe City, Qiqihar; Beilin district, Suihua City; Jiagedaqi district, Greater Khingan Range

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表 2 紫外诱变试验亚致死时间的确定

Table 2. Determination of sublethal time in ultraviolet mutagenesis test

紫外照射菌丝体时间Time of UV-irradiating mycelia/min	菌落生长直径Colony diameter/ mm	紫外照射孢子囊时间Time of UV-irradiating sporangia/s	菌丝生长疏密程度Degree of colony density
0	31.6 ± 0.54	0	+++++
10	27.6 ± 0.54	30	++++
15	21.6 ± 0.5	60	+++
20	17.9 ± 0.54	90	++
25	11.7 ± 0.6	120	+
30	6.3 ± 0.24	150	−
35	5 ± 0	180	−
注：“−” 表示没有菌丝生长；“+” 表示有极稀疏菌丝生长；“++” 表示有较稀疏的菌丝生长； “+++” 表示有中等密集的菌丝生长；“++++” 表示有较密集的菌丝生长；“+++++” 表示有很密集的菌丝生长.Note：“−” indicates no hypha growth. “+” indicates very thin hypha growth. “++” indicates thinner hypha growth. “+++” indicates mediumly dense hypha growth. “+++++” indicates the denser hypha growth. “+++++” indicates very dense hypha growth.

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表 3 致病疫霉亲本菌株GZGY经药剂驯化获得对缬菌胺抗性过程

Table 3. The acquisition of resistance to valifenalate in Phytophthora infestans parent strain GZGY

继代培养代数No. of subculture	最低抑制浓度MIC/ (mg/L)	亚致死浓度Sublethal concentration/ (mg/L)	亚致死浓度下抑制率Inhibition rate under sublethal concentration/%	有效抑制中浓度 EC₅₀/(mg/L)
G0	0.5	0.4	93.1	0.103
G1	0.5	0.4	91.9	0.113
G2	0.5	0.4	90.8	0.125
G3	1	0.8	91.3	0.143
G4	1	0.8	93.8	0.228
G5	2	1.5	90.7	0.319
G6	2	1.5	91.8	0.419
G7	3	2	93.8	0.467
G8	4	3	92.5	0.569
G9	4	3	92.4	0.658
G10	5	4	91.2	0.768
G11	6	5	92.3	0.973

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表 4 致病疫霉亲本菌株CFKLQ经药剂驯化获得对缬菌胺抗性过程

Table 4. The acquisition of resistance to valifenalate in P. infestans parent strain CFKLQ

继代培养代数No. of subculture	最低抑制浓度 MIC/(mg/L)	亚致死浓度Sublethal concentration/ (mg/L)	亚致死浓度下抑制率Inhibition rate under sublethal concentration/%	有效抑制中浓度 EC₅₀/(mg/L)
G0	0.4	0.3	91.7	0.109
G1	0.4	0.3	90.7	0.118
G2	0.4	0.3	90.6	0.124
G3	0.5	0.4	91.5	0.160
G4	0.5	0.4	92.9	0.212
G5	0.6	0.5	92.8	0.231
G6	0.6	0.5	92.8	0.245
G7	0.7	0.6	93.8	0.259
G8	0.8	0.7	92.3	0.272
G9	0.9	0.8	91.8	0.297
G10	1.2	1	91.5	0.310
G11	1.2	1	90.7	0.322

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表 5 致病疫霉对缬菌胺抗性突变体的抗性水平

Table 5. Resistance factor to valifenalate of the valifenalate-resistant mutants of P. infestans

菌株 Strain	毒力回归方程 Toxic regression equation (y=)	相关系数 Correlation coefficent, r	有效抑制中浓度 EC₅₀/(mg/L)	95% 置信区间 Confidence interval of 95%/(mg/L)	抗性水平 Resistance factor (RF)
GZGY^*	1.928x + 6.966	0.975	0.096	0.0762~0.120	—
GZGY^−UVS	1.978x + 5.208	0.977	0.785	0.683~0.904	8.2
GZGY^−FT	1.833x + 5.088	0.961	0.896	0.589~1.361	9.4
SCLS^*	1.826x + 6.926	0.982	0.088	0.0729~0.107	—
SCLS^−UVS	1.809x + 5.262	0.982	0.717	0.691~0.747	8.1
GZXF^*	2.335x + 7.274	0.988	0.106	0.0906~0.124	—
GZXF^−UVM	2.458x + 6.167	0.991	0.335	0.280~0.402	3.2
CFKLQ^*	2.317x + 7.266	0.990	0.105	0.0912~0.121	—
CFKLQ^−FT	2.056x + 6.012	0.976	0.322	0.239~0.433	3.1
CFKLQ^−UVM	1.612x + 4.689	0.956	1.560	0.932~2.612	14.8
ND^*	1.944x + 6.669	0.986	0.139	0.114~0.168	—
ND^−UVM	1.638x + 4.652	0.974	1.632	1.0959~2.431	11.8
QN^*	2.731x + 7.680	0.994	0.104	0.0947~0.115	—
QN^−UVM	1.498x + 4.711	0.959	1.559	0.948~2.562	14.9
注： ^代表亲本菌株；ZGY^−UVS、SCLS^−UVS、GZXF^−UVM、CFKLQ^−UVM、ND^−UVM和QN^−UVM分别代表敏感菌株GZGY、SCLS、GZXF、CFKLQ、 ND和QN通过紫外照射诱导获得的抗性突变体；GZGY^−FT和CFKLQ^−FT分别代表敏感菌株GZGY和CFKLQ通过药剂驯化获得的抗性突变体。Note: ^Indicates parental isolate. GZGY^−UVS, SCLS^−UVS, GZXF^−UVM, CFKLQ^−UVM, ND^−UVM and QN^−UVM represent resistant mutants induced by sensitive strains GZGY, SCLS, GZXF, CFKLQ, ND and QN through ultraviolet irradiation, respectively. GZGY^−FT and CFKLQ^−FT represent the resistant mutants obtained by drug domestication of sensitive strain GZGY and CFKLQ, respectively.

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