贡常委^,, 秦旖曼, 屈劲松, 王学贵^,四川农业大学农学院/无公害农药研究实验室,成都 611130

Resistance Detection and Mechanism of Strawberry Botrytis cinerea to Fludioxonil in Sichuan Province

GONG ChangWei^,, QIN YiMan, QU JinSong, WANG XueGui^,College of Agronomy/Biorational Pesticide Research Laboratory, Sichuan Agricultural University, Chengdu 611130

通讯作者: 王学贵,Tel：028-86290977;E-mail： wangxuegui@sicau.edu.cn

第一联系人: 联系方式：贡常委,Tel：028-86290977;E-mail： youguqiu@163.com。
收稿日期:2018-06-4接受日期:2018-07-27网络出版日期:2018-11-16

基金资助:

国家公益性行业（农业）科研专项.201303025

Received:2018-06-4Accepted:2018-07-27Online:2018-11-16


摘要
【目的】灰霉病是草莓生产过程中的一种重要病害,严重影响了其产量和品质。论文旨在明确四川省草莓灰霉病菌对咯菌腈的抗性频率以及抗性机制,为草莓灰霉病的药剂防治提供理论依据。【方法】2016—2017年从四川成都、德阳、眉山、乐山及雅安等地采集草莓灰霉病样本,并分离纯化得到188株草莓灰霉病菌菌株。采用区分计量法测定188株灰霉病菌对咯菌腈的敏感性水平,采用菌丝生长速率抑制法测定咯菌腈对代表性菌株的毒力和渗透压敏感性,采用甘油铜比色法测定经咯菌腈处理的抗性菌株和敏感菌株甘油含量,采用分段测序对抗性菌株和敏感菌株Ⅲ型组氨酸激酶基因BOS1（BC1G_00374）扩增测序,采用Swissmodle和I-TASSER预测和评价突变对BOS1的结构影响。【结果】188株灰霉病菌菌株中有8株表现为高抗,9株为中抗,43株为低抗,其余表现为敏感;咯菌腈对代表性菌株的EC₅₀介于0.03—0.62 μg·mL ^-1,代表性菌株的抗性倍数范围为2.2—45.9。NaCl浓度在1.25—10 g·L ^-1可刺激敏感菌株生长,浓度在1.25—20 g·L ^-1范围可刺激抗性菌株生长,但>40 g·L ^-1时则抑制菌丝生长,尤其对抗性菌株,抗性越高抑制作用越强;在正常条件下各代表性菌株甘油含量介于0.0025—0.0148 μg·mL ^-1,且菌株的甘油含量与咯菌腈抗性没有明显的关联性,但在使用咯菌腈处理(0.1 μg·mL ^-1)抗性菌株和敏感菌株后,甘油含量均上升,且抗性菌株甘油含量上升幅度明显低于敏感菌株。低抗菌株YAHY-13、CDCZ-2以及中抗菌株CDCZ-42在TAR和HAMP区域均发生突变,中抗菌株CDCZ-20和高抗菌株MYFC-10、CDCZ-43在TAR和REC区域均有突变,但CDCZ-20菌株在TAR区域位点是I365N,而两株高抗菌株在TAR区域位点是I365S。不同突变位置对BOS1区域结构有不同程度的影响,其中F127S、I365N、I365S、V1136I、A1259T均处于BOS1区域结构的无规则卷曲,但TAR区域I365N和I365S使区域结构无规则卷曲发生整体偏移。 【结论】四川省已有部分地区草莓灰霉病菌对咯菌腈产生了抗性;相比敏感菌株,田间抗性菌株对渗透压的耐受能力增加,但当浓度超过耐受范围后对渗透胁迫高度敏感,药剂胁迫下田间抗性菌株甘油含量增加量显著小于敏感菌株;组氨酸激酶BOS1突变的位置和方式与灰霉病菌菌株对咯菌腈的抗性水平存在必然联系。
关键词： 灰霉病菌;咯菌腈;抗药性;渗透压;甘油含量;BOS1

Abstract
【Objective】Grey mold is an important disease in strawberry production, which seriously affects the yield and quality of strawberry. The objective of this study is to clarify the resistance frequency and resistance mechanism of Botrytis cinerea in different strawberry-growing areas in Sichuan Province, and to provide theoretical basis for the fungicide control of strawberry grey mold.【Method】The disease samples were collected from Chengdu, Deyang, Meishan, Leshan and Yaan in Sichuan Province from 2016 to 2017, and 188 strains of B. cinerea were isolated and purified. The sensitivity of 188 strains of B. cinerea was classified with the distinguish measurement method. The toxicity and osmotic pressure sensitivity of fludioxonil to some representative strains were assayed using the method of mycelial growth-inhibition capacity. The glycerol content of the resistant and sensitive strains treated with fludioxonil was determined by the method of glycerol-copper colorimetric assay. The sequences of type III histidine kinase gene BOS1 (BC1G_00374) in the resistant- and sensitive-fludioxonil strains were piecewise amplified and sequenced. The effects of mutations on the structure of BOS1 were predicted and evaluated by Swissmodle and I-TASSER, respectively.【Result】Of 188 strains, 8 strains showed high resistance, 9 strains showed medium resistance, 43 strains showed low resistance and the rest were sensitive. The EC₅₀ of representative strains ranged from 0.03 to 0.62 μg·mL ^-1, and the resistance multiple of the representative strains ranged from 2.2 to 45.9. The concentrations of 1.25-10 g·L ^-1 and 1.25-20 g·L ^-1 NaCl could stimulate the hypha growth of the sensitive- and resistant-fludioxonil strains, respectively, whereas the concentration of >40 g·L ^-1 inhibited the hypha growth, especially in the resistant strains, and the higher the resistant level, the stronger the inhibition rate. The glycerol content of representative strains ranged from 0.0025 to 0.0148 μg·mL ^-1 under normal conditions, and there was no significant correlation between glycerol content and fludioxonil resistance of the strain, but the glycerol content of the resistant and sensitive strains increased after the treatment of fludioxonil (0.1 μg·mL ^-1). The increase of glycerol content in resistant strains was significantly lower than that in sensitive strains. The low resistance strains YAHY-13, CDCZ-2 and medium resistance strain CDCZ-42 mutated in the TAR and HAMP regions, meanwhile the medium resistance strain CDCZ-20 and high resistance strains MYFC-10 and CDCZ-43 mutated in TAR and REC regions, whereas the mutation site of TAR region in CDCZ-20 was I365N, and which of MYFC-10 and CDCZ-43 was I365S. Different mutation positions showed different effects on the region structure of BOS1, in which the F127S, I365N, I365S, V1136I, A1259T were all in the irregular curl of BOS1 structure, but the I365N and I365S in TAR region made the overall deviation of the region structure irregular curl. 【Conclusion】In some areas of Sichuan Province, B. cinerea has developed resistance to fludioxonil. Compared with the sensitive strains, the tolerance ability of field resistant strains to osmotic pressure increased, but when the concentrations exceeded the tolerance range, they were highly sensitive to osmotic stress and the increase of glycerol content in the field resistant strains under the fludioxonil stress was significantly lower than that of the sensitive strains. The mutation position and mode of histidine kinase BOS1 are closely related to the resistance level of B. cinerea to fludioxonil.
Keywords：Botrytis cinerea;fludioxonil;resistance;osmotic pressure;glycerol content;BOS1


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本文引用格式
贡常委, 秦旖曼, 屈劲松, 王学贵. 四川省草莓灰霉病菌对咯菌腈的抗性测定及其机制[J]. 中国农业科学, 2018, 51(22): 4277-4287 doi:10.3864/j.issn.0578-1752.2018.22.006
GONG ChangWei, QIN YiMan, QU JinSong, WANG XueGui. Resistance Detection and Mechanism of Strawberry Botrytis cinerea to Fludioxonil in Sichuan Province[J]. Scientia Agricultura Sinica, 2018, 51(22): 4277-4287 doi:10.3864/j.issn.0578-1752.2018.22.006

0 引言

【研究意义】灰霉病（主要致病菌为灰葡萄孢,Botrytis cinerea）危害多种作物,现已被列入世界十大重要作物真菌病害^[1,2]。灰霉病菌寄主广泛,可侵染200多种植物^[3],不仅在生长期通过植物表面或植物伤口侵染,还会在蔬菜和水果的运输、储藏期进行危害,影响其产量和质量。由于该病原菌具有繁殖速度快、变异频率高等特点,在杀菌剂长期选择压力下,极易造成抗药性的暴发,且由于菌株多抗性特点,导致灰霉病的防治十分困难^[4,5]。咯菌腈（fludioxonil）是瑞士先正达公司开发的一种新型苯基吡咯类杀菌剂,对灰霉病菌有特效,抑制孢子萌发的同时也能够抑制菌丝体的生长^[6]。检测灰霉病菌对咯菌腈的抗性频率以及抗性机制,可为灰霉病的药剂防治提供理论依据。【前人研究进展】防治灰霉病的化学药剂主要有7大类：苯并咪唑类、N-苯基氨基甲酸酯类、二甲酰亚胺类、苯胺基嘧啶类、甲氧基丙烯酸酯类、琥珀酸脱氢酶抑制剂类（SDHIs）、苯基吡咯类。苯并咪唑类杀菌剂是防治灰霉病历史最长的一类药剂,但引入我国后不久就出现了抗性报道^[7];二甲酰亚胺类杀菌剂在使用几年后便在田间发现了高抗菌株^[8];张玮等^[9]研究发现,我国葡萄灰霉病菌对嘧霉胺的抗性频率为22.22%—62.50%,且以高抗和中抗菌株为主,高抗菌株频率达44.23%。咯菌腈杀菌谱广,毒性低,可用于防治由担子菌、子囊菌、半知菌等引起的多种植物病害^[6]。徐建强等^[10]研究表明,咯菌腈对牡丹的4种叶片真菌,即牡丹黑斑病菌（Alternaria suffruticosae）、黄斑病菌（Phyllosticta commonsii）、腔孢叶斑病菌（Hainesia lythri）和叶霉病菌（Cladosporium paeoniae）的菌丝生长、孢子萌发、芽管伸长及产孢均具有很强的抑制活性。咯菌腈作用靶标为Ⅲ型组氨酸激酶,可抑制Ⅲ型组氨酸激酶磷酸化,激活HOG-MAPK途径,然后由它磷酸化下游目标Ypd1,最终可合成大量的甘油致使细胞死亡^[11,12];灰霉病菌Ⅲ型组氨酸激酶BOS1通过基因敲除的方法被克隆鉴定,敲除突变体对苯基吡咯类杀菌剂的抗性显著提升^[13];为了揭示BOS1对HOG-MAPK途径的具体功能,SEGMüLLER等构建了BOS1和Sak1（Hog1-like基因）双突变体,BOS1负调控SAK1磷酸化,参与对甘油合成和渗透压的调节功能;同时BOS1独立于HOG-MAPK途径,调节杀菌剂敏感性、在高渗条件下的适应性^[14,15]。在典型的Ⅲ型组氨酸激酶结构域的N-末端有5—6个重复HAMP结构域^[16],HAMP亚基含有两个螺旋,由约14个柔性残基连接,每一个螺旋都由典型的七肽重复序列（A-G）组成,在A和D的位置具有疏水残基^[17],根据NCBI对灰霉病菌B05.10菌株BOS1的注释,它除了具有5个重复的HAMP结构域外,还具有RAP、TAR、HisKA、HATPase_c、G-X-G motif、REC等结构。目前有关咯菌腈药剂的抗性报道较少,在国外部分地区有低、中水平抗性的报道,VIGNUTELLI等^[18]在法国监测到一株低抗菌株,FERNáNDEZORTU?O等^[19]在美国弗吉尼亚州分离得到一株低抗菌株,这也是美国在咯菌腈生产并使用10年后首次监测到咯菌腈的抗性菌株。【本研究切入点】国内外虽陆续有咯菌腈抗性菌株的报道,但对灰霉病菌咯菌腈抗性机制研究相对较少,且不同区域使用药剂类型及使用频率亦有差异,导致灰霉病菌对其产生抗性机制的原因可能有所不同;本文通过测定四川省草莓灰霉病菌对咯菌腈的抗性水平和频率,分析渗透压、甘油含量以及组氨酸激酶BOS1突变的位置和方式与咯菌腈抗性水平之间的关系,从而探索灰霉病菌对咯菌腈的抗性机制。【拟解决的关键问题】明确四川省草莓灰霉病菌对咯菌腈抗性频率、抗性水平和部分生理指标,通过抗性菌株靶标基因扩增测序,明确四川省草莓灰霉病菌对咯菌腈抗性机制,为科学使用咯菌腈提供理论依据。

1 材料与方法

1.1 试验材料

1.1.1 供试菌株 采用课题组2016—2017年在四川省草莓主产区采集、分离的草莓灰霉病菌菌株（表1）。

Table 1
表1
表1四川省草莓灰霉病菌供试菌株
Table 1The tested strains of strawberry B. cinerea in Sichuan Province

采样地区 Sampling area	采样时间 Sampling time	经纬度 Latitude and longitude	菌株编号 Strain code	菌株数 Strain number
CDCZ1（成都崇州市Chongzhou, Chengdu）	2017-02-16	103o41′, 30o34′	CDCZ (1-15)	52
CDCZ2（成都崇州市Chongzhou, Chengdu）	2017-02-16	103o37′, 30o36′	CDCZ (16-30)
CDCZ3（成都崇州市Chongzhou, Chengdu）	2017-02-16	103o44′, 30o40′	CDCZ (31-52)
CDSL（成都双流市Shuangliu, Chengdu）	2016-04-10	103o35′, 30o14′	CDSL (1-9)	9
CDPZ（成都彭州市Pengzhou, Chengdu）	2017-01-05	104o10′, 30o59′	CDPZ (1-10)	10
DYGH（德阳广汉市Guanghan, Deyang）	2017-03-02	104o20′, 31o3′	DYGH (1-11)	11
MSRS1（眉山仁寿县Renshou, Meishan）	2017-03-14	104o5′, 29o33′	MSRS (1-10)	26
MSRS2（眉山仁寿县Renshou, Meishan）	2017-03-14	104o4′, 29o31′	MSRS (11-26)
MSDP1（眉山东坡区Dongpo, Meishan）	2017-03-14	102o36′, 29o30′	MSDP (1-8)	18
MSDP2（眉山东坡区Dongpo, Meishan）	2017-03-22	103o36′, 30o0′	MSDP (9-18)
LSJY1（乐山井研县Jingyan, Leshan）	2017-03-14	104o1′, 29o24′	LSJY (1-4)	11
LSJY2（乐山井研县Jingyan, Leshan）	2017-03-14	103o34′, 29o18′	LSJY (5-11)
YAHY（雅安汉源县Hanyuan, Yaan）	2017-03-25	102o36′, 29o30′	YAHY (1-16)	16
MYFC（绵阳涪城区Fucheng, Mianyang）	2017-03-27	104o49′, 31o20′	MYFC (1-17)	17
MYJY（绵阳江油市Jiangyou, Mianyang）	2017-03-27	104o44′, 31o47′	MYJY (1-18)	18

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1.1.2 供试药剂与仪器 95%咯菌腈原药（山东亿嘉生物科技有限公司）;甘油、氢氧化钠、氯化钠、琼脂粉等化学试剂均购置于成都浩搏优科技有限公司,DNA提取试剂盒购置于上海生工生物工程股份有限公司（上海生工）;引物合成和测序均在上海生工完成。

SHP-250恒温培养箱（上海三发科学仪器有限公司）;SW-OJ-2FD超净工作台（苏州安泰空气技术有限公司）;UV3000紫外分光光度计（上海美普达仪器有限公司）;D37520离心机（美国科峻仪器公司）;Powerpac 200型电泳仪（Bio-rad公司）;C1000 PCR扩增仪（Bio-rad公司）。

1.2 试验方法

1.2.1 灰霉病菌对咯菌腈的抗性水平检测 按照区分剂量法^[20],对2016—2017年采集于四川省的188株草莓灰霉病菌对咯菌腈的抗性进行评价,即将采集并分离的菌株分别接种在含0.1、0.5、1 μg·mL^-1 3种咯菌腈浓度的PDA培养基上培养。将能在含0.1、0.5、1 μg·mL^-1咯菌腈浓度的PDA培养基生长菌丝的菌株视为高抗菌株,将能在含0.1、0.5 μg·mL^-1咯菌腈浓度的PDA培养基生长菌丝,而不能在1 μg·mL^-1咯菌腈浓度的培养基上生长菌丝的菌株视为中抗菌株,将能在含0.1 μg·mL^-1咯菌腈浓度的PDA培养基生长菌丝,而不能在0.5和1 μg·mL^-1咯菌腈浓度的培养基上生长菌丝菌株视为低抗菌株,将不能在含有咯菌腈的培养基上生长菌丝的菌株视为敏感菌株,统计抗性和敏感菌株的发生频率,并对抗性类型进行统计分析^[21]。

1.2.2 咯菌腈抗性菌株毒力测定 采用菌丝生长速率法^[22]。将原药分别配制成0.025、0.05、0.1、0.5、1 μg·mL^-1 5个浓度,每个浓度设3个重复,均匀混入PDA培养基中,然后将供试菌原接种于含毒培养基上,同时设置空白对照,在22℃恒温培养箱中培养。培养4 d后,采用“十字交叉法”测定菌落直径,根据菌落直径的平均值计算咯菌腈对菌丝生长的抑制率。

抑制率=（对照菌落直径-药剂处理菌落直径）/（对照菌落直径-菌饼直径）×100%。

根据抑制率查表得到相对应的机率值,将药剂浓度转换为对数值。以机率值为纵坐标,浓度对数值为横坐标,得到毒力回归方程,根据毒力回归方程,计算杀菌剂的有效中浓度（EC₅₀）,参考敏感基线EC₅₀=0.0135 μg·mL^-1[23],计算抗性倍数,抗性倍数=抗性菌株EC₅₀/敏感菌株EC₅₀。

1.2.3 咯菌腈抗性及敏感菌株渗透压敏感性测定 采用菌丝生长速率法^[22]。将NaCl溶液配制为0、1.25、2.5、5、10、20、40、80 g·L^-1,每个浓度设3个重复,均匀混入PDA培养基中,然后将供试菌原接种于含NaCl的培养基上,同时设置空白对照,在22℃恒温培养箱中培养。培养5 d后,采用“十字交叉法”测定菌落直径,并计算不同浓度下的菌丝生长抑制率,计算公式参考1.2.2。

1.2.4 咯菌腈抗性及敏感菌株甘油含量测定 采用甘油铜比色法^[24]。标准曲线制定：在50 mL的锥形瓶中依次加入0.05 g·mL^-1的CuSO₄溶液1 mL,0.05 g·mL^-1的NaOH溶液3.5 mL,再分别加入10 mL浓度为0.0025、0.003、0.004、0.005、0.006、0.008、0.01 g·mL^-1的甘油,振荡反应12 min后,用三层滤纸过滤,在630 nm下测定吸光度,记录数据并利用Excel 2016软件求得标准曲线。

将敏感菌株和抗性菌株分别接种于液体培养基中,每个菌种接种6瓶,每瓶接种5个菌饼,25℃,175 r/min培养60 h后,每个菌株取3瓶,加入0.1 μg·mL^-1的咯菌腈1 mL,培养4 h后,过滤收集菌丝,用超纯水洗净,滤纸吸干水分并风干。每个样品取0.5 g菌丝,在研钵中加入20 mL无菌水和少量石英砂,低温研磨,将上清液转移至50 mL离心管中,80℃加热15 min,然后在8 500 r/min下离心10 min,取上清液在630 nm下测定吸光度。以蒸馏水作为空白对照。将测得的吸光度代入标准曲线,求得菌株的甘油含量^[25]。

1.2.5 咯菌腈抗性菌株的分子检测 灰霉病菌DNA提取：采用真菌基因组DNA快速抽提试剂盒的方法提取灰霉病菌DNA。将新鲜菌株接种到含有100 mL PDA培养液的250 mL三角瓶中（每瓶5—7个菌饼）,然后置于摇床（25℃、120 r/min）振荡培养。将培养4 d后的菌丝取出后,放置4层纱布于布氏漏斗上,在无菌条件下抽干菌丝上的培养液;取50—100 mg菌丝用液氮研磨成粉末,加入到1.5 mL离心管中。加入400 μL Buffer Digestion和4 μL巯基乙醇,振荡混匀。将离心管置于65℃水浴锅中水浴1 h至细胞完全裂解,每10 min颠倒混匀一次;加入200 μL Buffer PF,充分颠倒混匀,-20℃冰箱放置5 min;室温10 000 r/min离心5 min,将上清液转移到新的1.5 mL离心管中;加入等体积的异丙醇,颠倒5—8次使之充分混匀,室温放置2—3 min。室温10 000 r/min离心5 min,弃上清液;加入1 mL 75%乙醇,颠倒漂洗1—3 min,室温10 000 r/min离心2 min,弃上清液,重复步骤1次;开盖,室温倒置5—10 min至残留的乙醇完全挥发;得到的DNA用50 μL TE Buffer溶解,置于-20℃下保存备用。

靶标基因扩增^[20]：根据杀菌剂的靶标基因BOS1（BC1G_00374）序列设计引物,采用50 μL的反应体系进行BOS1分段扩增,PCR反应程序如下：95℃ 3 min;95℃ 30 s,55℃ 30 s,72℃ 2 min,35个循环;72℃ 5 min;16℃ 2 min。取5 μL PCR产物于1%琼脂糖凝胶中进行电泳（120 V,15 min）,EB染色,在凝胶成像仪下观察条带大小。引物序列见表2,扩增反应体系：Template DNA（100 ng·μL^-1）1.0 μL,2×Phanta Max Buffer 25 μL,Dntp Mix（10 mmol·L^-1 each）2.0 μL,Phanta Max Super-Fidelity DNA Polymerase（1 U·μL^-1）1.0 μL,BF（10 pmol·L^-1） 2.0 μL,BR（10 pmol·L^-1）2.0 μL,ddH₂O 17 μL。

Table 2
表2
表2靶标基因BOS1分段扩增所需引物
Table 2The primers for the segment amplification of target gene BOS1

引物 Primer	序列 Sequence
BFl	TACCGATCGAAAAACCCAAC
BRl	TGGGCTGGTCTCTCAATCTT
BF2	CAACGTTATGGCACAAAATCTCA
BR2	AAGTTTCTGGCCATGGTGTTCA
BF3	GGTCGGAACTGATGGAACTC
BR3	CGCGGTAAGTGAGGTCTAGG
BF4	GCAAACCGTATGATCATGGA
BR4	AGCTCGATTCTCCAAAGCAG
BF5	TCCCGTTATTCATGTCAGCTT
BR5	AAGTACTCGCAGTCGGTGGT

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靶标基因测序分析：将PCR产物测序,测得菌株基因序列用DNAMAN序列分析软件进行拼接并翻译氨基酸序列,采用Swissmodle（https://swissmodel.expasy.org/）和I-TASSER（https://zhanglab.cmmb.med.umich.edu/I-TASSER/）对BOS1的三级结构进行预测分析;采用ENDscript 3.0^[26]对比对代表性菌株BOS1的氨基酸序列以及空间结构进行比较。

2 结果

2.1 草莓灰霉病菌对咯菌腈抗性频率测定

采用区分计量法对2016—2017年采集于四川省不同地区的188株草莓灰霉病菌进行抗性频率测定,结果见表3。在188株菌株中,高抗菌株有8株（4.25%）,中抗菌株有9株（4.78%）,低抗菌株有43株（22.87%）,敏感菌株128株（68.08%）;其中绵阳涪城区的高抗菌株比例最大（23.53%）,而成都崇州、成都彭州、德阳广汉、眉山仁寿的中抗菌株比例也分别达到7.84%、10.00%、9.09%和7.69%;敏感菌株比例高于80%的仅有成都双流（88.89%）和眉山东坡区（88.88%）。

Table 3
表3
表3四川省不同采集地点的草莓灰霉病菌对咯菌腈的抗性水平
Table 3Resistance level of strawberry B. cinerea to fludioxonil in different sampling sites of Sichuan Province

地区 Area	菌株数 Strain number	敏感菌株数 Number of sensitive strains	敏感菌株比例 Proportion of sensitive strains (%)	低抗菌株数 Number of low resistance strains	低抗菌株比例 Proportion of low resistance strains (%)	中抗菌株数Number of medium resistance strains	中抗菌株比例Proportion of medium resistance strains (%)	高抗菌株数Number of high resistance strains	高抗菌株比例Proportion of high resistance strains (%)
CDCZ	52	37	70.59	8	15.69	4	7.84	3	5.88
CDSL	9	8	88.89	1	11.11	0	0	0	0
CDPZ	10	3	30.00	6	60.00	1	10.00	0	0
DYGH	11	6	54.55	4	36.36	1	9.09	0	0
MSRS	26	20	76.92	3	11.54	2	7.69	1	3.85
MSDP	18	16	88.88	2	11.11	0	0	0	0
LSJY	11	6	54.54	5	45.45	0	0	0	0
YAHY	16	9	56.25	7	43.75	0	0	0	0
MYFC	17	10	58.82	2	11.76	1	5.88	4	23.53
MYJY	18	13	72.22	5	27.78	0	0	0	0
总计Total	188	128	68.08	43	22.87	9	4.78	8	4.25

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2.2 咯菌腈对草莓灰霉病菌毒力测定

基于草莓灰霉病菌区分计量法检测的结果,从其中筛选敏感菌株CDPZ-8和CDCZ-11,低抗菌株CDCZ-2和YAHY-13,中抗菌株CDCZ-42和CDCZ-20,高抗菌株MYFC-10和CDCZ-43进行了毒力测定（表4）。结果表明,低抗菌株及中抗菌株,抗性倍数均<10,高抗菌株抗性倍数>10。其中敏感菌株的EC₅₀在0.03—0.05 μg·mL^-1,抗性倍数为2.2—3.7;低抗菌株和中抗菌株EC₅₀在0.08—0.11 μg·mL^-1,抗性倍数为5.9—8.1,毒力结果相差不大;高抗菌株EC₅₀在0.43—0.62 μg·mL^-1,抗性倍数为31.9—45.9。

Table 4
表4
表4咯菌腈对抗性及敏感草莓灰霉病菌菌株毒力
Table 4Toxicity of fludioxime to resistant and sensitive strains of strawberry B. cinerea

菌株编号 Strain code	回归方程 Regression equation (y=a+bx)			EC50（95%置信区间） 95% confidence interval	抗性倍数 Resistance multiple
	a	b	r
CDCZ-2	5.7333	0.7442	0.9011	0.10 (0.06-0.17)	7.4
CDCZ-11	6.5566	1.1984	0.9801	0.05 (0.03-0.08)	3.7
CDCZ-20	5.8440	0.7696	0.9631	0.08 (0.05-0.12)	5.9
CDCZ-42	6.4192	1.4323	0.8460	0.10 (0.07-0.14)	7.4
CDCZ-43	5.4371	1.1802	0.8918	0.43 (0.27-0.68)	31.9
CDPZ-8	6.2100	0.8161	0.9945	0.03 (0.02-0.06)	2.2
YAHY-13	6.5496	1.6160	0.9865	0.11 (0.09-0.14)	8.1
MYFC-10	5.1480	0.7054	0.9456	0.62 (0.31-1.23)	45.9

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2.3 咯菌腈抗性及敏感菌株渗透压敏感性测定

将筛选菌株接种至分别含0、1.25、2.5、5、10、20、40、80 g·L^-1 NaCl溶液的PDA中培养,结果见图1。敏感菌株在1.25—10 g·L^-1 NaCl浓度范围内可刺激生长,抗性菌株在1.25—20 g·L^-1 NaCl浓度范围内刺激生长（MYFC-10菌株除外）,在高浓度40 g·L^-1以上生长被抑制,在80 g·L^-1 NaCl溶液的PDA中培养抗性越高的菌株其抑制率越高。

图1

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图1代表性菌株在不同浓度NaCl下的抑制率

Fig. 1The inhibition rate of the representative strains at different concentrations of NaCl

2.4 咯菌腈抗性及敏感菌株的甘油含量测定

甘油铜比色法标准曲线：y=5.427x+0.0012,R2=0.9929,线性相关性良好。由表5可知,在正常条件下各菌株之间甘油含量范围是0.0025—0.0148 μg·mL^-1,且与对咯菌腈抗性没有明显的相关性,在加入0.1 μg·mL^-1的咯菌腈1 mL处理4 h后,敏感菌株甘油含量明显上升,抗性菌株甘油含量虽然增加,但变化不明显,增加幅度低于敏感菌株。结果表明,咯菌腈抗性菌株在有药剂胁迫的作用下,甘油的合成受到了影响,且菌株抗性越高,抑制作用越明显。

Table 5
表5
表5咯菌腈对代表性菌株甘油含量影响
Table 5Effect of fludioxonil on the glycerol content of representative strains (μg·mL^-1)

菌株编号 Strain code	未加咯菌腈No fludioxonil added	加入0.1 μg·mL-1咯菌腈1 mL Add 1 mL fludioxonil (0.1 μg·mL-1)
CDCZ-2	0.0067±0.0008f	0.0143±0.0001c
CDCZ-11	0.0108±0.0001d	0.0300±0.0012e
CDCZ-20	0.0058±0.0002b	0.0099±0.0001b
CDCZ-42	0.0070±0.0005bc	0.0109±0.0001b
CDCZ-43	0.0081±0.0002c	0.0096±0.0001b
CDPZ-8	0.0077±0.0015g	0.0246±0.0001d
YAHY-13	0.0025±0.0002a	0.0057±0.0001a
MYFC-10	0.0148±0.0003e	0.0155±0.0016c

Data are mean±SE. Different lowercases in the same column indicate significant difference at P<0.05 level
表中数据为平均数±标准误,同列数据后不同字母表示差异显著（P<0.05）

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2.5 代表性菌株Ⅲ型组氨酸激酶基因BOS1测序

将待测菌株进行培养,并提取DNA,对双组份组氨酸激酶基因BOS1（Broad Institute登录号：BC1G_00374）进行测序。由图2可知,不同突变位置对BOS1区域结构有不同程度的影响,F127S、I365N、I365S、V1136I、A1259T均处于BOS1区域结构的无规则卷曲,但只有TAR区域I365N和I365S与野生型区域结构有很大差异,其中I365N使365 aa附近下部无规则卷曲发生整体偏移,而I365S使365 aa附近下部和上部无规则卷曲都发生整体偏移;V287G处于α螺旋,且两者螺旋结构差异不大;将拼接后的DNA序列翻译氨基酸序列,然后进行比对,发现在6个待测菌株中有5种突变类型,3种变异菌株。所有抗性菌株均在TAR区域有突变,且低抗菌株YAHY-13、CDCZ-2以及中抗菌株CDCZ-42在HAMP区域亦发生突变;而中抗菌株CDCZ-20和高抗菌株MYFC-10和CDCZ-43在TAR、REC区域均有突变,但CDCZ-20菌株在TAR区域突变位点是I365N,而两株高抗菌株突变位点是I365S（图3、表6）。

图2

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图2BOS1蛋白不同突变部分三级结构图

绿色Green：没有突变位点的结构Structure without mutation site;红色Red：突变位点结构Structure of mutation site;1、3、5、8、10：野生型BOS1蛋白在125—233、267—347、289—628、1111—1228、1229—1310 aa区域的三级结构Tertiary structure of wild type BOS1 protein in 125-233, 267-347, 289-628, 1111-1228, 1229-1310 aa regions;2：BOS1蛋白^F127S在125—233 aa的三级结构Tertiary structure of BOS1 protein^F127S in 125-233 aa;4：BOS1蛋白^V287G在267—347 aa的三级结构Tertiary structure of BOS1 protein^V287G in 267-347 aa;6、7：BOS1蛋白^I365N及BOS1蛋白^I365S在289—628 aa的三级结构Tertiary structure of BOS1 protein^I365N and BOS1 protein^I365S in 289-628 aa;9：BOS1蛋白^V1136I在1111—1228 aa的三级结构Tertiary structure of BOS1 protein^V1136I in 1111-1228 aa;11：BOS1蛋白^A1259T在1229—1310 aa的三级结构Tertiary structure of BOS1 protein^A1259T in 1229-1310 aa
Fig. 2Tertiary structure diagram of different mutation parts of BOS1 protein

图3

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图3BOS1氨基酸序列对比

Fig. 3Sequence comparison of BOS1 amino acids



Table 6
表6
表6代表性菌株Ⅲ型组氨酸激酶基因BOS1测序结果
Table 6The sequencing results of type III histidine kinase gene BOS1 of representative strains

菌株编号 Strain code	抗性水平 Resistance level	突变点 Discontinuity	突变结构域 Mutant domain
YAHA-13	低抗 Low resistance	127 F-S 287 V-G 365 I-N	— HAMP TAR
CDCZ-2	低抗 Low resistance	365 I-S 287 V-G 1136 V-I (+) 1259 A-T	TAR HAMP REC —
CDCZ-42	中抗 Medium resistance	127 F-S 287 V-G 365 I-N	— HAMP TAR
CDCZ-20	中抗 Medium resistance	365 I-N 1136 V-I (+) 1259 A-T	TAR REC —
MYFC-10	高抗 High resistance	365 I-S 1136 V-I (+) 1259 A-T	TAR REC —
CDCZ-43	高抗 High resistance	365 I-S 1136 V-I (+) 1259 A-T	TAR REC —

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3 讨论

同种病害在不同区域对同一种药剂表现出不同的抗性频率和水平,与药剂使用历史和频率有关。本研究表明,四川省多个地区的草莓灰霉病菌对咯菌腈已产生不同程度的抗性,其抗性频率为31.92%,其中绵阳涪城区的高抗菌株比例最大,成都崇州、成都彭州、德阳广汉、眉山仁寿的中抗菌株比例亦达到7%以上,而成都双流和眉山东坡区的敏感菌株比例在80%以上。1984年,瑞士先正达作物保护有限公司通过修饰硝吡咯菌素合成咯菌腈,1996年被美国等国家允许登记使用,LI等^[27]对在2009—2012年间从美国、德国采集的11株接触过咯菌腈的灰霉病菌菌株进行抗性监测,发现2株低抗菌株（EC₅₀范围为0.16—0.26 μg·mL^-1）,2株中抗菌株（EC₅₀范围为0.32—0.38 μg·mL^-1）;LIU等^[28]从河南不同地点采集分离了274株番茄灰霉病菌,敏感性测定发现这些菌株对咯菌腈EC₅₀在0.0033—0.0415 μg·mL^-1（平均值为0.005 μg·mL^-1）,并通过对敏感野生型菌株用咯菌腈持续培养,获得3个咯菌腈抗性突变株;禾丽菲等^[29]2016年从山东济南、泰安、聊城、潍坊、莱芜及淄博等地分离了126株番茄叶霉病菌,采用菌丝生长速率法测定这些菌株的敏感基线,发现敏感基线上升至0.64 μg·mL^-1;SANG等^[30]在2015—2017年间从施用咯菌腈防治灰霉病的浙江部分区域草莓灰霉病果和病杆上分离了242株菌株,采用区分剂量法发现3株高抗菌株和13株低抗菌株。由此可见,病原菌在咯菌腈的长期选择压力下会导致抗性的产生。

本研究发现,在高渗作用下,与敏感菌株相比田间抗性菌株对渗透压的耐受能力增加,但是当浓度超过耐受范围后对渗透胁迫高度敏感,菌株抗性越高越敏感,抑制率越高;在药剂胁迫作用下,抗性菌株产生甘油增加量显著低于敏感菌株,且抗性越强,甘油增加量越少;这与REN等研究结果一致^[31]。LI等^[32]也发现与敏感菌核病菌菌株相比,药剂筛选、化学诱变及紫外诱变的抗性菌株对氯化钠和葡萄糖形成的高渗透压均敏感;ZHANG等^[33]研究发现,粗糙脉孢菌（OsS）突变体对高渗透压敏感,不能在含4% NaCl的培养基上生长,但OS突变体对苯吡咯类杀菌剂表现抗性;随后研究发现OsS编码一个与酿酒酵母HOG1同源丝裂原活化蛋白（MAP）激酶,突变体阻碍甘油在细胞内的积累从而导致抗性的产生;KOJIMA等^[34]通过基因缺失突变的方法,敲除HOG1后发现灰葡萄孢对咯菌腈的抗性增加;在酿酒酵母中,渗透感应蛋白组氨酸激酶Slnl通过磷酸传递到YPD1,响应调控蛋白Sskl,从而对HOG1途径起负调控作用,不会产生大量甘油;但在高渗作用下,Slnl的活性暂时受到抑制,引发HOG1的途径激活,并且表达渗透响应基因,产生大量的甘油,而细胞是通过甘油的合成来平衡细胞内外的压强以维持正常生长^[35,36]。

咯菌腈的作用靶标为Ⅲ型双组分组氨酸激酶,是酿酒酵母Slnl同源基因,通过抑制Ⅲ型组氨酸激酶的活性,产生大量的甘油使真菌细胞胀裂死亡;咯菌腈抗性菌株因为Ⅲ型组氨酸激酶基因的突变,使咯菌腈与靶标Ⅲ型组氨酸激酶亲和性下降而正常表达,从而维持细胞形态^[16]。本文通过对咯菌腈的靶标基因BOS1（BC1G_00374）测序,发现四川省的低抗及中抗菌株各一株在HAMP区域发生突变,BOS1的三维结构由α螺旋和β折叠组成跨膜结构,高抗菌株均在C端存在突变,敏感菌株和低抗菌株（CDCZ-2除外）在此位置不存在突变;CDCZ-2除了此位置的突变,还存在N段V287G的突变,另一株低抗菌株也存在N段V287G的突变;因此BOS1突变的位置以及方式与灰霉病菌菌株对咯菌腈的抗性水平存在必然的联系。REN等^[31]发现田间BOS1^G311R、BOS1^G265D、BOS1^N609T菌株以及BOS1^G545E对咯菌腈达到高抗水平;SANG等^[30]发现田间BOS1^{F127S, I365N, S426P}、BOS1^{G538R, A1259T}、BOS1^{R319K, V336M, D337N, V346I, A350S, Q369P, N373S}菌株以及BOS1^{G262S, Q369P, N373S}对咯菌腈均达到高抗水平,采用分子模拟对接评价不同突变对BOS1空间结构的影响,发现这些BOS1突变体与咯菌腈的亲和力比野生型BOS1亲和力显著降低;FILLINGER等^[37]利用HAMP结构建模发现BOS1的HAMP结构域中疏水残基的置换能够改变HAMP的螺旋结构,从而可能终止信号转导;氨基酸残基E529、T581或E692的突变加强信号转导能力,而不影响HAMP的螺旋结构。因此,下一步可采用Surflex-Dock分子模拟^[38]对接技术进行模拟或者评估本研究中所得抗性菌株BOS1与咯菌腈亲和性,明确其抗性机制。

2016年,尚岩^[39]报道四川、重庆、云南、湖北的15个地区桃、樱桃的灰霉病菌对咯菌腈抗性频率为0,由于咯菌腈与多菌灵、乙霉威、腐霉利、嘧霉胺和啶酰菌胺之间均不存在交互抗性^[40],可考虑与其他类型杀菌剂混用、交替轮换等方法来防治灰霉病,减缓抗性菌株产生的频率,做到合理、科学用药。

4 结论

四川省部分地区草莓灰霉病菌已对咯菌腈产生了抗性;与敏感菌株相比,田间抗性菌株对渗透压的耐受能力增加,但当浓度超过耐受范围后对渗透胁迫高度敏感;在高渗作用下,菌株抗性越高抑制率越高,说明抗性菌株对高渗敏感;在咯菌腈胁迫作用下,抗性菌株产生甘油增加量显著低于敏感菌株,且抗性越强,甘油增加量越少;BOS1突变的位置及方式与灰霉病菌菌株对咯菌腈的抗性水平存在必然联系。

（责任编辑 岳梅）

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[4] MYRESIOTIS C K, KARAOGLANIDIS G S, TZAVELLAKLONARI K . Resistance of Botrytis cinerea isolates from vegetable crops to anilinopyrimidine, phenylpyrrole, hydroxyanilide, benzimidazole, and dicarboximide fungicides.
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During February 2005, 55 single-spore isolates of Botrytis cinerea were collected at the end of the season from vegetable crops grown in 18 greenhouses on the island of Crete, Greece. They were tested for sensitivity to the anilinopyrimidine fungicides pyrimethanil and cyprodinil, the hydroxyanilide fungicide fenhexamid, the phenylpyrrole fungicide fludioxonil, the dicarboximide fungicide iprodione, and the benzimidazole fungicide carbendazim. Results of the study showed the existence of benzimidazole- and dicarboximide-resistant strains at frequencies of 61.8 and 18%, respectively. Moreover, for first time, the development of resistance to anilinopyrimidine fungicides by B. cinerea was detected in greenhouse vegetable crops on the island of Crete. High resistance frequencies of 49.1 and 57.4% were observed for pyrimethanil and cyprodinil, respectively. In addition, one isolate was found to be resistant to the hydroxyanilide fungicide fenhexamid, while no strains resistant to the phenylpyrrole fungicide were detected. Among the 55 isolates tested, 13 were resistant only to carbendazim, 6 were resistant only to anilinopyrimidines, 3 were resistant to both benzimidazoles and dicarboximides, 17 were resistant to both benzimidazoles and anilinopyrimidines, 6 were resistant to both dicarboximides and anilinopyrimidines, 1 was simultaneously resistant to benzimidazoles, dicarboximides, and anilinopyrimidines, 1 was resistant to both anilinopyrimidines and hydroxyanilides, and 8 were sensitive to all fungicides tested. A strong cross-resistance relationship was found between the two anilinopyrimidine fungicides tested when log transformed EC

[5] FERNáNDEZ-ORTU?O D, BRYSON P K, GRABKE A, SCHNABEL G . Monitoring for resistance in Botrytis cinerea from strawberry to seven chemical classes of fungicides in the eastern United States
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[6] SCHIRRA M, D’AQUINO S, PALMA A, MARCEDDU S, ANGIONI A, CABRAS P, SCHERM B, MIGHELI Q . Residue level, persistence, and storage performance of citrus fruit treated with fludioxonil
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DOI:10.1021/jf051004wURLPMID:16104790 [本文引用: 2]
Abstract The potential of postharvest dip treatments with fludioxonil (FLU) (a synthetic analogue of the bacterial metabolite of pyrrolnitrin), in controlling postharvest decay caused by Penicillium digitatum and Penicillium italicum of citrus fruit was investigated in comparison with the conventional fungicide imazalil (IMZ). The ultrastructural changes of fruit epicuticular wax was investigated as a function of water dip temperature, and the possible role of these changes was related to residue accumulation under FLU treatment. Residues retained by fruit were determined as a function of fungicide concentration, dip temperature, and fruit storage conditions. Scanning electron microscopy analysis revealed that fruit dipping in water at 30 or 40 degrees C did not cause differences in cuticular wax's ultrastructure in comparison to control fruit, while treatments at 50, 55, or 60 degrees C caused the disappearance of wax platelets, resulting in relatively homogeneous skin surface, due to partial "melting" of epicuticular wax. Residues of FLU in fruit treated at 20 or 50 degrees C were significantly correlated with the doses of fungicide applied. When equal amounts of fungicide were employed, the residue concentrations were notably higher (from 2.6- to 4-fold) in fruit treated at 50 degrees C than in fruit treated at 20 degrees C. The dissipation rate of FLU in "Salustiana" and "Tarocco" oranges was lower in fruit subjected to treatment at 50 degrees C. The minimal FLU concentration for almost complete decay control in artificially wounded fruit during 7-d storage at 20 degrees C was 400 mg/L active ingredient (ai) in fruit treated at 20 degrees C and 100 mg/L ai in fruit treated at 50 degrees C. Results on nonwounded Tarocco oranges subjected to 3 weeks of simulated quarantine conditions at 1 degrees C, plus 6 weeks of standard storage at 8 degrees C and an additional two weeks of simulated marketing period (SMP) at 20 degrees C revealed that almost complete decay control with FLU applications of 100 mg/L at 50 degrees C and 400 mg/L at 20 degrees C resulted in ca. 0.8 mg/kg FLU fruit residues, in agreement with results on wounded citrus fruit. When equal concentrations and temperatures were applied, FLU treatments were as effective as IMZ. In vitro trials showed a low sensitivity to FLU against P. digitatum and P. italicum isolates. MIC values for the complete inhibition of mycelium growth were >or=100 microg/mL, while ED(50) values ranged from 0.1 to 1 microg/mL for P. digitatum and from 1 to >100 microg/mL for P. italicum. The latter result suggests that care should be taken to avoid exclusive application of FLU in a sustainable program for management of fruit decay. However, integrating fungicide application and hot water dip may reduce the possibility of selecting fungicide-resistant populations of the pathogen, by increasing the effectiveness of the treatment.

[7] 乔广行, 严红, 么奕清, 黄金宝, 李兴红 . 北京地区番茄灰霉病菌的多重抗药性检测
植物保护, 2011,37(5):176-180.
DOI:10.3969/j.issn.0529-1542.2011.05.035URL [本文引用: 1]
From December of 2009 to May of 2010, 150 Botrytis cinerea samples were collected from 12 suburban counties in Beijing, and 109 isolates were purified from them. The isolates were detected of their multiple fungicide resistance to benzimidazole (carbendazim), dicarboximides (procymidone) and carbamate (diethofencarb) by minimal inhibitory concentration (MIC) method. The results showed that the percentages of carbendazim resistance (BenR), procymidone resistance (DicsR) and diethofencarb resistance (NPCR) were 96.3%, 80.7% and 58.7%, respectively, and the resistance types were BenRDicSNPCS, BenSDicSNPCR, BenRDicRNPCS and BenRDicRNPCR, accounting for 19.3%, 3.7%, 21.1% and 56.0%, respectively. These results suggested that benzimidazole (carbendazim), dicarboximides (procymidone) and carbamate (diethofencarb) resistances were common and severe in Beijing. It is necessary to use new types of fungicides or bio fungicides to control Botrytis cinerea.
QIAO G H, YAN H, YAO Y Q, HUANG J B, LI X H . Detection of multiple fungicide resistance in Botrytis cinerea from tomato in Beijing.
Plant Protection, 2011,37(5):176-180. (in Chinese)
DOI:10.3969/j.issn.0529-1542.2011.05.035URL [本文引用: 1]
From December of 2009 to May of 2010, 150 Botrytis cinerea samples were collected from 12 suburban counties in Beijing, and 109 isolates were purified from them. The isolates were detected of their multiple fungicide resistance to benzimidazole (carbendazim), dicarboximides (procymidone) and carbamate (diethofencarb) by minimal inhibitory concentration (MIC) method. The results showed that the percentages of carbendazim resistance (BenR), procymidone resistance (DicsR) and diethofencarb resistance (NPCR) were 96.3%, 80.7% and 58.7%, respectively, and the resistance types were BenRDicSNPCS, BenSDicSNPCR, BenRDicRNPCS and BenRDicRNPCR, accounting for 19.3%, 3.7%, 21.1% and 56.0%, respectively. These results suggested that benzimidazole (carbendazim), dicarboximides (procymidone) and carbamate (diethofencarb) resistances were common and severe in Beijing. It is necessary to use new types of fungicides or bio fungicides to control Botrytis cinerea.

[8] BARDAS G A, VELOUKAS T, KOUTITA O, KARAOGLANIDIS G S . Multiple resistance of Botrytis cinerea from kiwifruit to SDHIs, QoIs and fungicides of other chemical groups.
Pest Management Science, 2010,66(9):967-973.
DOI:10.1002/ps.1968URLPMID:20730988 [本文引用: 1]
BACKGROUND:Botrytis cinerea Pers.: Fr. is a high-risk pathogen for fungicide resistance development that has caused resistance problems on many crops throughout the world. This study investigated the fungicide sensitivity profile of isolates from kiwifruits originating from three Greek locations with different fungicide use histories. Sensitivity was measured by in vitro fungitoxicity tests on artificial nutrient media.RESULTS: Seventy-six single-spore isolates were tested for sensitivity to the SDHI fungicide boscalid, the QoI pyraclostrobin, the anilinopyrimidine cyprodinil, the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the dicarboxamide iprodione and the benzimidazole carbendazim. All isolates from Thessaloniki showed resistance to both boscalid and pyraclostrobin, while in the other two locations the fungal population was sensitive to these two fungicides. Sensitive isolates showed EC50 values to boscalid and pyraclostrobin ranging from 0.9 to 5.2 and from 0.04 to 0.14 mg L611 respectively, while the resistant isolates showed EC50 values higher than 50 mg L611 for boscalid and from 16 to > 50 mg L611 for pyraclostrobin. All QoI-resistant isolates carried the G143A mutation in cytb. Sensitivity determinations to the remaining fungicides revealed in total eight resistance phenotypes. No isolates were resistant to the fungicides fenhexamid and fludioxonil.CONCLUSION: This is the first report of B. cinerea field isolates with resistance to both boscalid and pyraclostrobin, and it strongly suggests that there may be a major problem in controlling this important pathogen on kiwifruit. Copyright 08 2010 Society of Chemical Industry

[9] 张玮, 乔广行, 黄金宝, 王忠跃, 李兴红 . 中国葡萄灰霉病菌对嘧霉胺的抗药性检测
中国农业科学, 2013,46(6):1208-1212.
DOI:10.3864/j.issn.0578-1752.2013.06.014URLMagsci [本文引用: 1]
【目的】检测中国葡萄灰霉病菌对苯胺基嘧啶类杀菌剂嘧霉胺的抗药性，明确中国不同葡萄产区灰霉病菌对嘧霉胺的抗药性及抗性频率，为葡萄灰霉病的药剂防治提供理论基础。【方法】从中国葡萄主产区采集、分离纯化104个葡萄灰霉病菌（Botrytis cinerea Pers.）的单孢菌株，采用菌丝生长速率法测定其对嘧霉胺的抗药性。【结果】中国葡萄灰霉病菌对嘧霉胺的抗性频率为22.22%&mdash;62.5%，且以高抗和中抗菌株为主，其中高抗菌株频率达44.23%；葡萄不同气候栽培区的灰霉病菌对嘧霉胺抗药性不同。【结论】中国葡萄灰霉病菌对嘧霉胺的抗药性较为普遍，且存在交互抗性，据此，在葡萄灰霉病的防治中应限制嘧霉胺的使用次数，可与二甲酰亚胺类、氨基甲酸酯类杀菌剂交替使用；引进防治葡萄灰霉病的新型杀菌剂或生物农药。
ZHANG W, QIAO G H, HUANG J B, WANG Z Y, LI X H . Evaluation on resistance of grape gray mold pathogen Botrytis cinerea to pyrimethanil in China.
Scientia Agricultura Sinica, 2013,46(6):1208-1212. (in Chinese)
DOI:10.3864/j.issn.0578-1752.2013.06.014URLMagsci [本文引用: 1]
【目的】检测中国葡萄灰霉病菌对苯胺基嘧啶类杀菌剂嘧霉胺的抗药性，明确中国不同葡萄产区灰霉病菌对嘧霉胺的抗药性及抗性频率，为葡萄灰霉病的药剂防治提供理论基础。【方法】从中国葡萄主产区采集、分离纯化104个葡萄灰霉病菌（Botrytis cinerea Pers.）的单孢菌株，采用菌丝生长速率法测定其对嘧霉胺的抗药性。【结果】中国葡萄灰霉病菌对嘧霉胺的抗性频率为22.22%&mdash;62.5%，且以高抗和中抗菌株为主，其中高抗菌株频率达44.23%；葡萄不同气候栽培区的灰霉病菌对嘧霉胺抗药性不同。【结论】中国葡萄灰霉病菌对嘧霉胺的抗药性较为普遍，且存在交互抗性，据此，在葡萄灰霉病的防治中应限制嘧霉胺的使用次数，可与二甲酰亚胺类、氨基甲酸酯类杀菌剂交替使用；引进防治葡萄灰霉病的新型杀菌剂或生物农药。

[10] 徐建强, 平忠良, 刘莹, 马世闯, 许道超, 杨岚, 郑伟, 刘圣明, 夏彦飞, 林晓民 . 咯菌腈对四种牡丹叶片病原真菌的抑制活性
中国农业科学, 2017,50(20):4036-4045.
DOI:10.3864/j.issn.0578-1752.2017.20.018URL [本文引用: 1]
[目的]探究咯菌腈对牡丹黑斑病菌（Alternaria suffruticosae）、黄斑病菌（Phyllosticta commonsii）、腔孢叶斑病菌（Hainesia lythri）和叶霉病菌（Cladosporium paeoniae）菌丝生长、孢子萌发、芽管伸长及产孢的抑制活性,分析咯菌腈在牡丹病害化学防治上的应用前景。[方法]采用菌丝生长速率法测定咯菌腈对菌丝生长的抑制活性,采用涂布平板法测定咯菌腈对孢子萌发、产孢时间及产孢量、芽管伸长及芽管和孢子形态的影响。[结果]咯菌腈对腔孢叶斑病菌菌丝生长的抑制作用最强,EC_（50）为0.01μg·mL~（-1）,其次为黑斑病菌和黄斑病菌,分别为0.07和0.35μg·mL~（-1）;咯菌腈对4种病菌的孢子萌发均有较强的抑制作用,对腔孢叶斑病菌的抑制作用最强,其EC_（50）为1.26μg·mL~（-1）,对其他3种病菌孢子萌发的EC_（50）在3.27—3.45μg·mL~（-1）;0.1μg·mL~（-1）咯菌腈对4种病菌分生孢子芽管伸长的抑制率在40%—70%,表明咯菌腈对4种病菌分生孢子芽管伸长均有明显的抑制作用,浓度越大抑制作用越强,各浓度间差异显著,其中对腔孢叶斑病菌芽管伸长抑制的EC_（50）为0.04μg·mL~（-1）,抑制作用最强,对其他3种病菌芽管伸长的EC_（50）在0.08—0.22μg·mL~（-1）;咯菌腈对黑斑病菌和黄斑病菌分生孢子和芽管的致畸作用强烈,可导致孢子及芽管膨大、过度分枝,而对叶霉病菌和腔孢叶斑病菌致畸作用较弱,芽管及孢子形态基本正常;咯菌腈可推迟黑斑病菌、黄斑病菌及叶霉病菌的产孢时间,对黑斑病菌产孢量的抑制作用最强,EC_（50）为0.05μg·mL~（-1）,对叶霉病菌次之,EC_（50）为0.38μg·mL~（-1）,但对腔孢叶斑病菌产孢有促进作用。[结论]咯菌腈对牡丹黑斑病菌及腔孢叶斑病菌的菌丝生长、孢子萌发及芽管伸长均有很强的抑制作用,但对腔孢叶斑产孢具有一定的促进作用;对叶霉病菌和黄斑病菌孢17
XU J Q, PING Z L, LIU Y, MA S C, XU D C, YANG L, ZHENG W, LIU S M, XIA Y F, LIN X M . Inhibitory activity of fludioxonil to four pathogenic fungi of peony leaves
Scientia Agricultura Sinica, 2017,50(20):4036-4045. (in Chinese)
DOI:10.3864/j.issn.0578-1752.2017.20.018URL [本文引用: 1]
[目的]探究咯菌腈对牡丹黑斑病菌（Alternaria suffruticosae）、黄斑病菌（Phyllosticta commonsii）、腔孢叶斑病菌（Hainesia lythri）和叶霉病菌（Cladosporium paeoniae）菌丝生长、孢子萌发、芽管伸长及产孢的抑制活性,分析咯菌腈在牡丹病害化学防治上的应用前景。[方法]采用菌丝生长速率法测定咯菌腈对菌丝生长的抑制活性,采用涂布平板法测定咯菌腈对孢子萌发、产孢时间及产孢量、芽管伸长及芽管和孢子形态的影响。[结果]咯菌腈对腔孢叶斑病菌菌丝生长的抑制作用最强,EC_（50）为0.01μg·mL~（-1）,其次为黑斑病菌和黄斑病菌,分别为0.07和0.35μg·mL~（-1）;咯菌腈对4种病菌的孢子萌发均有较强的抑制作用,对腔孢叶斑病菌的抑制作用最强,其EC_（50）为1.26μg·mL~（-1）,对其他3种病菌孢子萌发的EC_（50）在3.27—3.45μg·mL~（-1）;0.1μg·mL~（-1）咯菌腈对4种病菌分生孢子芽管伸长的抑制率在40%—70%,表明咯菌腈对4种病菌分生孢子芽管伸长均有明显的抑制作用,浓度越大抑制作用越强,各浓度间差异显著,其中对腔孢叶斑病菌芽管伸长抑制的EC_（50）为0.04μg·mL~（-1）,抑制作用最强,对其他3种病菌芽管伸长的EC_（50）在0.08—0.22μg·mL~（-1）;咯菌腈对黑斑病菌和黄斑病菌分生孢子和芽管的致畸作用强烈,可导致孢子及芽管膨大、过度分枝,而对叶霉病菌和腔孢叶斑病菌致畸作用较弱,芽管及孢子形态基本正常;咯菌腈可推迟黑斑病菌、黄斑病菌及叶霉病菌的产孢时间,对黑斑病菌产孢量的抑制作用最强,EC_（50）为0.05μg·mL~（-1）,对叶霉病菌次之,EC_（50）为0.38μg·mL~（-1）,但对腔孢叶斑病菌产孢有促进作用。[结论]咯菌腈对牡丹黑斑病菌及腔孢叶斑病菌的菌丝生长、孢子萌发及芽管伸长均有很强的抑制作用,但对腔孢叶斑产孢具有一定的促进作用;对叶霉病菌和黄斑病菌孢17

[11] FURUKAWA K, RANDHAWA A, KAUR H, MONDAL A K, HOHMANN S . Fungal fludioxonil sensitivity is diminished by a constitutively active form of the group III histidine kinase
FEBS Letters, 2012,586(16):2417-2422.
DOI:10.1016/j.febslet.2012.05.057URLPMID:22687241 [本文引用: 1]
The fungicide fludioxonil is used to control plant-pathogenic fungi by causing improper activation of the Hog1-type MAPK. However, the appearance of fludioxonil resistant mutants, mostly caused by mutations in the group III histidine kinases, poses a serious problem. Moreover, such mutations cause also hyperosmotic sensitivity and the underlying mechanism has been elusive for a long time. Using Saccharomyces cerevisiae as an experimental host, we show that those phenotypes are conferred by a constitutively active form of the group III histidine kinase. Our results explain the different reasons for fludioxonil resistance conferred by its deletion and missense mutation.

[12] LAWRY S M, TEBBETS B, KEAN I, STEWART D, HETELLE J, KLEIN B S . Fludioxonil induces Drk1, a fungal group III hybrid histidine kinase, to dephosphorylate its downstream target, Ypd1
Antimicrobial Agents and Chemotherapy, 2017,61(2):e01414-16.
DOI:10.1128/AAC.01414-16URLPMID:27872062 [本文引用: 1]
Abstract Novel antifungal drugs and targets are urgently needed. Group III hybrid histidine kinases (HHKs) represent an appealing new therapeutic drug target because they are widely expressed in fungi but absent from humans. We investigated the mode of action of the widely utilized, effective fungicide fludioxonil. The drug acts in an HHK-dependent manner by constitutive activation of the HOG (high-osmolarity glycerol) pathway, but its mechanism of action is poorly understood. Here, we report a new mode of drug action that entails conversion of the HHK from a kinase into a phosphatase. We expressed Drk1 (dimorphism-regulating kinase), which is an intracellular group III HHK from the fungal pathogen Blastomyces dermatitidis, in Saccharomyces cerevisiae Drk1 engendered drug sensitivity in B. dermatitidis and conferred sensitivity upon S. cerevisiae In response to fludioxonil, Drk1 behaved as a phosphatase rather than as a kinase, leading to dephosphorylation of its downstream target, Ypd1, constitutive activation of the HOG pathway, and yeast cell death. Aspartic acid residue 1140 in the Drk1 receiver domain was required for in vivo phosphatase activity on Ypd1, and Hog1 was required for drug effect, indicating fidelity in HHK-dependent drug action. In in vitro assays with purified protein, intact Drk1 demonstrated intrinsic kinase activity, and the Drk1 receiver domain exhibited intrinsic phosphatase activity. However, fludioxonil failed to induce intact Drk1 to dephosphorylate Ypd1. We conclude that fludioxonil treatment in vivo likely acts on an upstream target that triggers HHK to become a phosphatase, which dephosphorylates its downstream target, Ypd1.

[13] VIAUD M, FILLINGER S, LIU W, POLEPALLI JS, LE PêCHEUR P, KUNDURU AR, LEROUX P, LEGENDRE L . A class III histidine kinase acts as a novel virulence factor in Botrytis cinerea.
Molecular Plant-Microbe Interactions, 2006,19(9):1042-1050.
URL [本文引用: 1]

[14] SEGMüLLER N, ELLENDORF U, TUDZYNSKI B, TUDZYNSKI P . BcSAK1, a stress-activated mitogen-activated protein kinase, is involved in vegetative differentiation and pathogenicity in Botrytis cinerea.
Eukaryotic Cell, 2007,6(2):211-221.
DOI:10.1128/EC.00153-06URLPMID:1797955 [本文引用: 1]
The gene bcsak1, encoding a mitogen-activated protein kinase (MAPK) of Botrytis cinerea, was cloned and characterized. The protein has high homology to the yeast Hog1 and to corresponding MAPKs from filamentous fungi, but it shows unique functional features. The protein is phosphorylated under osmotic stress, specific fungicides, and oxidative stress mediated by H(2)O(2) and menadione. Northern blot analyses indicate that only a subset of typical oxidative stress response genes is regulated by BcSAK1. In contrast to most other fungal systems, Deltabcsak1 mutants are significantly impaired in vegetative and pathogenic development: they are blocked in conidia formation, show increased sclerotial development, and are unable to penetrate unwounded plant tissue. These data indicate that in B. cinerea the stress-activated MAPK cascade is involved in essential differentiation programs.

[15] LIU W, LEROUX P, FILLINGER S . The HOG1-like MAP kinase Sak1 of Botrytis cinerea is negatively regulated by the upstream histidine kinase Bos1 and is not involved in dicarboximide- and phenylpyrrole-resistance.
Fungal Genetics and Biology, 2008,45(7):1062-1074.
DOI:10.1016/j.fgb.2008.04.003URLPMID:18495505 [本文引用: 1]
In filamentous ascomycetes, HOG-like signal transduction cascades are involved in the resistance to hyper-osmotic conditions and to dicarboximides and phenylpyrroles. The histidine kinase (HK) Bos1 and the mitogen-activated protein kinase (MAPK) Sak1 are important for the adaptation to hyper-osmotic and oxidative stress, development, and pathogenicity in the phytopathogenic fungus Botrytis cinerea. However, bos1Δ and sak1Δ mutants created previously, also presented different phenotypes, especially the sak1Δ mutants were not resistant to high fungicide concentrations. Since both single mutants were constructed in different parental strains, phenotypic variations due to the genetic background might be suspected. In order to establish the relationship between both protein kinases, we analyzed Sak1 phosphorylation under the control of the Bos1 HK and we realized epistasis analysis between bos1Δ and sak1Δ mutations through the construction of isogenic single and double mutants. Our results show that Bos1 negatively regulates Sak1 phosphorylation and that Bos1 regulates certain phenotypes independently of Sak1. They include fungicide susceptibility, adaptation and conidiation on high neutral osmolarity.

[16] PARKINSON J S . Signaling mechanisms of HAMP domains in chemoreceptors and sensor kinases
Annual Review of Microbiology, 2010,64:101-122.
DOI:10.1146/annurev.micro.112408.134215URLPMID:20690824 [本文引用: 2]
HAMP domains mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and some phosphatases. HAMP subunits have two 16-residue amphiphilic helices (AS1, AS2) joined by a 14- to 15-residue connector segment. Two alternative HAMP structures in these homodimeric signaling proteins have been described: HAMP(A), a tightly packed, parallel, four-helix bundle; and HAMP(B), a more loosely packed bundle with an altered AS2/AS2' packing arrangement. Stimulus-induced conformational changes probably modulate HAMP signaling by shifting the relative stabilities of these opposing structural states. Changes in AS2/AS2' packing, in turn, modulate output signals by altering structural interactions between output helices through heptad repeat stutters that produce packing phase clashes. Output helices that are too tightly or too loosely packed most likely produce kinase-off output states, whereas kinase-on states require an intermediate range of HAMP stabilities and dynamic behaviors. A three-state, dynamic bundle signaling model best accounts for the signaling properties of chemoreceptor mutants and may apply to other transducers as well.

[17] AIROLA M V, WATTS K J, BILWES A M, CRANE B R . Structure of concatenated HAMP domains provides a mechanism for signal transduction
Structure, 2010,18(4):436-448.
DOI:10.1016/j.str.2010.01.013URLPMID:2892831 [本文引用: 1]
78 Presents the first poly-HAMP structure and identifies a novel HAMP domain conformation 78 HAMP conformations differ by changes in helical register, rotation, and crossing angle 78 Proposes a new signal transduction model consistent with known signal inputs to HAMP 78 Provides an output mechanism for HAMP domains involving stutter compensation

[18] VIGNUTELLI A, HIBER-BODMER M, HIBER U W . Genetic analysis of resistance to the phenylpyrrole fludioxonil and the dicarboximide vinclozolin in Botryotinia fuckeliana (Botrytis cinerea).
Mycological Research, 2002,106(3):329-335.
[本文引用: 1]

[19] FERNáNDEZORTU?O D, BRYSON P K, GRABKE A, SCHNABEL G . First report of fludioxonil resistance in Botrytis cinerea from a strawberry field in Virginia.
Plant Disease, 2013,97(6):848-849.
DOI:10.1094/PDIS-01-13-0012-PDNURL [本文引用: 1]
In 2012, a total of 790 strains of were collected from 76 strawberry fields in Arkansas, Florida, Georgia, Kansas, Maryland, North Carolina, South Carolina and Virginia, USA. Strains were collected from sporulating flowers and fruits, and sensitivity to fludioxonil was determined using a conidial germination assay. Only one isolate from a farm in Westmoreland County, Virginia, grew on medium amended with 0.1 08g fludioxonil/ml, indicating a low level of resistance. The isolate did not grow on 10 08g fludioxonil/ml. All other 789 isolates did not grow at either of the two doses. This assay was repeated twice with a single-spore culture of the same strain. In both cases, residual growth was observed on the medium containing 0.1 08g fludioxonil/ml. To assess resistance , ripe strawberry fruits were inoculated with either water or 2.5 ml fludioxonil/litre to runoff using a hand mister. The sensitive and low-resistance (LR) isolates were indistinguishable in pathogenicity on detached, unsprayed fruits. The LR isolate induced grey mold disease on all treated and untreated fruits (100% disease incidence). The sensitive isolates only developed disease on untreated fruits. The ECvalues determined in microtitre assays with concentrations of 0.01, 0.03, 0.1, 0.3, 1.0, 3.0 and 10.0 08g fludioxonil/ml were 0.01 08g fludioxonil/ml for the sensitive isolates and 0.26 08g fludioxonil/ml for the resistant isolate. This is thought to be the first report of fludioxonil resistance in from strawberry in North America.

[20] 武东霞 . 灰葡萄孢菌(Botrytis cinereal)对苯噻菌酯和咯菌睛的抗药性风险研究
[D]. 南京: 南京农业大学, 2015.
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WU D X . Resistance risk for benzothiostrobin and fludioxonil against Botrytis cinerea
[D]. Nanjing: Nanjing Agricultural University, 2015. (in Chinese)
[本文引用: 2]

[21] YOSHIDA H, ANO H, ISHIDA C, TANIGAWA N, KIKUI M, TAKASHIMA T, TSUYUGUCHI I . A study of INH 0.1 microgram/ml resistant M. tuberculosis strains assessed by BrothMIC MTB-1 method.
Kekkaku(Tuberculosis), 2002,77(7):533-535.
URLPMID:12187818 [本文引用: 1]
In the antimycobacterial susceptibility test for INH using the egg-based Ogawa media, 3 concentrations (0.1, 1, or 5 micrograms/ml) of INH were used, and 1 microgram/ml was used as a critical concentration for INH resistance. However, it was controversial whether INH 0.1 microgram/ml resistant M. tuberculosis was clinically significant or not. We investigated the MIC values of INH 0.1 microgram/ml resistant strains by using BrothMIC MTB-1 method, and 115 strains of M. tuberculosis confirmed by DNA-prove test were used. The distribution of MIC values of 115 strains determined by Ogawa INH susceptibility test was shown in figure. By BrothMIC MTB-1 method, they were classified into 3 groups; susceptible, low resistant and high resistant groups. The mean MIC value of INH 0.1 microgram/ml resistant M. tuberculosis was estimated to be 4.53 micrograms/ml with its 95% confidence interval 3.21-5.85 micrograms/ml, and they were determined as "resistant" in BrothMIC MTB-1 method. These results supported the idea that patients with INH 0.1 microgram/ml resistant M. tuberculosis strains should be regarded as clinically "resistant".

[22] 慕立义 . 植物化学保护研究方法. 北京: 中国农业出版社, 1994.
[本文引用: 2]

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[本文引用: 2]

[23] 赵建江, 张小风, 马志强, 王文桥, 韩秀英 . 番茄灰霉病菌对咯菌腈的敏感基线及其与不同杀菌剂的交互抗性
农药, 2013,52(9):684-685.
URL [本文引用: 1]
【目的】明确番茄灰霉病菌对咯菌腈的敏感性及其与其他杀菌剂之间是否存在交互抗性。【方法】采用菌丝生长速率法测定灰霉病菌对咯菌腈、多菌灵、乙霉威、腐霉利、嘧霉胺和啶酰菌胺的敏感性。【结果】咯菌腈对106株野生菌株EC50的平均值为（0．0135±0．0096）mg／L。咯菌腈与其他5种杀菌剂之间均不存在交互抗性。【结论】野生菌株对咯菌腈的敏感性的平均值，可作为番茄灰霉病菌对咯菌腈的敏感基线。咯菌腈可广泛用于番茄灰霉病的防治。
ZHAO J J, ZHANG X F, MA Z Q, WANG W Q, HAN X Y . Baseline-sensitivity of Botrytis cinerea on tomato to fludioxonil and cross-resistance against diverse fungicides.
Agrochemicals, 2013,52(9):684-685. (in Chinese)
URL [本文引用: 1]
【目的】明确番茄灰霉病菌对咯菌腈的敏感性及其与其他杀菌剂之间是否存在交互抗性。【方法】采用菌丝生长速率法测定灰霉病菌对咯菌腈、多菌灵、乙霉威、腐霉利、嘧霉胺和啶酰菌胺的敏感性。【结果】咯菌腈对106株野生菌株EC50的平均值为（0．0135±0．0096）mg／L。咯菌腈与其他5种杀菌剂之间均不存在交互抗性。【结论】野生菌株对咯菌腈的敏感性的平均值，可作为番茄灰霉病菌对咯菌腈的敏感基线。咯菌腈可广泛用于番茄灰霉病的防治。

[24] 仇骏, 王大兵, 黄得庆 . 甘油铜比色法测定水中甘油的含量方法研究
中国化工贸易, 2014,30(6):140.
DOI:10.3969/j.issn.1674-5167.2014.30.125URL [本文引用: 1]
利用甘油与铜离子在碱性溶液中生成深蓝色络合物(甘油铜)，该络合物在一定波长下存在最大吸光度的特点，建立了比色法测定甘油含量的方法。结果表明，最佳测定条件为：取CuS04溶液(0.05 g/mL)1 mL与碱液(0.05g/mL)3.5 mL，摇匀，加入处理后的样品，振荡12 min，过滤，然后在波长630 nnl处测定吸光度。实测显示，所建立的测定方法操作简单、速度快。
QIU J, WANG D B, HUANG D Q . Determination of glycerin in water by glycerol copper colorimetric method
China Chemical Trade, 2014,30(6):140. (in Chinese)
DOI:10.3969/j.issn.1674-5167.2014.30.125URL [本文引用: 1]
利用甘油与铜离子在碱性溶液中生成深蓝色络合物(甘油铜)，该络合物在一定波长下存在最大吸光度的特点，建立了比色法测定甘油含量的方法。结果表明，最佳测定条件为：取CuS04溶液(0.05 g/mL)1 mL与碱液(0.05g/mL)3.5 mL，摇匀，加入处理后的样品，振荡12 min，过滤，然后在波长630 nnl处测定吸光度。实测显示，所建立的测定方法操作简单、速度快。

[25] DUAN Y B, GE C G, LIU S G, CHEN C J, ZHOU M G . Effect of phenylpyrrole fungicide fludioxonil on morphological and physiological characteristics of Sclerotinia sclerotiorum.
Pesticide Biochemistry and Physiology, 2013,106(1/2):61-67.
DOI:10.1016/j.pestbp.2013.04.004URL [本文引用: 1]
In this study, the effect of fludioxonil on morphological and physiological characteristics of Sclerotinia sclerotiorum has been investigated. The results indicated that fludioxonil had a strong inhibition on mycelia growth of S. sclerotiorum. After fludioxonil treatment, cell membrane permeability, glycerol content, POD and PAL activity increased markedly, but oxalate and EPS content significantly decreased. The protective and curative test of fludioxonil suggested that protective effect was better than curative either on leaves or on stems of oilseed rape. These results will increase our understanding of action mechanism of fludioxonil against S. sclerotiorum.

[26] ROBERT X, GOUET P . Deciphering key features in protein structures with the new ENDscript server
Nucleic Acids Research, 2014,42(Web Server Issue):W320-W324.
DOI:10.1093/nar/gku316URLPMID:24753421 [本文引用: 1]
Abstract ENDscript 2 is a friendly Web server for extracting and rendering a comprehensive analysis of primary to quaternary protein structure information in an automated way. This major upgrade has been fully re-engineered to enhance speed, accuracy and usability with interactive 3D visualization. It takes advantage of the new version 3 of ESPript, our well-known sequence alignment renderer, improved to handle a large number of data with reduced computation time. From a single PDB entry or file, ENDscript produces high quality figures displaying multiple sequence alignment of proteins homologous to the query, colored according to residue conservation. Furthermore, the experimental secondary structure elements and a detailed set of relevant biophysical and structural data are depicted. All this information and more are now mapped on interactive 3D PyMOL representations. Thanks to its adaptive and rigorous algorithm, beginner to expert users can modify settings to fine-tune ENDscript to their needs. ENDscript has also been upgraded as an open platform for the visualization of multiple biochemical and structural data coming from external biotool Web servers, with both 2D and 3D representations. ENDscript 2 and ESPript 3 are freely available at http://endscript.ibcp.fr and http://espript.ibcp.fr, respectively. The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

[27] LI X, FERNáNDEZ-ORTU?O D, GRABKE A, SCHNABEL G . Resistance to fludioxonil in Botrytis cinerea isolates from blackberry and strawberry.
Phytopathology, 2014,104(7):724-732.
DOI:10.1094/PHYTO-11-13-0308-RURLPMID:24423402 [本文引用: 1]
Site-specific fungicides, including the phenylpyrrole fludioxonil, are frequently used for gray mold control but are at risk for the development of resistance. In this study, field isolates that were low-resistant (LR) and moderately resistant (MR) to fludioxonil from blackberry and strawberry fields of North Carolina, South Carolina, and Virginia were characterized. Genes involved in osmoregulation, including bcsak1, BcOS4, bos5, and BRRG-1, were cloned and sequenced to detect potential target gene alterations; however, none were found. A previously described mutation (R632I) in transcription factor Mrr1, which is known to increase the expression of ATP-binding cassette transporter AtrB, was found in MR but not in sensitive (S) or LR isolates. Expression of atrB in MR isolates was 200-fold increased compared with an S isolate; however, 30- to 100-fold overexpression was also detected in LR isolates. Both MR isolates exhibited increased sensitivity to salt stress in the form of mycelial growth inhibition at 4% NaCl, indicating a disruption of osmoregulatory processes in those strains. However, the glycerol content was indistinguishable between S, LR, and MR isolates with and without exposure to fludioxonil, suggesting that the glycerol synthesis pathway may not be a part of the resistance mechanism in LR or MR strains. An investigation into the origin of LR and MR isolates from blackberry revealed two insertions in the mrr1 gene consistent with those found in the Botrytis clade group S. The emergence of strains overexpressing atrB in European and now in North American strawberry fields underscores the importance of this resistance mechanism for development of resistance to fludioxonil in Botrytis cinerea.

[28] LIU S, HAI F, JIANG J . Sensitivity to fludioxonil of Botrytis cinerea isolates from tomato in Henan Province of China and characterizations of fludioxonil-resistant mutants.
Journal of Phytopathology, 2017,165(2):98-104.
DOI:10.1111/jph.12542URL [本文引用: 1]
Abstract Grey mould, caused by Botrytis cinerea Pers ex Fr., is one of the most common diseases of tomato worldwide. Fludioxonil belongs to the phenylpyrrole fungicides, which have high activity against B. cinerea. The sensitivity of fludioxonil was evaluated on the basis of the level of inhibition of mycelium growth in 274 B. cinerea isolates collected from different locations (untreated with this fungicide) in Henan Province, China. The EC50 values for fludioxonil ranged from 0.0033 to 0.0415 mg/l, and the average EC50 values were 0.0156 卤 0.0078 mg/l. Three fludioxonil-resistant mutants were obtained by subculturing fludioxonil-sensitive wild-type isolates on continuously increasing fludioxonil concentrations. For the cross-resistance assay, fludioxonil revealed positive cross-resistance with procymidone but did not reveal cross-resistance with pyrimethanil, boscalid and trifloxystrobin. Mycelial growth, conidial production, hyphal dry weight and pathogenicity were diminished significantly between the fludioxonil-resistant mutants and their sensitive wild-type parental isolates. This study shows for the first time that fludioxonil-resistant isolates of B. cinerea are still not present in Henan Province because this fungicide is an attractive and effective fungicide for chemical control. Recommendations can be made to growers to use fludioxonil to control grey mould and to consider the potential moderate resistance risk of using this fungicide.

[29] 禾丽菲, 陈乐乐, 肖斌, 赵时峰, 李秀环, 慕卫, 刘峰 . 番茄叶霉病菌对咯菌腈敏感基线的建立及田间防治效果评价
中国农业科学, 2018,51(8):1475-1483.
URL [本文引用: 1]
【目的】番茄叶霉病是危害温室番茄的重要病害之一,其致病菌对常用杀菌剂已产生不同程度的抗性,亟待开发高效替代药剂。研究旨在探索番茄叶霉病菌(Fulvia fulva)对咯菌腈的敏感性,建立敏感性基线,明确咯菌腈在田间的防治效果。【方法】在山东济南、泰安、聊城、潍坊、莱芜、淄博6个蔬菜产区采集感叶霉病的番茄叶片,经分离纯化后,培养得到126株番茄叶霉菌株;采用菌丝生长速率法和孢子萌发法,测定咯菌腈对3株代表菌株菌丝生长、分生孢子萌发和芽管伸长3个生长发育阶段的抑制活性;利用菌丝生长速率法测定不同地区的126株菌株对咯菌腈的敏感性,并建立敏感性基线。通过两年的田间试验,评价咯菌腈的保护作用和治疗作用。【结果】咯菌腈对番茄叶霉病菌芽管伸长和菌丝生长均有较强的抑制活性,平均EC50值分别为0.30和0.80μg·m L~(-1),而对孢子萌发抑制作用较弱,平均EC50值均100μg·m L~(-1)。不同地区番茄叶霉病菌群体间对咯菌腈的敏感性均无显著差异。其中,潍坊地区叶霉菌株对咯菌腈敏感性最高,平均EC50值为0.43μg·m L~(-1);淄博地区叶霉菌株敏感性相对较低,平均EC50值为0.79μg·m L~(-1)。泰安、济南、聊城、莱芜地区的叶霉菌株敏感性无显著差异,平均EC50值分别为0.65、0.75、0.71、0.58μg·m L~(-1)。咯菌腈对供试菌株菌丝生长的EC50值在0.16—1.69μg·m L~(-1),平均值为0.64μg·m L~(-1),敏感性频率呈单峰曲线分布,符合正态分布,可作为番茄叶霉病菌对咯菌腈的敏感性基线。2016—2017年田间试验表明,咯菌腈对番茄叶霉病均具有较好的防治效果,咯菌腈在60.75 g a.i./hm~2剂量下对番茄叶霉病保护作用和治疗作用防治效果分别为72.21%—75.02%和61.94%—70.65%,均显著高于对照药剂苯醚甲环唑100 g a.i./hm~2、代森锰锌700 g a.i./hm~2和甲基硫菌灵540 g a.i./hm~2,与氟吡菌酰胺150 g a.i./hm~2防治效果差异不显著;咯菌腈有效成分为40.50 g a.i./hm~2时对番茄叶霉病保护作用和治疗作用的防治效果分别高于对照药剂甲基硫菌灵540 g a.i./hm~2的保护和治疗防治效果。2017年咯菌腈有效成分为20.25 g a.i./hm~2时对番茄叶霉病保护作用和治疗作用的防治效果分别显著高于对照药剂代森锰锌700 g a.i./hm~2和甲基硫菌灵540 g a.i./hm~2的保护作用和治疗作用的防治效果。咯菌腈对番茄叶霉病的保护作用均高于治疗作用。【结论】咯菌腈对番茄叶霉病菌菌丝生长和芽管伸长具有显著的抑制作用,而对孢子萌发基本无抑制作用,山东省6个蔬菜产区番茄叶霉病菌对咯菌腈相对比较敏感。该药剂在番茄叶霉病的田间防治中具有一定的应用潜力。
HE L F, CHEN L L, XIAO B, ZHAO S F, LI X H, MU W, LIU F . Establishment of sensitivity baseline and evaluation of field control efficacy of fludioxonil against Fulvia fulva.
Scientia Agricultura Sinica, 2018,51(8):1475-1483. (in Chinese)
URL [本文引用: 1]
【目的】番茄叶霉病是危害温室番茄的重要病害之一,其致病菌对常用杀菌剂已产生不同程度的抗性,亟待开发高效替代药剂。研究旨在探索番茄叶霉病菌(Fulvia fulva)对咯菌腈的敏感性,建立敏感性基线,明确咯菌腈在田间的防治效果。【方法】在山东济南、泰安、聊城、潍坊、莱芜、淄博6个蔬菜产区采集感叶霉病的番茄叶片,经分离纯化后,培养得到126株番茄叶霉菌株;采用菌丝生长速率法和孢子萌发法,测定咯菌腈对3株代表菌株菌丝生长、分生孢子萌发和芽管伸长3个生长发育阶段的抑制活性;利用菌丝生长速率法测定不同地区的126株菌株对咯菌腈的敏感性,并建立敏感性基线。通过两年的田间试验,评价咯菌腈的保护作用和治疗作用。【结果】咯菌腈对番茄叶霉病菌芽管伸长和菌丝生长均有较强的抑制活性,平均EC50值分别为0.30和0.80μg·m L~(-1),而对孢子萌发抑制作用较弱,平均EC50值均100μg·m L~(-1)。不同地区番茄叶霉病菌群体间对咯菌腈的敏感性均无显著差异。其中,潍坊地区叶霉菌株对咯菌腈敏感性最高,平均EC50值为0.43μg·m L~(-1);淄博地区叶霉菌株敏感性相对较低,平均EC50值为0.79μg·m L~(-1)。泰安、济南、聊城、莱芜地区的叶霉菌株敏感性无显著差异,平均EC50值分别为0.65、0.75、0.71、0.58μg·m L~(-1)。咯菌腈对供试菌株菌丝生长的EC50值在0.16—1.69μg·m L~(-1),平均值为0.64μg·m L~(-1),敏感性频率呈单峰曲线分布,符合正态分布,可作为番茄叶霉病菌对咯菌腈的敏感性基线。2016—2017年田间试验表明,咯菌腈对番茄叶霉病均具有较好的防治效果,咯菌腈在60.75 g a.i./hm~2剂量下对番茄叶霉病保护作用和治疗作用防治效果分别为72.21%—75.02%和61.94%—70.65%,均显著高于对照药剂苯醚甲环唑100 g a.i./hm~2、代森锰锌700 g a.i./hm~2和甲基硫菌灵540 g a.i./hm~2,与氟吡菌酰胺150 g a.i./hm~2防治效果差异不显著;咯菌腈有效成分为40.50 g a.i./hm~2时对番茄叶霉病保护作用和治疗作用的防治效果分别高于对照药剂甲基硫菌灵540 g a.i./hm~2的保护和治疗防治效果。2017年咯菌腈有效成分为20.25 g a.i./hm~2时对番茄叶霉病保护作用和治疗作用的防治效果分别显著高于对照药剂代森锰锌700 g a.i./hm~2和甲基硫菌灵540 g a.i./hm~2的保护作用和治疗作用的防治效果。咯菌腈对番茄叶霉病的保护作用均高于治疗作用。【结论】咯菌腈对番茄叶霉病菌菌丝生长和芽管伸长具有显著的抑制作用,而对孢子萌发基本无抑制作用,山东省6个蔬菜产区番茄叶霉病菌对咯菌腈相对比较敏感。该药剂在番茄叶霉病的田间防治中具有一定的应用潜力。

[30] SANG C, REN W, WANG J, XU C, ZHANG Z H, ZHOU M G, CHEN C J, WANG K . Detection and fitness comparison of target-based highly fludioxonil-resistant isolates of Botrytis cinerea, from strawberry and cucumber in China.
Pesticide Biochemistry and Physiology, 2018,147:110-118.
DOI:10.1016/j.pestbp.2018.01.012URLPMID:29933980 [本文引用: 2]
Botrytis cinerea has a high risk of developing resistance to fungicides. Fludioxonil, belonging to phenylpyrroles, has been used for more than three decades, however, only few cases of field resistance against phenylpyrroles have been reported. In this study, the highly fludioxonil-resistant (HR) isolates of Botrytis cinerea were firstly detected in the commercial greenhouses of strawberry in China in 2015, and biochemical characterization differences in high fludioxonil-resistance from strawberry and cucumber were compared. All of the five HR isolates from greenhouses of strawberry and cucumber could grow on PDA amended with 10062μg/mL fludioxonil, and exhibited a positive correlation between the resistance of dicarboximide fungicides and fludioxonil. Sporulation and sclerotium production of the strawberry-originated HR isolates were increased in comparison with the cucumber-originated HR isolates. No matter how the HR isolates were from strawberry and cucumber, all the HR isolates showed enhanced sensitivity to the osmotic agents, but with significant difference. Based on sequence alignment of the BcOS1 which codes protein bound by fludioxonil, two genotypes of the strawberry-originated HR isolates were observed, i.e., (F127S62+62I365N62+62S426P) and (G538R62+62A1259T), which were totally different from those of the cucumber-originated HR isolates. Molecular docking of fludioxonil to the binding site of BcOS1 protein from the five HR isolates illustrated that all the HR isolates had less affinity than the sensitive isolates. Our data indicated that genotypes of the HR isolates match the corresponding fludioxonil-selection pressure on the field populations of B. cinerea in the commercial greenhouses of the two host plants.

[31] REN W C, SHAO W Y, HAN X, ZHOU M G, CHEN C J . Molecular and biochemical characterization of laboratory and field mutants of Botrytis cinerea resistant to fludioxonil.
Plant Disease, 2016,100(7):1414-1423.
DOI:10.1094/PDIS-11-15-1290-REURL [本文引用: 2]
Abstract Botrytis cinerea is a filamentous phytopathogen with a high risk of developing resistance to fungicides. The phenylpyrrole fungicide fludioxonil has been reported to have excellent activity against B. cinerea and increasingly has been applied to control gray mold in China. In this study, molecular and biochemical characteristics of laboratory and field mutants of B. cinerea resistant to fludioxonil has been investigated. During 2012 to 2014, B. cinerea isolates collected from Jiangsu and Shandong Provinces in China were tested in vitro for sensitivity to fungicides commonly used to suppress gray mold of cucumber and tomato. Among the 75 isolates collected from cucumber in 2013, two were highly resistant (HR) to fludioxonil. Of the 308 isolates collected from tomato in 2014, four were fludioxonil-HR. This was the first time that B. cinerea isolates HR to fludioxonil had been detected in the field. Six fludioxonil-resistant mutants were obtained in the laboratory by selection on fungicide-amended media. These mutants exhibited stable resistance to fludioxonil, as indicated by resistance factor values that ranged from 34.38 to >10,000. In comparison with fludioxonil-sensitive isolates of B. cinerea, all field and laboratory mutants showed reduced fitness, as defined by mycelial growth, sporulation, virulence, and sensitivity to osmotic stress. When treated with fludioxonil at 1 mu g/ml, sensitive isolates showed increased glycerol contents in mycelium and expression levels of BchogI, while levels in field and laboratory HR mutants increased only slightly. Sequences of the Bos1 gene of field and laboratory fludioxonil-HR mutants showed that mutations in field mutants were located in the histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase (HAMP) domains of the N-terminal region, whereas mutations in the laboratory mutants were distributed in HAMP domains or in the HATPase_c domain of the C-terminal region. These results will enhance our understanding of the resistance mechanism of B. cinerea to fludioxonil.

[32] LI J L, WU F C, ZHU F X . Fitness is recovered with the decline of dimethachlon resistance in laboratory-induced mutants of Sclerotinia sclerotiorum after long-term cold storage.
The Plant Pathology Journal, 2015,31(3):305-309.
DOI:10.5423/PPJ.OA.04.2015.0066URLPMID:4564156 [本文引用: 1]
After four years of cold storage, dimethachlon resistance of two laboratory-induced resistant Sclerotinia sclerotiorum isolates SCG7 and LA50 declined by 99.5% and 98.9%, respectively, and cross resistance to iprodione and procymidone also declined dramatically. Along with the decline of fungicide resistance, osmotic sensitivity to sodium chloride and glucose decreased tremendously; mycelial growth rate, sclerotia number and weight per potato dextrose agar (PDA) plate increased on average by 118.6%, 85. 5% and 64.5%, respectively; and virulence to detached leaves of oilseed rape increased by 72.7% on average. Significant negative correlations were detected between dimethachlon resistance levels and mycelial growth rate on PDA (r = 610.980, P = 0.021), and between resistance levels and lesion diameters on detached leaves of oilseed rape plants (r = 610.997, P = 0.002). These results have profound implications for assessing the potential risk for resistance development to dicarboximide fungicides in S. sclerotiorum.

[33] ZHANG Y, LAMM R, PILLONEL C, LAM S, XU J R . Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue.
Applied and Environmental Microbiology, 2002,68(2):532-538.
DOI:10.1128/AEM.68.2.532-538.2002URLPMID:11823187 [本文引用: 1]
Abstract Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway.

[34] KOJIMA K, TAKANO Y, YOSHIMI A, TANAKA C, KIKUCHI T, OKUNO T . Fungicide activity through activation of a fungal signalling pathway
Molecular Microbiology, 2004,53(6):1785-1796.
DOI:10.1111/j.1365-2958.2004.04244.xURLPMID:15341655 [本文引用: 1]
Summary Fungicides generally inhibit enzymatic reactions involved in fungal cellular biosynthesis. Here we report, for the first time, an example of fungicidal effects through hyperactivation of a fungal signal transduction pathway. The OSC1 gene, encoding a MAP kinase (MAPK) related to yeast Hog1, was isolated from the fungal pathogen Colletotrichum lagenarium that causes cucumber anthracnose. The osc1 knockout mutants were sensitive to high osmotic stress and showed increased resistance to the fungicide fludioxonil, indicating that Osc1 is involved in responses to hyperosmotic stress and sensitivity to fludioxonil. The Osc1 MAPK is phosphorylated under high osmotic conditions, indicating activation of Osc1 by high osmotic stress. Importantly, fludioxonil treatment also activates phosphorylation of Osc1, suggesting that improper activation of Osc1 by fludioxonil has negative effects on fungal growth. In the presence of fludioxonil, the wild-type fungus was not able to infect the host plant because of a failure of appressorium-mediated penetration, whereas osc1 mutants successfully infected plants. Analysis using a OSC1- GFP fusion gene indicated that Osc1 is rapidly translocated to the nucleus in appressorial cells after the addition of fludioxonil, suggesting that fludioxonil impairs the function of infection structures by activation of Osc1. Furthermore, fludioxonil activates Hog1-type MAPKs in the plant pathogenic fungi Cochliobolus heterostrophus and Botrytis cinerea . These results strongly suggest that fludioxonil acts as a fungicide, in part, through activation of the MAPK cascade in fungal pathogens.

[35] HOHMANN S . Osmotic stress signaling and osmoadaptation in yeasts
Microbiology and Molecular Biology Reviews, 2002,66(2):300-372.
DOI:10.1128/MMBR.66.2.300-372.2002URL [本文引用: 1]

[36] CHEN R E, THOMER J . Function and regulation in MAPK signaling pathways: lessons learned from the yeast Saccharomyces cerevisae.
Biochimica et Biophysica Acta, 2007,1773(8):1311-1340.
[本文引用: 1]

[37] FILLINGER S, AJOUZ S, NICOT P C, LEROUX P, BARDIN M . Functional and structural comparison of pyrrolnitrin- and iprodione- induced modifications in the class III histidine-kinase Bos1 of Botrytis cinerea.
PLoS ONE, 2012,7(8):e42520.
DOI:10.1371/journal.pone.0042520URLPMID:22912706 [本文引用: 1]
Dicarboximides and phenylpyrroles are commonly used fungicides against plant pathogenic ascomycetes. Although their effect on fungal osmosensing systems has been shown in many studies, their modes-of-action still remain unclear. Laboratory- or field-mutants of fungi resistant to either or both fungicide categories generally harbour point mutations in the sensor histidine kinase of the osmotic signal transduction cascade. In the present study we compared the mechanisms of resistance to the dicarboximide iprodione and to pyrrolnitrin, a structural analogue of phenylpyrrole fungicides, in Botrytis cinerea. Pyrrolnitrin-induced mutants and iprodione-induced mutants of B. cinerea were produced in vitro. For the pyrrolnitrin-induced mutants, a high level of resistance to pyrrolnitrin was associated with a high level of resistance to iprodione. For the iprodione-induced mutants, the high level of resistance to iprodione generated variable levels of resistance to pyrrolnitrin and phenylpyrroles. All selected mutants showed hypersensitivity to high osmolarity and regardless of their resistance levels to phenylpyrroles, they showed strongly reduced fitness parameters (sporulation, mycelial growth, aggressiveness on plants) compared to the parental phenotypes. Most of the mutants presented modifications in the osmosensing class III histidine kinase affecting the HAMP domains. Site directed mutagenesis of the bos1 gene was applied to validate eight of the identified mutations. Structure modelling of the HAMP domains revealed that the replacements of hydrophobic residues within the HAMP domains generally affected their helical structure, probably abolishing signal transduction. Comparing mutant phenotypes to the HAMP structures, our study suggests that mutations perturbing helical structures of HAMP2-4 abolish signal-transduction leading to loss-of-function phenotype. The mutation of residues E529, M427, and T581, without consequences on HAMP structure, highlighted their involvement in signal transduction. E529 and M427 seem to be principally involved in osmotic signal transduction.

[38] YANG Y, LI M X, DUAN Y B, LI T, SHI Y Y, ZHAO D L, ZHOU Z H, XIN W J, WU J, PAN X Y, LI Y J, ZHU Y Y, ZHOU M G . A new point mutation in β₂-tubulin confers resistance to carbendazim in Fusarium asiaticum.
Pesticide Biochemistry and Physiology, 2018,145:15-21.
[本文引用: 1]

[39] 尚岩 . 桃、樱桃灰霉病菌对七种杀菌剂的抗药性研究
[D]. 武汉: 华中农业大学, 2016.
[本文引用: 1]

SHANG Y . Study on resistance of Botrytis cinerea from peach and cherry to seven fungicides
[D]. Wuhan: Huazhong Agricultural University, 2016. (in Chinese)
[本文引用: 1]

[40] 贾娇, 苏前富, 孟玲敏, 张伟, 李红, 刘婉丽, 晋齐鸣 . 禾谷镰孢菌对咯菌腈的抗药性诱导及对不同药剂的交互抗性
//中国植物病理学会会议论文集, 2015: 553.
URL [本文引用: 1]
咯菌腈是一种新型苯基吡咯类杀菌剂,对番茄灰霉病、马铃薯早疫病和大豆菌核病等病害有良好的防效。研究发现,该药剂对玉米茎腐病也具有较好的防治效果。然而,目前未见该药剂对玉米茎腐病病原菌抗药性的研究报道。因此,为评估玉米茎腐病病原菌禾谷镰孢菌(Fusarium graminearum)对咯菌腈的抗药性风险,本研究通过室内紫外照射获得抗咯菌腈突变体,测定抗性突变体对咯菌腈、戊唑醇、苯醚甲环唑和嘧菌酯的敏感性,研究了亲本菌株与抗性突变体之间的生物学特性。结果表明,经紫外照射5 min后,共产生17株抗咯菌腈突变菌株,其对咯菌腈的EC_(50)值是亲本菌株的4 000~17 000倍;300μg/mL咯菌腈浓度下,多数抗性突变菌株分生孢子萌发率与亲本菌株无明显差别,且亲本菌株和抗性突变菌株分生孢子均可以正常萌发,未产生芽管畸形;相关分析显示,所得抗咯菌腈突变体对苯醚甲环唑无交互抗性,对戊唑醇和嘧菌酯产生正交互抗性。相关研究报道,经药剂驯化对咯菌腈产生抗药性的核盘菌菌株对咯菌腈的抗药性是其亲本菌株的2000倍以上;经紫外诱变的抗咯菌腈突变菌株灰霉菌和早疫病菌对咯菌腈的抗药性是野生型菌株的310倍和34.87倍,表明不同菌株对咯菌腈产生抗药性的程度不同,且咯菌腈主要通过抑制禾谷镰孢菌的菌丝生长来起到防治玉米茎腐病的作用。研究发现,应用咯菌腈防治番茄灰霉病时,咯菌腈与多菌灵、乙霉威、腐霉利、嘧霉胺和啶酰菌胺无交互抗性;在防治马铃薯早疫病时与吡唑醚菌酯和苯醚甲环唑无交互抗性,与异菌脲和腐霉利之间存在正交互抗性;在防治核盘菌引起的病害时与二甲酰亚胺之间存在正交互抗性,表明同一药剂在防治不同病害时与其他药剂之间产生的交互抗性不同,尽管产生交互抗性的药剂与咯菌腈不属于同一类型杀菌剂,其之间仍然可能会产生抗药性。综合以上研究结果表明禾谷镰孢菌对咯菌腈存在中等抗性风险。
JIA J, SU Q F, MENG L M, ZHANG W, LI H, LIU W L, JIN Q M . The resistance of Fusarium graminearum to fludioxonil and the interaction resistance to different medicaments//
Proceeding of the Chinese Society of Plant Pathology, 2015: 553. (in Chinese)
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咯菌腈是一种新型苯基吡咯类杀菌剂,对番茄灰霉病、马铃薯早疫病和大豆菌核病等病害有良好的防效。研究发现,该药剂对玉米茎腐病也具有较好的防治效果。然而,目前未见该药剂对玉米茎腐病病原菌抗药性的研究报道。因此,为评估玉米茎腐病病原菌禾谷镰孢菌(Fusarium graminearum)对咯菌腈的抗药性风险,本研究通过室内紫外照射获得抗咯菌腈突变体,测定抗性突变体对咯菌腈、戊唑醇、苯醚甲环唑和嘧菌酯的敏感性,研究了亲本菌株与抗性突变体之间的生物学特性。结果表明,经紫外照射5 min后,共产生17株抗咯菌腈突变菌株,其对咯菌腈的EC_(50)值是亲本菌株的4 000~17 000倍;300μg/mL咯菌腈浓度下,多数抗性突变菌株分生孢子萌发率与亲本菌株无明显差别,且亲本菌株和抗性突变菌株分生孢子均可以正常萌发,未产生芽管畸形;相关分析显示,所得抗咯菌腈突变体对苯醚甲环唑无交互抗性,对戊唑醇和嘧菌酯产生正交互抗性。相关研究报道,经药剂驯化对咯菌腈产生抗药性的核盘菌菌株对咯菌腈的抗药性是其亲本菌株的2000倍以上;经紫外诱变的抗咯菌腈突变菌株灰霉菌和早疫病菌对咯菌腈的抗药性是野生型菌株的310倍和34.87倍,表明不同菌株对咯菌腈产生抗药性的程度不同,且咯菌腈主要通过抑制禾谷镰孢菌的菌丝生长来起到防治玉米茎腐病的作用。研究发现,应用咯菌腈防治番茄灰霉病时,咯菌腈与多菌灵、乙霉威、腐霉利、嘧霉胺和啶酰菌胺无交互抗性;在防治马铃薯早疫病时与吡唑醚菌酯和苯醚甲环唑无交互抗性,与异菌脲和腐霉利之间存在正交互抗性;在防治核盘菌引起的病害时与二甲酰亚胺之间存在正交互抗性,表明同一药剂在防治不同病害时与其他药剂之间产生的交互抗性不同,尽管产生交互抗性的药剂与咯菌腈不属于同一类型杀菌剂,其之间仍然可能会产生抗药性。综合以上研究结果表明禾谷镰孢菌对咯菌腈存在中等抗性风险。