稻瘟病菌琥珀酸脱氢酶不同位点氨基酸替换对苯并烯氟菌唑敏感性的影响

doi:10.3864/j.issn.0578-1752.2025.24.006

摘要/Abstract

摘要：

【背景】稻瘟病由稻瘟病菌（Magnaporthe oryzae）引起，是严重威胁水稻产量的毁灭性真菌病害。先正达公司研发的苯并烯氟菌唑，属于全新结构的琥珀酸脱氢酶抑制剂（succinate dehydrogenase inhibitors，SDHIs）类杀菌剂，对稻瘟病菌抑制效果显著，但稻瘟病菌对其抗性机制研究比较匮乏。【目的】探究稻瘟病菌对苯并烯氟菌唑的抗性机制，为科学指导苯并烯氟菌唑在稻瘟病防治中的应用、延长其有效使用年限、保障防治效果提供理论依据。【方法】采用同源比对、分子对接和定点突变等方法，探究稻瘟病菌对不同SDHIs杀菌剂（苯并烯氟菌唑、联苯吡菌胺、氟唑菌酰胺、萎锈灵和氟吡菌酰胺）敏感性差异的原因，并分析不同位点氨基酸替换对苯并烯氟菌唑与稻瘟病菌亲和力的影响。【结果】75株稻瘟病菌对苯并烯氟菌唑高度敏感，平均EC₅₀值为0.041 μg·mL^-1，分布于0.018—0.068 μg·mL^-1；5种SDHIs杀菌剂与MoSdh之间的相互作用存在差异，包括氢键、π-π堆积和疏水相互作用，二者亲和力由高到低依次为苯并烯氟菌唑、联苯吡菌胺、氟唑菌酰胺、萎锈灵和氟吡菌酰胺，与这5种SDHIs杀菌剂对稻瘟病菌的EC₅₀值(从低到高)相对应。在稻瘟病菌琥珀酸脱氢酶复合体的8个位点中，共发现15种类型的氨基酸替换，其中，B亚基P198等7个位点氨基酸高度保守，C亚基S77位点存在物种差异。多数替换影响苯并烯氟菌唑与MoSdh的结合亲和力（主要与疏水相互作用及π-π堆积数量相关），其中MoSdhB^P198Q、MoSdhB^R243H等11种替换导致亲和力下降，SdhB^H245D/L/Y突变菌株对苯并烯氟菌唑敏感性降低，与亲和力变化一致。【结论】分子对接可用于筛选防控稻瘟病的SDHIs杀菌剂；稻瘟病菌琥珀酸脱氢酶的B、C、D亚基发生多种氨基酸替换，包括MoSdhB^P198Q、MoSdhB^R243H、MoSdhB^H245D/L/Y/Q、MoSdhB^I247V/N、MoSdhC^S77N和MoSdhD^D122G/N，这些替换均可导致稻瘟病菌对苯并烯氟菌唑产生抗性。

关键词: 稻瘟病菌, 苯并烯氟菌唑, 琥珀酸脱氢酶, 定点突变, 分子对接

Abstract:

【Background】Rice blast, caused by Magnaporthe oryzae, is a devastating fungal disease that seriously threatens rice yield. Benzovindiflupyr developed by Syngenta belongs to the new structure of succinate dehydrogenase inhibitors (SDHIs) fungicides, which has a significant inhibitory effect on M. oryzae, but the research on the resistance mechanism of M. oryzae is relatively scarce.【Objective】This study aims to explore the internal mechanism of M. oryzae against benzovindiflupyr, and to provide a theoretical basis for scientifically guiding the application of benzovindiflupyr in the prevention and control of rice blast, prolonging its effective service life and ensuring the control effect.【Method】Homology comparison, molecular docking and site-directed mutagenesis were used to explore the reasons for the difference in sensitivity of M. oryzae to different SDHIs fungicides (benzovindiflupyr, bixafen, fluxapyroxad, carboxin and fluopyram), and the effect of amino acid substitution at different sites on the affinity of benzovindiflupyr to M. oryzae was analyzed.【Result】75 strains of M. oryzae collected in the field were highly sensitive to benzovindiflupyr, with an average EC₅₀ value of 0.041 μg·mL^-1, which was distributed between 0.018 and 0.068 μg·mL^-1. The interaction between five SDHIs fungicides and MoSdh was different, including hydrogen bonding, π-π stacking and hydrophobic interaction. The affinity of these fungicides to MoSdh from high to low was benzovindiflupyr, bixafen, fluxapyroxad, carboxin and fluopyram, which corresponded to the EC₅₀values of these five SDHIs fungicides to M. oryzae (from low to high). A total of 15 types of amino acid substitutions were found in the 8 sites of the succinate dehydrogenase complex of M. oryzae. Among them, 7 sites such as B subunit P198 were highly conserved, while the amino acids at the S77 site in the C subunit were species-specific. Most of the substitutions affected the binding affinity of benzovindiflupyr to MoSdh (mainly related to hydrophobic interaction and π-π stacking number). Among them, 11 substitutions such as MoSdhB^P198Q and MoSdhB^R243H resulted in a decrease in affinity, and the sensitivity of SdhB^H245D/L/Y mutant to benzovindiflupyr decreased, which was consistent with the change of affinity.【Conclusion】Molecular docking technology can be used as an effective method to preliminarily screen SDHIs fungicides with prevention and control effects on M. oryzae. Amino acid substitutions in B, C and D subunits of M. oryzae, including MoSdhB^P198Q, MoSdhB^R243H, MoSdhB^H245D/L/Y/Q, MoSdhB^I247V/N, MoSdhC^S77N, MoSdhD^D122G/N, can lead to resistance of M. oryzae to benzovindiflupyr.

Key words: Magnaporthe oryzae, benzovindiflupyr, succinate dehydrogenase, site-directed mutation, molecular docking

王梦云, 邓力元, 余洋, 杨宇衡, 方安菲, 田斌年, 王静, 毕朝位. 稻瘟病菌琥珀酸脱氢酶不同位点氨基酸替换对苯并烯氟菌唑敏感性的影响[J]. 中国农业科学, 2025, 58(24): 5175-5189.

WANG MengYun, DENG LiYuan, YU Yang, YANG YuHeng, FANG AnFei, TIAN BinNian, WANG Jing, BI ChaoWei. Effects of Amino Acid Substitutions at Different Sites of Succinate Dehydrogenase on the Sensitivity of Magnaporthe oryzae to Benzovindiflupyr[J]. Scientia Agricultura Sinica, 2025, 58(24): 5175-5189.

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https://www.chinaagrisci.com/CN/Y2025/V58/I24/5175

图/表 9

表1

图1

图2

图3

表2

图4

表3

苯并烯氟菌唑与不同类型稻瘟病菌MoSdh的分子对接"

突变方式 Mutation mode	药剂结合腔 Binding pocket (≤5 Å)	结合模式Binding mode			MoSdh与苯并烯氟菌唑的结合亲和力 Docking affinity of MoSdh with benzovindiflupyr (kJ·mol^-1)	MoSdh与苯并烯氟菌唑的结合亲和力变化 Change of binding affinity between MoSdh and benzovindiflupyr (kJ·mol^-1)
突变方式 Mutation mode	药剂结合腔 Binding pocket (≤5 Å)	疏水作用 Hydrophobic interaction	氢键 Hydrogen bond	其他作用力 Other interaction
野生型Wild-type	B-W202, P198, I247	B-P198, I247	B-W202	D-Y123 (π-π stacking)	-30.209	0
	C-L66, V80	C-L66, V80	—
	D-Y123	D-Y123	D-Y123
SdhB^H245D	B-W202, P198	B-P198	B-W202	D-Y123 (π-π stacking)	-28.326	1.883
	C-L66, V80	C-L66, V80	—
	D-Y123	—	D-Y123
SdhB^H245L	B-W202, P198, I247	B-P198, I247	B-W202	—	-29.539	0.670
	C-L66, V80	C-L66, V80	—
	D-Y123	D-Y123	D-Y123
SdhB^H245Y	B-W202, P198	B-P198	B-W202	—	-29.999	0.209
	C-L66, V80	C-L66, V80	—
	D-Y123	D-Y123	D-Y123
SdhB^H245Q	B-W202, I247	B-I247	B-W202	—	-29.748	0.460
	C-L66, V80	C-L66, V80	—
	D-Y123	D-Y123	D-Y123
SdhB^P198Q	B-H245, W202	B-H245	B-W202	C-R83 (π-π stacking)	-29.414	0.795
SdhB^P198Q	C-Q82, R83	C-Q82	—	C-R83 (π-π stacking)	-29.414	0.795
SdhB^N203H	B-W202, P198, I247	B-P198, I247	B-W202	—	-30.376	-0.167
	C-L66, V80	C-L66, V80	—
	D-Y123	D-Y123	D-Y123
SdhB^N203K	B-W202, P198, I247	B-P198, I247	B-W202	D-Y123 (π-π stacking)	-30.167	0.042
	C-L66, V80	C-L66, V80	—
	D-Y123	D-Y123	D-Y123
SdhB^R243H	B-W202, P198, I247	B-P198, I247	B-W202	D-Y123 (π-π stacking)	-27.949	2.259
	C-L66, V80	C-L66, V80	—
	D-Y123	—	D-Y123
SdhB^I247V	B-W202, P198, I247	B-P198	B-W202	D-Y123 (π-π stacking)	-28.870	1.339
	C-L66, V80	C-L66, V80	—
	D-Y123	D-Y123	D-Y123
SdhB^I247N	B-W202, P198	B-P198	B-W202	—	-29.455	0.753
	C-L66, V80	C-L66, V80	N247
	D-Y123	D-Y123	D-Y123
SdhC^L66F	B-W202, P198, I247	B-P198, W202, I247	B-W202	D-Y123 (π-π stacking)	-32.593	-2.385
	C-F66, V80	C-F66, V80	—
	D-Y123	D-Y123	D-Y123
SdhC^L66R	B-P198, W201, W202, I247	B-P198, W201, I247	B-W202	D-D122 (Halogen bond)	-31.840	-1.632
	C-R66, Y71, L76, V80	C-R66, Y71, L76, V80	C-R66
	D-D122	—	—
SdhC^S77N	B-W202, P198, I247	B-P198, I247	B-W202	—	-29.957	0.251
	C-L66, V80	C-L66, V80	—
	D-Y123	D-Y123	D-Y123
SdhD^D122G	B-W202, I247	B-I247	B-W202	—	-27.656	2.552
	C-L66, V80	C-L66, V80	—
	D-Y123	—	D-Y123
SdhD^D122N	B-W202, P198, I247	B-P198, I247	B-W202	—	-28.033	2.176
	C-L66, V80	C-L66, V80	—
	D-Y123	—	D-Y123

表3

图5

表4

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引物名称 Primer name	序列 Sequence (5′-3′)	产物 Production description
P1F	CGAGCTCCTCACATCTTGCCATCCTCGGTAC	扩增构建pSKH载体的上游片段（1568 bp）Amplify the upstream fragment (1568 bp) for constructing the pSKH vector
P1R	TTGCGGCCGCTCATACGAAAGCCATCTCCTTCTTGAT
P2F	CAAGCTTGGTGTGTGAGGAAGACGGTGATAGA	扩增构建pSKH载体的下游片段（1128 bp）Amplify the downstream fragment (1128 bp) for constructing the pSKH vector
P2R	CCCCGGGACGCTTGTTGTTGTTAGGTTGTATTCG
Jsac	TTAACCCTCACTAAAGGGAAC	验证构建pSKH载体的上游片段（1649 bp）Verify the upstream fragment (1649 bp) of the constructed pSKH vector
Jnot	TTCAATATCATCTTCTGTCGAC
Jsma	CCAGAATGCACAGGTACACTTGTT	验证构建pSKH载体的下游片段（1276 bp）Verify the downstream fragment (1276 bp) of the constructed pSKH vector
Jhind	ACGACTCACTATAGGGCGAATTGG
L1F	CATGAGCCTGTACCGTTGCCTCACCATTCTTAACTGCACAAG	构建MoSdhB^H245L质粒载体 Construct the MoSdhB^H245L plasmid vector
L1R	CTTGTGCAGTTAAGAATGGTGAGGCAACGGTACAGGCTCATG
Y1F	CCATGAGCCTGTACCGTTGCTACACCATTCTTAACTGCAC	构建MoSdhB^H245Y质粒载体 Construct the MoSdhB^H245Y plasmid vector
Y1R	GTGCAGTTAAGAATGGTGTAGCAACGGTACAGGCTCATGG
DF	TGATCCTCGGTGGCTACTG	扩增原生质体转化所需的大片段（4597 bp） Amplify the large fragment (4597 bp) required for protoplast transformation
DR	CGCTTGTTGTTGTTAGGTTGTA
JP1F	TCCACCATTGAATGCGACCATGAA	验证转化子的上游片段（1859 bp） Verify the upstream fragment (1859 bp) of the transformant
JP1R	ATGTCCTCGTTCCTGTCTGCTAATAAG
JP2F	CGGCGAAGCAGAAGAATAGC	验证转化子的下游片段（2030 bp） Verify the downstream fragment (2030 bp) of the transformant
JP2R	CTGGAAGGGCGTGTTGAAC

菌株 Strain	基因型 Genotype	回归方程 Equation of regression (y=)	R²	EC₅₀(μg·mL^-1)	RF	抗性水平 Resistance level
wz-1	—	7.4741+1.5842x	0.992	0.027	—	—
H1	—	8.8909+2.5357x	0.972	0.029	1	S
D3	SdhB^H245D	4.2376+0.9175x	0.953	6.776	247	HR
D5	SdhB^H245D	4.3753+0.6525x	0.947	9.066	331	HR
Y8	SdhB^H245Y	5.8840+1.3997x	0.972	0.234	7	LR
Y9	SdhB^H245Y	5.8630+1.4149x	0.973	0.246	9	LR
Y10	SdhB^H245Y	6.2961+1.6271x	0.994	0.160	6	LR
L12	SdhB^H245L	3.5038+0.9303x	0.999	40.579	1481	VHR
L14	SdhB^H245L	3.5434+0.9548x	0.981	33.539	1224	VHR
L15	SdhB^H245L	3.3332+1.0327x	0.989	41.117	1501	VHR
N1	SdhB^N203H	5.8713+1.4021x	0.923	0.239	9	LR
R1	SdhB^R243H	7.8322+2.9574x	0.917	0.110	4	S