【作者】 赵平；

【导师】 纪明山；

【作者基本信息】 沈阳农业大学 ， 农药学， 2023， 博士

【摘要】 SY-6316为沈阳中化农药化工研发有限公司自主创制的化合物,根据其化学结构初步推测为琥珀酸脱氢酶抑制剂（SDHI）,对水稻纹枯病、玉米锈病、大豆锈病、小麦叶锈病等有较好的防效;其杀菌谱、敏感基线、作用特性及作用机制等研究均为空白,其抗性风险和作用机理也尚不明确。本研究针对我国自主创制农药SY-6316开展生物活性、作用特性及作用机制等研究,明确SY-6316的杀菌谱,并以水稻纹枯病菌为靶标,初步探索SY-6316对水稻纹枯病菌的作用特性及作用机制,取得如下结果:1)生物活性研究结果表明:SY-6316对子囊菌、担子菌和半知菌等病原菌引起的病害生物活性较好,如对水稻纹枯病、大豆锈病、小麦叶锈病、小麦全蚀病、黄瓜枯萎病、番茄早疫病、黄瓜白粉病等20种测定靶标均有一定的杀菌活性;SY-6316对卵菌纲引起的病害无效,对黄瓜疫霉病和黄瓜霜霉病没有杀菌活性。对水稻纹枯病菌、黄瓜枯萎病菌、番茄早疫病菌、番茄叶霉病菌、小麦全蚀病菌离体EC₅₀值分别为0.04、0.54、0.62、0.69、0.80 mg/L;SY-6316对水稻纹枯病菌、大豆锈病菌、玉米锈病菌、小麦叶锈病菌、黄瓜白粉病菌的活体盆栽EC₉₀值分别为9.35、0.77、2.09、4.00、49.76 mg/L。SY-6316具有优异的保护活性和一定的治疗、内吸活性,持效性与对照药剂氟唑酰菌胺、嘧菌酯接近。获得了物性和生物活性良好的10%SY-6316悬浮剂（SC）配方,并进行了田间试验验证。10%SY-6316 SC田间试验结果表明,在45～90 g a.i./hm²处理剂量下对水稻纹枯病防效良好,同等剂量下防效优于10%联苯吡菌胺SC,与15%氟唑菌酰胺SC和24%噻呋酰胺SC相当,对水稻幼苗生长无不良影响,对作物安全。2)测定了全国13个省360株水稻纹枯病菌对SY-6316和氟唑菌酰胺的敏感性,对SY-6316均敏感,EC₅₀值在0.00079～0.020 mg/L之间,平均EC₅₀值为0.0067±0.0048mg/L,敏感性频率呈单峰曲线分布。因此,0.0067 mg/L可作为敏感性基线用来监测田间水稻纹枯病菌对SY-6316敏感性未来变化。氟唑菌酰胺对水稻纹枯病菌的菌丝生长有明显的抑制作用,EC₅₀值为0.010～0.13 mg/L,平均EC₅₀值为0.066±0.025 mg/L,敏感性频率呈单峰分布,这表明这些菌株中不存在抗性亚群。因此0.066 mg/L可以作为敏感性基线用于监测水稻纹枯病菌对氟唑菌酰胺的敏感性变化。3)水稻纹枯病菌抗SY-6316菌株获得较难,仅单独使用紫外诱变或者药剂驯化无法获得抗性菌株,最后选择紫外诱导结合药剂驯化,从亲本菌株PJ2与SY3诱导获得5株抗性菌株,其他菌株未能诱导成功。然而抗性菌株抗性遗传并不稳定,抗性菌株PJ2R1在第一代时抗性倍数为3.487,而连续转接10代之后抗性倍数明显下降,EC₅₀值反而低于敏感菌株。结果表明SY-6316不易使水稻纹枯病菌产生抗药性,其抗性风险较低。4)SY-6316与氟唑菌酰胺对水稻纹枯病菌的作用方式类似,都使水稻纹枯病菌菌丝表面变得褶皱,浓度越大褶皱变得越明显。同时使病原菌菌丝碎片化、弯曲、变薄。SY-6316在高浓度时具有的菌丝致畸作用强于对照药剂氟唑菌酰胺,结果与菌丝生长速率法测定的结果一致。SY-6316对琥珀酸脱氢酶（SDH）活性的抑制IC₅₀值为0.30 mg/L,而水稻纹枯病菌SDH对氟唑菌酰胺较不敏感,体外活性稍差,IC₅₀值为1.23 mg/L,结果与前述实验结果一致。从分子对接结果可知,SY-6316的烷基链延伸向SDH疏水腔内,形成疏水作用力,并与关键氨基酸形成2个氢键,由此可以进一步稳定SY-6316与SDH的结合作用;相比于SY-6316,对照药剂氟唑菌酰胺与SDH的亲和力较弱,其与SDH上关键氨基酸仅形成一个氢键。通过分子动力学模拟,运用MM-PBSA和MM-GBSA方法计算得到了SY-6316和氟唑菌酰胺与受体SDH之间的结合自由能,进一步证实了SY-6316相比于氟唑菌酰胺与SDH之间具有更强的结合作用。5)通过对SY-6316、氟唑菌酰胺处理的两株水稻纹枯病菌HZ1、YZ1进行转录组测序,18个样品共计获得Clean base为120.24 Gb,Q30均大于93%,与参考基因组比对率良好、各个样本重复性良好、测序结果可靠。基于表达量结果对表达量差异显著的基因进行了GO富集分析、KEGG富集分析。对SY-6316与氟唑菌酰胺处理后的水稻纹枯病菌基因表达差异分析发现,SY-6316、氟唑菌酰胺诱导的差异表达基因显著富集于GO通路中的代谢生物过程以及氧化还原酶活性分子功能。相比氟唑菌酰胺,SY-6316诱导的差异表达基因与血红素结合、氧化还原酶活性、四吡咯结合相关并且大部分定位到生物膜上,更多地参与到外源性跨膜转运、外源运输生物过程。KEGG分析发现氟唑菌酰胺、SY-6316诱导的表达差异显著的基因主要集中在代谢途径、谷胱甘肽代谢、ABC转运蛋白、次生代谢产物的生物合成、类固醇生物合成等途径。相比氟唑菌酰胺,SY-6316诱导的差异基因参与到次生代谢产物的生物合成、类固醇生物合成、ABC转运蛋白和谷胱甘肽代谢的基因更加显著。采用实时荧光定量PCR对转录组进一步验证,结果表明细胞色素P450家族蛋白、血红素过氧化物酶、谷胱甘肽S转移酶、过氧化氢酶等代谢相关基因以及与靶标相关的琥珀酸脱氢酶泛醌黄素蛋白亚基等基因上调表达,参与水稻纹枯病菌对SY-6316的代谢。推断琥珀酸脱氢酶泛醌黄素蛋白亚基是SY-6316的重要作用靶标。更多还原

【Abstract】 SY-6316 is a compound independently developed by Shenyang Sinochem Agrochemical R&D Co.,Ltd.It is preliminarily speculated to be a succinate dehydrogenase inhibitor（SDHI）based on its chemical structure,and has good efficacy against rice sheath blight,corn rust,soybean rust and wheat rust.The research on its biological activity,sensitivity baseline,and mechanism of action is blank,and its resistance risk and mode of action are not yet clear.This study aims to conduct a series of studies on the independently developed pesticide SY-6316 in China,clarifying its bactericidal spectrum.The biological activity,characteristics,and mechanism of action of SY-6316 were studied using Rhizoctonia solani as the target,and the following results were obtained:1)The results of the biological activity study showed that SY-6316 had good activity against pathogenic fungi such as Ascomycetes,Basidiomycetes and Deuteromycetes.For example,it had certain fungicidal activity against twenty measured targets such as Rhizoctonia solani,Phakopsora pachyrhizi,Puccina triticina,Gaeumammomyces graminis,Fusarium oxysporum,Alternaria solani,and Podosphaera xanthii.SY-6316 was ineffective against Oomycetes and had no fungicidal activity against Phytophthora drechsleri and Pseudoperonospora cubensis.The EC₅₀values for SY-6316 against R.solani,F.oxysporum,A.solani,Fulvia fulva and G.graminis were 0.04,0.546,0.62,0.69,and 0.80 mg/L,respectively;The EC₉₀ values for SY-6316 in vivo pot tests against R.solani,P.pachyrhizi,Puccinia sorghi,P.recondite,and P.xanthii were 9.35,0.77,2.09,4.00,and 49.76 mg/L,respectively.SY-6316had excellent preventive activity and certain curative and systemic activities,with efficacy similar to that of fluxapyroxad.SY-6316 10%SC with good physical properties and biological activity was obtained and field tested.The results of the field trials showed that at a dosage of45-90 g a.i./ha,SY-6316 had good efficacy in controlling R.solani,with better efficacy than that of bixafen 10%SC at the same dose,similar efficacy to that of fluxapyroxad 15%SC and thifluzamide 24%SC,and no adverse effects on the growth of rice seedlings or crop safety.2)The sensitivity of 360 isolates of R.solani from 13 provinces across China to SY-6316and fluxapyroxad was determined.The results showed that R.solani was highly sensitive to SY-6316,with EC₅₀ values ranging from 0.00079 to 0.020 mg/L,and an average EC₅₀ value of0.0067±0.0048 mg/L.Sensitivity frequency was distributed in a unimodal curve.Therefore,0.00667 mg/L can be used as the sensitivity baseline to monitor changes in the sensitivity of R.solani to SY-6316 in the future.R.solani was less sensitive to fluxapyroxad,with EC₅₀ values ranging from 0.010 to 0.130 mg/L and an average EC₅₀ value of 0.066±0.025 mg/L.Sensitivity frequency was also distributed in a unimodal curve,indicating the absence of resistant subgroups in these isolates.Therefore,0.066 mg/L can be used as the sensitivity baseline to monitor changes in the sensitivity of R.solani to fluxapyroxad.3)It was difficult to obtain resistant strains of R.solani to SY-6316,and it was not possible to obtain resistant strains by solely using UV mutagenesis or drug domestication.Finally,UV mutagenesis combined with drug domestication was selected to induce 5 resistant strains from parent strains PJ2 and SY3,while other strains were not induced successfully.However,the resistance inheritance of the resistant isolates was unstable.The resistance multiple of PJ2R1was 3.487 in the first generation,but it decreased significantly after ten transfers,and the EC₅₀value was lower than that of the sensitive isolate.The results showed that SY-6316 was less likely to cause resistance to R.solani,and its risk of resistance was lower.4)The action of SY-6316 and fluxapyroxad on R.solani was similar,both of which make the surface of R.solani mycelium wrinkled,and the higher the concentration,the more obvious the wrinkling becomes.At the same time,the pathogen mycelia were fragmentation,bent and thinned.The mycelial teratogenic effect of SY-6316 at high concentrations is stronger than that of the control agent fluxapyroxad,and the results are consistent with those measured by the mycelial growth rate method.The IC₅₀ value of inhibition of succinate dehydrogenase（SDH）activity by SY-6316 was 0.30 mg/L,while the SDH of R.solani was less sensitive to fluxapyroxad and has slightly lower in vitro activity.The IC₅₀ value is 1.23 mg/L,which was consistent with the previous experimental results.From the results of molecular docking,it can be seen that the alkyl chain of valerylamide extends into the hydrophobic cavity of SDH,forming hydrophobic forces and forming two hydrogen bonds with key amino acids,which can further stabilize the binding effect between SY-6316 and SDH;Compared to SY-6316,the control agent fluxapyroxad had a weaker affinity with SDH,forming only one hydrogen bond with the key amino acid on SDH.Molecular dynamics simulations using MM-PBSA and MM-GBSA methods were used to calculate the binding free energies between compounds and the receptor SDH,further confirming that SY-6316 had a stronger binding affinity to SDH than fluxapyroxad.5)Transcriptome sequencing was performed on two isolates of R.solani,HZ1 and YZ1,treated with SY-6316 and fluxapyroxad.A total of 18 samples were sequenced,generating120.24 Gb of clean data with Q30 scores all above 93%.The mapping rate to the reference genome was good,and the repeatability of each sample was good,indicating reliable sequencing results.Based on the expression level results,GO enrichment analysis and KEGG enrichment analysis were conducted on genes with significant differences in expression levels.Analysis of the differential gene expression of R.solani treated with SY-6316 and fluxapyroxad showed that the differentially expressed genes induced by SY-6316 and fluxapyroxad were significantly enriched in the metabolic biological processes and oxidoreductase activity molecular functions in the GO pathway.Compared to fluxapyroxad,differentially expressed genes induced by SY-6316 were more involved in heme binding,oxidoreductase activity,and tetrapyrrole binding,most of which were located on biological membranes,and participated more in exogenous transmembrane transport and exogenous transport biological processes.KEGG analysis showed that the significantly differentially expressed genes induced by both compounds mainly affected metabolic pathways,glutathione metabolism,ABC transporters,secondary metabolite biosynthesis,and steroid biosynthesis pathways.Compared to fluxapyroxad,the differentially expressed genes induced by SY-6316 were more significantly involved in secondary metabolite biosynthesis,steroid biosynthesis,ABC transporters,and glutathione metabolism genes.Real-time fluorescence quantitative PCR was used for further validation of the transcriptome,and the results showed that metabolic-related genes such as cytochrome P450 family proteins,heme peroxidases,glutathione S-transferases,catalases,and target-related genes such as succinate dehydrogenase flavin adenine dinucleotide protein subunits were upregulated,and involved in the metabolism of R.solani to SY-6316.It is concluded that succinate dehydrogenase flavin adenine dinucleotide protein subunit is an important target for the action of SY-6316.更多还原