西瓜蔓枯病菌啶酰菌胺抗性突变体的生物学特性研究及其Sdh B基因位点检测

为评价西瓜蔓枯病菌对啶酰菌胺的抗性风险,了解其抗性机理,室内通过药剂驯化方法获得2株啶酰菌胺的抗性突变体XF21-3和YC60-1,测定了抗性突变体的生物学特性,并通过对Sdh B基因片段的测序比对,分析了西瓜蔓枯病菌对啶酰菌胺的抗性机理。生物测定结果表明:啶酰菌胺对2株抗性突变体的EC50值分别为108和124 μg/mL,抗性倍数（RR）分别为1 007和1 347,均为高抗菌株;抗性突变体的菌丝生长速率和产孢量均大于亲本菌株,但其致病性与亲本菌株无显著差异,对外界环境渗透压的敏感性低于亲本菌株;此外,啶酰菌胺与萎锈灵、戊唑醇、乙霉威及醚菌酯之间均不存在交互抗性,但与噻呋酰胺之间存在交互抗性。Sdh B基因片段测序及比对结果表明,高抗性突变体中Sdh B亚基277位上的氨基酸所对应的碱基由CAC突变为TAC,即由组氨酸（His）突变为酪氨酸（Tyr）。研究表明,西瓜蔓枯病菌在药剂选择压力下容易形成啶酰菌胺的抗性群体,且抗性突变体的离体适合度高于亲本菌株,此外,啶酰菌胺与同类型杀菌剂噻呋酰胺之间存在交互抗性,因此认为西瓜蔓枯病菌对啶酰菌胺具有中等抗性风险;同时进一步验证了Sdh B亚基277位上的氨基酸突变（His→Tyr,CAC→TAC）是西瓜蔓枯病菌对啶酰菌胺产生抗性的原因。     

抗啶酰菌胺蔬菜灰霉病菌突变体的诱导及其生物学性状

为评价蔬菜灰霉病菌对啶酰菌胺的抗性风险,采用紫外线照射和药剂驯化相结合的诱导方法,对蔬菜灰霉病菌D9、L17、J9亲本敏感菌株进行了室内啶酰菌胺抗性诱导,并采用菌落直径法比较了抗、感菌株在生物学性状方面的差异。结果显示,共获得不同抗性水平的突变体23株,其中突变体D9-4的抗性倍数为253.76倍,达高抗水平。突变体菌丝生长速率较敏感菌株有所下降,抗、感菌株的适宜生长温度均为25 ℃,敏感菌株最适pH为5,突变体最适pH为7,突变体适应酸碱度和温度变化的能力较敏感菌株差;低抗突变体产孢量和孢子萌发率与敏感菌株无显著差异,但高抗突变体产孢量和孢子萌发率较敏感菌株显著降低;在无药条件下,敏感菌株致病力强于抗性突变体,而在药剂胁迫下,突变体表现出一定的耐药性,致病力强于敏感菌株;6株突变体无药继代培养15代后,突变体抗性稳定遗传。此外,蔬菜灰霉病菌对啶酰菌胺与其它5种杀菌剂之间没有交互抗性。研究表明,蔬菜灰霉病菌抗啶酰菌胺突变体适合度较低,但在药剂选择压力下容易形成啶酰菌胺抗性群体,在生产中需引起注意。     

死亡谷芽胞杆菌NBIF-001防治番茄棒孢叶斑病研究

为明确死亡谷芽胞杆菌NBIF-001菌株对棒孢叶斑病的防治效果,采用平板对峙结合显微观察确定其对多主棒孢的抑菌活性,并采用活体盆栽试验研究该菌株对番茄棒孢叶斑病防治效果。结果表明,在平板对峙条件下NBIF-001对多主棒孢具有良好的抑菌活性,显微镜下观察到菌丝发生扭曲、肿胀和变形;盆栽试验结果表明NBIF-001对棒孢叶斑病的防治效果达到71.46%。NBIF-001和50%啶酰菌胺WG联用防治番茄棒孢叶斑病的效果明显好于单独施用50%啶酰菌胺WG,具有较明显的增效作用,化学杀菌剂50%啶酰菌胺WG对NBIF-001菌株生长无明显影响,两者具有较好的相容性。     

樱桃褐腐病菌对啶酰菌胺的敏感性及其对4种琥珀酸脱氢酶抑制剂的交互抗性

采用菌丝生长速率法测定了樱桃褐腐病菌Monilinia fructicola对啶酰菌胺的敏感性,同时研究了不同敏感性菌株的生物学性状,探究了琥珀酸脱氢酶B亚基的氨基酸突变与其对啶酰菌胺产生抗性的相关性,并分析了樱桃褐腐病菌对啶酰菌胺与其他3种琥珀酸脱氢酶抑制剂(SDHIs)氯苯醚酰胺、氟唑菌苯胺和氟吡菌酰胺之间的交互抗...     

浙江省果蔬灰葡萄孢对啶酰菌胺的抗性

以2004—2006年从浙江、江苏等地采集的灰葡萄孢对啶酰菌胺的敏感性基线[EC₅₀ = (1.07 ± 0.11) mg/L]为依据,采用菌丝生长速率法连续监测了浙江省果蔬灰葡萄孢群体对啶酰菌胺的敏感性变化。结果表明:浙江省果蔬灰葡萄孢对啶酰菌胺的抗性发展迅速,2012—2013年和2017—2018年的平均EC₅₀值分别为 (5.23 ± 7.79) 和 (24.30 ± 49.33) mg/L。其中,2012—2013年的抗药性菌株频率为15.3%,且均为低水平抗性 (LR) 菌株;而2017—2018年的抗药性频率上升至53.2%,并出现了7.5%的中等水平抗性 (MR) 菌株和1.3%的高水平抗性 (HR) 菌株。啶酰菌胺抗性菌株的菌丝生长速率、产孢量、产菌核数和致病力与敏感菌株相比均无显著差异。抗药性分子机制研究表明:啶酰菌胺抗性菌株的琥珀酸脱氢酶B亚基 (SDH B) 均发生了点突变,共包括H272R、P225F和N230I 3种类型,其中H272R型突变占88.5%;其SDH A和SDH D均未发生点突变;而SDH C的突变 (G85A + I93V + M158V + V168I) 与对药剂敏感性之间无明显联系。     

重庆地区辣椒疫霉对氟吡菌胺的敏感性及抗性突变体的生物学性状

为明确重庆地区辣椒疫霉对氟吡菌胺的抗性风险,采用菌丝生长速率法测定了采自重庆未使用过氟吡菌胺地区的110株辣椒疫霉菌株对氟吡菌胺的敏感性,并对辣椒疫霉抗氟吡菌胺突变体的诱导方法及抗性突变体的主要生物学性状进行了研究。结果表明:110株辣椒疫霉对氟吡菌胺的EC₅₀平均值为(0.32 ± 0.11) μg/mL,不同菌株的敏感性频率呈连续单峰曲线分布,未出现敏感性明显下降的亚群体,因此可将该EC₅₀平均值作为重庆地区辣椒疫霉对氟吡菌胺田间抗性监测的敏感基线。通过紫外诱导菌丝体的方法,共获得3株可稳定遗传的抗氟吡菌胺突变体,抗性倍数介于69.5~98.5之间,突变频率为0.86%;抗性突变体BS11-5-1与亲本菌株BS11-5在菌丝生长速率、温度适应性、产孢子囊能力及致病力方面均无显著差异,而抗性突变体JLP11-4-2和JLP11-4-3在菌丝生长速率、温度适应性、产孢子囊能力及致病力方面均显著低于亲本菌株JLP11-4;不同抗性突变体对渗透压的敏感性与亲本菌株之间均存在不同程度差异;3个抗性突变体对Biolog PM1中95种碳源的利用情况与亲本菌株基本相似。交互抗性测定表明,辣椒疫霉抗氟吡菌胺突变体对甲霜灵、霜脲氰、烯酰吗啉、丁吡吗啉及嘧菌酯之间均不存在交互抗性,建议可将氟吡菌胺与上述几种杀菌剂交替或混合使用。     

番茄灰霉病菌对咯菌腈的抗性诱变及抗药突变体的生物学特性

为评估番茄灰霉病菌Botrytis cinerea对咯菌腈的抗性风险,就室内经紫外照射获得抗药突变体的方法及抗性突变体的生物学性状进行了研究。结果表明:番茄灰霉病菌分生孢子的紫外照射亚致死时间为90~120 s;经亚致死时间紫外照射后,4个亲本菌株中有2个菌株共产生了6个抗咯菌腈的突变体,其EC50值是亲本菌株的310倍以上,抗性突变频率为3.13×10-7;经紫外照射诱变获得的所有抗性突变体在菌丝生长速率、产孢量、产菌核能力及其在番茄果实上的致病性方面均比其亲本菌株明显降低。相关分析显示,所得抗咯菌腈突变体对氟啶胺、啶菌唑、啶酰菌胺和嘧霉胺无交互抗性。表明番茄灰霉病菌对咯菌腈的抗药性风险较低。     

抗氰烯菌酯假禾谷镰孢突变体的诱导及其生物学特性

为评估引起小麦茎基腐病的病原菌假禾谷镰孢Fusarium pseudograminearum对氰烯菌酯的抗性风险,对5株敏感菌株进行了室内药剂驯化,获得33株抗性突变体,突变频率为16.5%,其对氰烯菌酯的抗性水平范围为7.39～1 665.76倍,3株表现低抗,4株表现中抗,26株表现高抗;发现在myosin-5基因上存在11种抗性突变类型,其中217位的丝氨酸突变为亮氨酸(S217L)、420位的谷氨酸突变为赖氨酸(E420K)和135位的丙氨酸突变为苏氨酸(A135T)为主要突变类型,其比例分别为45.5%、15.2%和9.1%。S217L型抗性突变体的产孢量显著下降,菌丝生长速率和致病力与亲本菌株无显著差异。E420K型抗性突变体的菌丝生长速率和致病力显著下降,产孢量与亲本菌株无显著差异。A135T型抗性突变体的菌丝生长速率和产孢量与亲本菌株无显著差异。研究结果表明假禾谷镰孢在药剂选择压力下易形成氰烯菌酯的抗性群体,对氰烯菌酯存在中到高等的潜在抗性风险,其myosin-5的点突变与其对氰烯菌酯的抗性相关。     

五种琥珀酸脱氢酶抑制剂类杀菌剂与灰葡萄孢琥珀酸脱氢酶的结合模式及抗性机制分析

为探究琥珀酸脱氢酶抑制剂 (SDHI) 类杀菌剂与灰葡萄孢Botrytis cinerea琥珀酸脱氢酶 (SDH) (以下简称BcSDH) 的结合方式,阐明BcSDH对SDHI类杀菌剂产生抗性的结构生物学机制,通过同源建模构建了BcSDH的三维模型,通过分子对接预测了5种SDHI (异丙噻菌胺、氟吡菌酰胺、氟唑菌酰胺、吡噻菌胺和啶酰菌胺) 与野生型和突变型BcSDH的亲和力及结合模式之间的变化,分析其抗药性机制,对相关突变位点进行保守性预测,并分析突变类型。结果表明:5种SDHI与BcSDH 具有较强的亲和力,其中酸部分插入BcSDH活性腔底,胺部分在活性腔口,能够形成牢固的疏水作用、氢键、卤键、π-π堆积作用和π-阳离子等相互作用。B-P225F氨基酸残基突变 (以下简称突变) 会造成活性腔口变窄,使得SDHI酸部分不能进入活性腔;B-P225L突变会造成异丙噻菌胺、氟吡菌酰胺和吡噻菌胺与靶标蛋白的结合模式发生变化,亲和力降低;B-H272R突变后,活性腔底变窄,与SDHI的亲和力下降。另外,保守性分析结果表明,B-P225和B-H272均位于BcSDH的保守区域,B-P225F、B-H272R和B-H272L突变可能为随机突变。因此推测BcSDH的B-P225F和B-H272R突变可能是引起灰葡萄孢对5种杀菌剂产生抗性的主要原因,也可能是引起SDHI类杀菌剂之间交互抗性的主要原因之一;B-P225L突变可能降低灰葡萄孢对部分杀菌剂的敏感性,而不是引起BcSDH对SDHI类杀菌剂产生交互抗性的主要原因。在实际生产中,应采取合理有效的抗性监测治理策略来延缓灰葡萄孢对SDHI类杀菌剂抗性的产生,在SDHI分子设计时也应考虑该位点氨基酸残基突变,避免产生交互抗性。     

番茄早疫病菌抗啶酰菌胺菌株的适合度及生物学特性

为了评价番茄早疫病菌Alternaria solani对啶酰菌胺的抗性风险,采用菌丝直径法和孢子萌发法探讨番茄早疫病菌抗啶酰菌胺菌株的适合度变化和生物学性状差异。结果表明:番茄早疫病菌对啶酰菌胺的抗性可稳定遗传;与敏感菌株相比,抗性菌株的菌丝生长减慢,产孢量明显增多,病斑直径达8.5 mm以上,致病力显著增强,产毒量也有增加的趋势,但孢子萌发率无显著差异;啶酰菌胺对抗性菌株的防效显著下降为64.17%~33.49%;抗性菌株的最适p H范围变宽为5~9;中抗菌株的最适温度(25℃)、最佳碳源(淀粉和乳糖)和氮源(蛋白胨)没发生变化,但高抗和极高抗菌株的最适温度范围变宽为25~30℃,最适碳、氮源发生了变化。其中,高抗菌株的最适碳、氮源分别为乳糖和大豆粉;极高抗菌株的最适碳源为淀粉,最适氮源为玉米粉和蛋白胨。表明番茄早疫病菌对啶酰菌胺产生抗性后,较易存活而形成优势群体,因此需采取一定措施延缓抗性的发展、延长啶酰菌胺的使用期限。     

Characterization of fenarimol-resistant mutants of Aspergillus nidulans

The fitness of twelve fenarimol-resistant mutants of Aspergillus nidulans was tested with respect to spore germination, germ tube elongation, hyphal growth and sporulation. Half of the strains tested were isolated on triarimolcontaining media. The other strains were selected on imazalil-or cycloheximide-containing media (Van Tuyl, 1977).A number of mutant strains produced spores with unimpaired viability on synthetic medium. In other strains a reduction in spore viability was noticed. The rate of germ tube elongation of all resistant mutants was significantly lower than that of the wild-type strain. Mutant strains with a low degree of resistance always had an almost normal mycelial growth rate, whereas growth of some of the strains with a relatively high degree of resistance was significantly slower. Spore production on malt agar was highest in the wild-type strain and was found to be lower in fenarimol-resistant mutants. In most of the mutant strains tested a high degree of cross-resistance between fenarimol, imazalil and triadimefon was established; in some of them cross-resistance to these chemicals was low or even absent.Possible implications of the results described for the chance of development of resistance in phytopathogenic fungi to sterol biosynthesis-inhibiting fungicides are discussed.     

Occurrence of Corynespora cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture, Japan

A total of 651 isolates of cucumber corynespora leaf spot fungus ( Corynespora cassiicola ) collected from cucumber in Japan, either with (438 isolates) or without (213 isolates) a prior history of boscalid use, were tested for their sensitivity to boscalid by using a mycelial growth inhibition method on YBA agar medium. Additionally, seven isolates of C. cassiicola obtained from tomato, soybean, eggplant (aubergine) and cowpea in different locations in Japan were tested before boscalid registration. Minimum inhibitory concentration (MIC) and 50% effective concentration (EC₅₀) values for 220 isolates from crops without a prior history of boscalid use ranged from 0·5 to 7·5 μg mL⁻¹ and from 0·04 to 0·59 μg mL⁻¹, respectively. Two hundred and fourteen out of 438 isolates collected from ten cucumber greenhouses in Ibaraki Prefecture, Japan, which received boscalid spray applications showed boscalid resistance, with MIC values higher than 30 μg mL⁻¹. Moreover, resistant isolates were divided into two groups: a moderately resistant (MR) group consisting of 189 isolates with EC₅₀ values ranging from 1·1 to 6·3 μg mL⁻¹, and a very highly resistant (VHR) group consisting of 25 isolates with EC₅₀ values higher than 24·8 μg mL⁻¹. MR isolates were detected from all ten greenhouses, but VHR isolates were detected from only three. As a result of fungus inoculation tests which used potted cucumber plants, control failures of boscalid were observed against resistant isolates. Efficacy of boscalid was remarkably low against VHR isolates in particular. This is the first known report on boscalid resistance in Japan.     

Impact of fludioxonil resistance on fitness and cross-resistance profiles of Altrenaria solani laboratory mutants

Sensitivity and inherent resistance risk of Alternaria solani to fludioxonil, cross-resistance profiles and the potential implications of resistance mutations on fitness parameters were investigated. Fludioxonil was highly effective against a wild type A. solani field strain both in vitro (EC₅₀?=?0.05 μg/mL) and in preventive applications on artificially inoculated tomato fruit. Mutants with low [Resistance factor (Rf): 15 based on EC₅₀], medium (Rf: 150–300) and high (Rf: > 1000) levels of phenylpyrrole resistance were isolated from the wild type strain at high frequencies following mutagenesis with UV irradiation and selection on fludioxonil containing medium. Resistant isolates retained their resistance levels even after 9 subcultures on fungicide-free growth medium while they could express their resistant phenotypes in planta. Investigation of cross-resistance relationships showed that fludioxonil resistance mutations also reduce the sensitivity of mutant strains to the aromatic hydrocarbon fungicide quintozene as well as the dicarboximides iprodione and vinclozolin. No cross-resistance was observed between fludioxonil and fungicides with different modes of action such as the sterol biosynthesis inhibitors (DMIs) imazalil and flusilazole and the carboxamide boscalid. All fludioxonil resistant isolates were more sensitive to the anilinopyrimidine pyrimethanil, while only two isolates were less sensitive to the QoI pyraclostrobin compared to the wild-type strain. Study of fitness determining parameters showed that resistance mutation(s) had no adverse effects on mycelial growth, conidial germination and sensitivity to osmotic stress while they had a pleiotropic effect on virulence and conidia production in resistant mutants. Results of the present study indicate that fludioxonil is a highly effective fungicide against A. solani, while the risk of resistance development to this fungicide is considered to be medium making fludioxonil an ideal alternative to high risk fungicides such as boscalid and pyraclostrobin whose performance against early blight has already been compromised by resistance development.

     

橡胶树棒孢霉落叶病病原菌的生物学特性

通过对橡胶多主棒孢进行生物学特性研究,结果表明:菌丝生长的适宜温度范围为25～30℃,最适温度为30℃;最适pH范围为4～8;菌丝在以麦芽糖为碳源和以KNO3为氮源的培养基上生长最好。分生孢子萌发的适宜温度范围为20～35℃,最适温度为25℃;水滴是孢子萌发的必要条件;在不同的pH条件下分生孢子萌发无明显差异;分生孢子在果糖中萌发率最低。     

Characterization of mutations in the iron-sulphur subunit of succinate dehydrogenase correlating with Boscalid resistance in Alternaria alternata from California pistachio

Thirty-eight isolates of Alternaria alternata from pistachio orchards with a history of Pristine (pyraclostrobin + boscalid) applications and displaying high levels of resistance to boscalid fungicide (mean EC(50) values >500 microg/ml) were identified following mycelial growth tests. A cross-resistance study revealed that the same isolates were also resistant to carboxin, a known inhibitor of succinate dehydrogenase (Sdh). To determine the genetic basis of boscalid resistance in A. alternata the entire iron sulphur gene (AaSdhB) was isolated from a fungicide-sensitive isolate. The deduced amino-acid sequence showed high similarity with iron sulphur proteins (Ip) from other organisms. Comparison of AaSdhB full sequences from sensitive and resistant isolates revealed that a highly conserved histidine residue (codon CAC in sensitive isolates) was converted to either tyrosine (codon TAC, type I mutants) or arginine (codon CGC, type II mutants) at position 277. In other fungal species this residue is involved in carboxamide resistance. In this study, 10 and 5 mutants were of type I and type II respectively, while 23 other resistant isolates (type III mutants) had no mutation in the histidine codon. The point mutation detected in type I mutants was used to design a pair of allele-specific polymerase chain reaction (PCR) primers to facilitate rapid detection. A PCR-restriction fragment length polymorphism (RFLP) assay in which amplified gene fragments were digested with AciI was successfully employed for the diagnosis of type II mutants. The relevance of these modifications in A. alternata AaSdhB sequence in conferring boscalid resistance is discussed.     

海南橡胶树棒孢霉落叶病菌毒素蛋白基因检测与致病性分析

Cassiicolin毒素蛋白是橡胶树棒孢霉落叶病菌的主要致病因子。本研究对分离自海南省5个市县的橡胶树棒孢霉落叶病菌进行了鉴定,形态学鉴定结合分子生物学鉴定结果表明,病原菌为多主棒孢。利用Cassiicolin毒素不同亚型编码基因特异性引物和菌饼活体接种法分别鉴定了分离菌株的毒素蛋白类型和致病性,结果显示,分离的35个菌株都含有Cas5基因,未发现含其他类型的毒素基因,表明海南橡胶树棒孢霉落叶病菌为含Cas5毒素蛋白优势群体;所有菌株对橡胶树品种‘RRIM600’都能够致病,但是在不同抗性品种上致病性存在一定差异。     

两株橡胶多主棒孢霉对12种杀菌剂原药的敏感性

采用菌落生长速率法测定了两株橡胶多主棒孢霉［Corynespora cassiicola (Berk &; Curt.) Wei］对12种杀菌剂原药的敏感性。结果表明：两个菌株对杀菌剂的敏感性存在一定的差异;供试药剂中多菌灵、甲基硫菌灵、咪鲜胺和抑霉唑的EC50均小于5 μg/mL,抑菌活性高;在加入SHAM后嘧菌酯的抑菌活性也很高,与SHAM具有协同增效作用;其他供试杀菌剂均有较好的抑菌作用,依次为戊唑醇、腈菌唑、异菌脲、溴菌腈、代森锰锌、百菌清和三唑酮。     

江西省番茄绵疫病菌对嘧菌酯的敏感性检测及抗性风险分析

为明确番茄绵疫病菌对嘧菌酯的敏感性,采用菌丝生长速率法测定了2018—2019年从江西省番茄主产区采集分离得到的58株绵疫病菌Phytophthora capsici对嘧菌酯的敏感性,并以敏感菌株YD5为亲本,通过紫外线诱导获得了4株抗性突变体,研究了突变体的生物学性状;进而采用菌丝生长速率法测定了敏感菌株YD5及其4...