Susceptibility to Tebuconazole and Suitability of Resistant Strains of Magnaporthe oryzae in Hainan Province

doi:10.15933/j.cnki.1004-3268.2024.09.009

Abstract

Abstract: By detecting the susceptibility of Magnaporthe oryzae to tebuconazole in Hainan Province and studying the fitness of resistance strains,the resistance risk of M.oryzae to tebuconazole was understood,which would provide theoretical reference for the scientific use of fungicides and resistance management.The mycelial growth rate method was used to determine the susceptibility to tebuconazole of 144 strains of M.oryzae collected and isolated from Hainan Province in 2022,and the susceptibility baseline of M.oryzae in Hainan Province to tebuconazole was established based on the susceptibility frequency distribution of the strains.Fifteen susceptible strains with low effective inhibitory mid‑concentration(EC₅₀)measured during the establishment of the susceptibility baseline were selected for resistance domestication under continuous selection pressure，and the domesticated strains were classified into resistance levels,and the genetic stability and suitability of the resistant strains as well as the cross‑resistance to the fungicides pyraclostrobin,flucycloxazole,imidacloprid,fenpyroximate，and aliconazole were determined.The EC₅₀ values of the 144 strains tested ranged from 0.176 9 to 0.998 0 μg/mL.The EC50 value of the least sensitive strain was 5.64 times that of the most sensitive strain.The average EC₅₀ value was(0.476 3±0.174 5)μg/mL.The susceptibility frequencies of the strains were distributed in a continuous single‑peak curve,and the susceptibility baseline to tebuconazole of M.oryzae in Hainan Province was 0.476 3 μg/mL.Through indoor domestication of 15 sensitive strains,a total of two resistant strains were obtained.EC₅₀ values were 1.289 5 μg/mL and 1.868 4 μg/mL respectively,and resistance folds were 5.38 times and 6.65 times respectively,both of which were low resistance level.And after eight consecutive generations of transfer,the two resistant strains always maintained low‑level resistance.The EC₅₀ values of pyraclostrobin,epoxiconazole,prochloraz,difenoconazole,and diniconazole were measured and compared between the strains resistant to tebuconazole and their parental strains.The results showed that there was no cross‑resistance in M.oryzae in Hainan Province between tebuconazole and the above chemicals.Through the in vitro fitness test of the tebuconazole‑resistant strains,it was found that compared with the parental strains,the mycelial growth rate of the two resistant strains was significantly reduced,and the conidia production was reduced to varying degrees.In addition,they were more sensitive to strong acid and alkali environment，and their pathogenicity was greatly reduced.Therefore,the frequency of resistance mutations to tebuconazole in rice blast strains in Hainan Province is low,and the environmental suitability of their resistant strains is significantly reduced,resulting in a low risk of resistance.In addition,there is no cross‑resistance between tebuconazole and commonly used fungicides in Hainan Province,and the development of resistance can be delayed by strategies such as cross‑rotation or mixing with multi‑site‑of‑action protectants in production.

Key words: Magnaporthe oryzae, Tebuconazole, Susceptibility baseline, Resistance, Fitness

摘要： 通过检测海南省稻瘟病菌对戊唑醇的敏感性及抗性菌株的适合度，了解海南省稻瘟病菌对戊唑醇的抗药性风险，为该杀菌剂的科学使用及抗性治理提供理论依据和策略。采用菌丝生长速率法测定2022年采集分离自海南省的144株稻瘟病菌株对戊唑醇的敏感性，根据菌株的敏感性频率分布，建立海南省稻瘟病菌对戊唑醇的敏感基线。选取敏感基线建立过程中测得的有效抑制中浓度（EC₅₀）较低的敏感菌株15个，在连续选择压下进行抗药性驯化，对驯化出的菌株进行抗性水平划分，并测定抗性菌株的遗传稳定性及其对戊唑醇与杀菌剂吡唑醚菌酯、氟环唑、咪鲜胺、苯醚甲环唑、烯唑醇之间的交互抗性以及适合度。结果表明，供试144株菌株的EC₅₀值在0.176 9~0.998 0 μg/mL，其中最不敏感菌株的EC₅₀值是最敏感菌株的5.64倍，EC₅₀平均值为（0.476 3±0.174 5）μg/mL。菌株的敏感性频率呈连续性单峰曲线分布，海南省稻瘟病菌对戊唑醇的敏感基线为0.476 3 μg/mL。通过对15株敏感菌株进行药剂驯化共获得2株抗性菌株，其EC₅₀值分别为1.289 5 μg/mL和1.868 4 μg/mL，抗性倍数分别为5.38倍和6.65倍，均属低抗性水平。将上述2株抗性菌连续转接8代后，其始终保持低水平抗性。通过测定吡唑醚菌酯、氟环唑、咪鲜胺、苯醚甲环唑、烯唑醇对戊唑醇抗性菌株及亲本菌株的EC₅₀值并进行对比，结果显示，戊唑醇在对海南省稻瘟病菌的防治上与上述药剂均不存在交互抗性。对戊唑醇抗性菌株的离体适合度进行测定发现，相比于亲本菌株，2株抗性菌株的菌丝生长速率显著降低，且孢子产量均出现不同程度下降。此外，其对强酸和强碱环境更加敏感，致病力也大大减弱。因此，海南省稻瘟菌株对戊唑醇产生抗药性突变的频率较低，其抗性菌株的环境适合度显著下降，抗药性风险较低。此外，海南省稻瘟病菌对戊唑醇与常用杀菌剂无交互抗药性，可在生产上通过交互轮换使用或与多作用位点保护剂混合使用等策略来延缓抗药性的产生。

关键词: 稻瘟病菌, 戊唑醇, 敏感基线, 抗药性, 适合度

CLC Number:

S435.111.4⁺1

WANG Yanxia, ZHAO Yuhan, GU Xinyi, FU Zhongju, SUN Yujia, WU Chenglong, WU Weihuai, ZHANG Yaling. Susceptibility to Tebuconazole and Suitability of Resistant Strains of Magnaporthe oryzae in Hainan Province[J]. Journal of Henan Agricultural Sciences, 2024, 53(9): 88-96.

王艳霞, 赵羽涵, 顾欣怡, 付忠举, 孙宇佳, 吴成龙, 吴伟怀, 张亚玲. 海南省稻瘟病菌对戊唑醇的敏感性及抗性菌株适合度[J]. 河南农业科学, 2024, 53(9): 88-96.

References

［1］褚晋，闫晗，徐晗，等.辽宁省稻瘟病菌对肟菌酯敏感性监测［J］.植物病理学报，2021，51（1）：95‑103.
CHU J，YAN H，XU H，et al.Sensitivity detection of Magnaporthe oryzae to trifloxystrobin in Liaoning Province［J］.Acta Phytopathologica Sinica，2021，51（1）：95‑103.

［2］孟峰，张亚玲，靳学慧.黑龙江省稻瘟病菌无毒基因AVR‑Pita及其同源基因的检测与分析［J］.中国水稻科学，2020，34（2）：143‑149.

MENG F，ZHANG Y L，JIN X H.Detection and analysis of Magnaporthe oryzae avirulent gene AVR‑pita and its homologous genes in Heilongjiang Province［J］.Chinese Journal of Rice Science，2020，34（2）：143‑149.

［3］何桢锐，黄晓彤，舒灿伟，等.稻瘟病菌真菌病毒的研究进展［J］.热带生物学报，2021，12（3）：385‑392.

HE Z R，HUANG X T，SHU C W，et al.Research progress on mycoviruses of Magnaporthe oryzae［J］.Journal of Tropical Biology，2021，12（3）：385‑392.

［4］刘世江，赵琪君，丁怡，等.稻瘟病菌对咪鲜胺和三环唑的敏感性及两者的协同作用［J］.河南农业科学，2020，49（9）：81‑87.

LIU S J，ZHAO Q J，DING Y，et al.Sensitivity of Magnaporthe oryzae to prochloraz and tricyclazole and the synergistic effect of two agents［J］.Journal of Henan Agricultural Sciences，2020，49（9）：81‑87.

［5］赵东磊.稻瘟病菌对咪鲜胺的抗药性遗传多样性及其抗性机制研究［D］.南京：南京农业大学，2018.

ZHAO D L.The genetic diversity and mechaniam of prochloraz resistance in Magnaporthe oryzae[D］.Nanjing：Nanjing Agricultural University，2018.

［6］阮宏椿，石妞妞，田佩玉，等.福建省稻瘟病菌对4种杀菌剂的敏感性分析［J］.西北农林科技大学学报（自然科学版），2022，50（2）：125‑134.

RUAN H C，SHI N N，TIAN P Y，et al.Sensitivity of Magnaporthe oryzae in Fujian to 4 fungicides［J］.Journal of Northwest A & F University（Natural Science Edition），2022，50（2）：125‑134.
［7］高静，周明国.SDHIs和Qo Is杀菌剂抗性研究进展［J］.现代农药，2022，21（5）：7‑12.

GAO J，ZHOU M G.Research progress of SDHIs and QoIs fungicide resistance［J］.Modern Agrochemicals，2022，21（5）：7‑12.

［8］SIEROTZKI H，FREY R，WULLSCHLEGER J，et al.Cytochrome b gene sequence and structure of Pyrenophora teres and P. tritici‑repentis and implications for QoI resistance［J］.Pest Management Science，2007，63（3）：225‑233.

［9］李富根，吴新平，刘乃炽.戊唑醇的作用特点及其应用概况［J］.农药科学与管理，2001（3）：40‑41.

LI F G，WU X P，LIU N C.Characteristics and application of tebuconazole［J］.Pesticide Science and Administration，2001（3）：40‑41.

［10］ HENRY M J. Mode of action of the fungicide flusilazole in ustilago maydis［J］.Pesticide Science，1990，28（1）：35‑42.

［11］郭胜，王小勇.高效三唑类杀菌剂：戊唑醇［J］.精细与专用化学品，2001（6）：19‑20.

GUO S，WANG X Y.Tebuconazole，a high efficiency triazole fungicide［J］.Fine and Specialty Chemicals，2001（6）：19‑20.

［12］石凤梅.25%戊唑醇WG对水稻稻瘟病的防治效果［J］.黑龙江农业科学，2014（6）：47‑49.

SHI F M.Control effect of 25% tebuconazole WG on rice blast［J］.Heilongjiang Agricultural Sciences，2014（6）：47‑49.

［13］黎永健.5种杀菌剂对稻瘟病的田间防效试验［J］.广西植保，2014，27（3）：18‑20.

LI Y J.Field experiment of five fungicides against rice blast［J］.Guangxi Plant Protection，2014，27（3）：18‑20.

［14］鲜菲，刘顺涛，李雨，等.西南地区稻瘟病菌对戊唑醇的敏感性基线建立及抗性监测［J］.农药学学报，2015，17（6）：753‑756.

XIAN F，LIU S T，LI Y，et al.Sensitivity base‑line and resistance detection of Magnaporthe grisea to tebuconazole in southwest of China［J］.Chinese Journal of Pesticide Science，2015，17（6）：753‑756.

［15］魏松红，刘伟，王海宁，等.辽宁稻瘟病病菌对戊唑醇的敏感性及抗性突变体生物学特性［J］.中国植保导刊，2018，38（3）：61‑65.

WEI S H，LIU W，WANG H N，et al.Sensitivity of Pyricularia oryzae to tebuconazole and biological characteristics of resistant mutants in Liaoning［J］.China Plant Protection，2018，38（3）：61‑65.

［16］刘召阳，王帅，高宇琪，等.2株不同地理来源的苹果树腐烂病菌对甾醇生物合成抑制剂类杀菌剂的交互抗药性及生物适合度分析［J］.西北林学院学报，2020，35（2）：119‑124.

LIU Z Y，WANG S，GAO Y Q，et al.The Cross‑Resistance of Two Geographically Different Valsa Mali Strains to SBIs Inhibitors and Their Biotic Fitness［J］.Journal of Northwest Forestry University，2020，35（2）：119‑124.

［17］陈明丽，纪明山，赵琳，等.辽宁省稻瘟病菌对烯肟菌酯敏感基线及抗药性风险研究［J］.现代农药，2008，7 （2）：23‑25.

CHEN M L，JI M S，ZHAO L，et al.Baseline sensitivity and resistance risk of Magnaporthe grisea to enestroburin in Liaoning Province［J］.Modern Agrochemicals，2008，7（2）：23‑25.

［18］赵东磊，辛文静，杨莹，等.稻瘟病菌对咪鲜胺的抗药性风险评估［J］.南京农业大学学报，2019，42（3）：440‑447.

ZHAO D L，XIN W J，YANG Y，et al.Risk assessment of resistance to prochloraz in Magnaporthe oryzae［J］.Journal of Nanjing Agricultural University，2019，42 （3）：440‑447.

［19］符美英，罗激光，曾向萍，等.海南水稻稻瘟病病原鉴定及系统进化分析［J］.基因组学与应用生物学，2021，40（3）：1214‑1218.

FU M Y，LUO J G，ZENG X P，et al.Pathogenic identification and phylogenetic analysis of rice blast pathogens in Hainan［J］.Genomics and Applied Biology，2021，40（3）：1214‑1218.

［20］刘瑞，赵羽涵，顾欣怡，等.黑龙江省和海南省稻瘟病菌中AVR‑Pita家族的分布及变异分析［J］.中国农业科学，2023，56（3）：466‑480.

LIU R，ZHAO Y H，GU X Y，et al.Distribution and variation analysis of AVR‑pita family in Magnaporthe oryzae from Heilongjiang Province and Hainan Province ［J］.Scientia Agricultura Sinica，2023，56（3）：466‑480.

［21］符尚娇，谢圣华，严婉荣，等.海南水稻稻瘟病化学防治现状与药剂筛选［C］//彭友良，缪卫国. 中国植物病理学会2015年学术年会论文集. 北京：中囯农业出版社，2015：549‑552.

FU S J，XIE S H，YAN W R，et al.Current situation of chemical control and chemical screening of Magnaporthe oryzea in Hainan Province［C］//PENG Y L，MIAO W G. Proceedings of the Annual Meeting of Chinese Society for Plant Pathology（2015）.Beijing：China Agriculture Press，2015：549‑552.

［22］傅宇航，马慧，李娟，等.戊唑醇与嘧菌酯复配对稻瘟病和水稻纹枯病的联合毒力及田间防效［J］.中国农学通报，2020，36（36）：113‑117.

FU Y H，MA H，LI J，et al.Mixtures of tebuconazole and azoxystrobin：Indoor toxicity and field control

effects on rice blast and rice sheath blight［J］.Chinese Agricultural Science Bulletin，2020，36（36）：113‑117.

［23］赵志祥，严婉荣，肖敏，等.热带油茶根腐病病原菌的分子鉴定［J］.分子植物育种，2020，18（19）：6433‑6440.

ZHAO Z X，YAN W R，XIAO M，et al.Molecular identification of pathogens causing root rot of Camellia oleifera in tropica［l J］.Molecular Plant Breeding，2020，18（19）：6433‑6440.

［24］兰波，李湘民，杨迎青，等.稻瘟病菌产孢技术研究［J］.江西农业学报，2012，24（2）：74‑76.

LAN B，LI X M，YANG Y Q，et al.Study on sporulation technology of Magnaporthe grisea［J］.Acta Agriculturae Jiangxi，2012，24（2）：74‑76.

［25］林福呈，李德葆.稻瘟病菌分生孢子发芽和附着胞形成的影响因素研究［J］.中国水稻科学，2001（4）：52‑58.

LIN F C，LI D B.Factors affecting on conidium germination and its appressorium formation of Magnaporthe grisea［J］.Chinese Journal of Rice Science，2001（4）：52‑58.

［26］金晓春，李志新，张海生，等.稻瘟病菌分离及保存方法研究［J］.安徽农业科学，2008（17）：7308，7390.

JIN X C，LI Z X，ZHANG H S，et al.The study on the isolation and preservation of rice blast fungus［J］.Journal of Anhui Agricultural Sciences，2008（17）：7308，7390.

［27］纪明山，祁之秋，王英姿，等.番茄灰霉病菌对嘧霉胺的抗药性［J］.植物保护学报，2003（4）：396‑400.

JI M S，QI Z Q，WANG Y Z，et al.Resistance of Botrytis cinerea to pyrimethanil in tomato［J］.Journal of Plant Protection，2003（4）：396‑400.

［28］SMITH F D，PARKER D M，KOLLER W.Sensitivity distribution of Venturia inaequalis to the sterol demethylation inhibitor flusilazole：Baseline sensitivity and implications for resistance monitoring［J］.Phytopathology，1991，81（4）：392‑396.

［29］陈明丽.辽宁省稻瘟病菌对烯肟菌酯的抗药性及其分子机制研究［D］.沈阳：沈阳农业大学，2008.

CHEN M L. Studies on resistance and mechanism of Magnapothe grisea to enestrobulin in Liaoning Province D］.Shenyang：Shenyang Agricultural University，2008.

［30］王文桥，马志强，张小风，等.植物病原菌对杀菌剂抗性风险评估［J］.农药学学报，2001（1）：6‑11.

WANG W Q，MA Z Q，ZHANG X F，et al.Evaluation of risk of resistance in plant pathogenous fungi to fungicides［J］.Chinese Journal of Pesticide Science，2001（1）：6‑11.

［31］ZIOGAS B N，MARKOGLOU A N，SPYROPOULOU V.Effect of phenylpyrrole‑resistance mutations on ecological fitness of Botrytis cinerea and their genetical basis in Ustilago maydis［J］.European Journal of Plant Pathology，2005，113（1）：83‑100.

［32］潘夏艳，朱凤，齐中强，等.江苏省稻瘟病菌对稻瘟灵的抗药性监测及特性分析［J］.植物病理学报，2022，52（3）：416‑424.

PAN X Y，ZHU F，QI Z Q，et al.Sensitivity test and characteristic analysis of Magnaporthe oryzae to isoprothiolane in Jiangsu Province［J］.Acta Phytopathologica Sinica，2022，52（3）：416‑424.

［33］李波涛，吴隆起，倪笑霞，等.水稻稻瘟病菌对烯肟菌胺的抗性风险评估及抗性机制初探［J］.植物病理学报，2014，44（1）：80‑87.

LI B T，WU L Q，NI X X，et al.Risk assessment and molecular mechanism of the resistance of Magnaporthe

oryzae from rice to SYP‑1620［J］.Acta Phytopathologica Sinica，2014，44（1）：80‑87.

［34］鲜菲.西南地区稻瘟病菌对戊唑醇的抗性监测及抗性分子机理研究［D］.重庆：西南大学，2016.

XIAN F.Monitoring and molecule mechanisms of resistance to tebuconazole by Magnaporthe grisea in

southwest China［D］.Chongqing：Southwest University，2016.

［35］徐汉虹. 植物化学保护学［M］.4版. 北京：中国农业出版社，2007.

XU H H.Plant Chemical Protection［M］.4th ed.Beijing：China Agriculture Press，2007.

[1]	DAI Ziju, LI Wenxu, YANG Huimin, ZHU Xiuhua, WANG Yahuan, XU Fuxin, LIU Dongyang, HOU Jinna, QIN Maomao, WU Zhengqing, ZHOU Zhengfu, LEI Zhensheng. Identification and Evaluation of Resistance to Stripe Rust of 480 Wheat Germplasms [J]. Journal of Henan Agricultural Sciences, 2024, 53(9): 1-15.
[2]	LI Zhimin, LI Zheng, DING Junqiang, TIAN Zhiqiang. Major QTL Mapping and Effect Analysis for Resistance to Southern Corn Rust [J]. Journal of Henan Agricultural Sciences, 2024, 53(7): 109-116.
[3]	GUO Wenwen, ZHUO Mecao, HAN Jiahua, FANG Jiangping. Anatomical Structure and Stress Resistance Evaluation of Leaves of Rhododendron L.in the Shergyla Mountain [J]. Journal of Henan Agricultural Sciences, 2024, 53(6): 111-119.
[4]	WANG Junmei, LI Yahong, XU Fei, YANG Gongqiang, FENG Chaohong, LI Haohai, SONG Yuli. Isolation，Identification of Golubevia albescens 17wy1 and Exploration of Its Control Effect on Wheat Powdery Mildew [J]. Journal of Henan Agricultural Sciences, 2024, 53(2): 101-107.
[5]	CAO Liru, LIANG Xiaohan, MA Chenchen, YE Feiyu, PANG Yunyun, LI Weiya, ZHANG Xin, LU Xiaomin. Expression and Functional Analysis of Maize Stress Response Gene ZmTPR1 [J]. Journal of Henan Agricultural Sciences, 2024, 53(1): 12-21.
[6]	YANG Chun, SU Shengfeng, YANG Daixing, LIANG Sihui, GUO Yan, GUO Can, CHEN Zhengwu. Comparison of Leaf Anatomical Atructure and Stress Resistance Analysis of Wild Tea Plants in Panzhou City and Sandu County，Guizhou Province [J]. Journal of Henan Agricultural Sciences, 2024, 53(1): 48-61.
[7]	REN Zhichao, LI Xiang, LI Xianfeng, WU Lili, WANG Jing, PENG Zhiliang, LIU Guoshun, YIN Quanyu. Effect of Biochar on Rhizosphere Soil Fungal Community Structure of Flue‑cured Tobacco Varieties with Different Resistance to Black Shank Disease [J]. Journal of Henan Agricultural Sciences, 2024, 53(1): 105-115.
[8]	LU Xiaomin, GUO Shulei, ZHANG Xin, WEI Liangming, ZHANG Qianjin, CAO Liru, LIU Haijing, DENG Yazhou, ZHANG Zhen, WANG Zhenhua. Analysis of Yield Composition of Stem Lodging Resistance Related Traits of Maize under Different Densities [J]. Journal of Henan Agricultural Sciences, 2023, 52(9): 44-55.
[9]	LI Haiyong, LI Mengyu, GAO Chuang, XU Hao, YIN Guihong, DONG Chunhao, NIU Jishan, LI Qiaoyun. Correlation Analysis between the Resisitance of Wheat to Black Point and Leaf Blight at Seedling Stage Caused by Bipolaris sorokiniana [J]. Journal of Henan Agricultural Sciences, 2023, 52(8): 96-104.
[10]	WEI Xin, WANG Sheng, WANG Xingdong, YANG Yuchun, LIU Youchun, LIU Cheng. Physiological Response and Cold Resistance Evaluation of Blueberry Varieties under Low Temperature Treatment [J]. Journal of Henan Agricultural Sciences, 2023, 52(8): 115-125.
[11]	LEI Mengyao, GAO Xiaofeng, BAI Qingmin, DENG Ke, ZUO Weifang, LI Yuying. Evaluation of Cold Resistance of Pomegranate Branches from Different Varieties [J]. Journal of Henan Agricultural Sciences, 2023, 52(6): 120-130.
[12]	HAO Hongyan, SANG Huitong, LÜ Shanhua, FAN Yinglun, LI Haiyun. New Research Progress of bZIP Transcription Factors in Improving Plant Stress Resistance [J]. Journal of Henan Agricultural Sciences, 2023, 52(4): 1-8.
[13]	WU Si, ZHOU Yingxin, LUO Wei, TAO Mingde, CHEN Pingping, LUO Hongbing, ZHOU Wenxin, YI Zhenxie. Effects of Cultivation Patterns on Yield and Lodging Resistance of Summer Maize [J]. Journal of Henan Agricultural Sciences, 2023, 52(4): 42-50.
[14]	ZHANG Lei, WU Xinjuan, SONG Penghui, ZHOU Shuang, WANG Teng, ZHANG Lili, GU Xuejia, WANG Yufeng. Study on Resistance and Control Technology of Nitrogen and Phosphorus Nutrient Loss in Freeze‐Thaw Farmland [J]. Journal of Henan Agricultural Sciences, 2023, 52(12): 88-96.
[15]	HE Zhanxue, ZHU Taifu, LI Xin, SU Xiaolan, LIU Huimin, WANG Lianchun. Analysis of Differences and Mechanism of Resistance to Canker among Different Kiwifruit Rootstock‑Scion Combinations [J]. Journal of Henan Agricultural Sciences, 2023, 52(1): 95-107.