棉铃疫病菌对甲霜灵的抗药性风险研究

在实验室条件下,对分离自安徽省芜湖、无为、宣城、望江、肥东、寿县、滁州等7个地区的棉铃疫病菌(Phytophthora boehmeriae Sawada)对甲霜灵的抗药性进行了测定。结果显示,甲霜灵对各供试菌株的EC50分布为0.008 3～0.079 1μg／mL,平均为0.026 1 μg／mL,远小于1 μg／mL,且供试菌株在含甲霜灵质量浓度为1μg／mL的LBA平板上菌丝生长几乎完全被抑制,表明各供试菌株对甲霜灵十分敏感,但其敏感程度地区间存在较大差异。通过室内药剂直接诱变,获得抗甲霜灵菌株,经抗性测定结果表明,抗性菌株的抗性水平较其敏感亲本菌株高13 000倍以上,提示安徽省棉铃疫病菌对甲霜灵具有潜在的抗药性风险。     

苎麻疫霉抗甲霜灵突变株对棉苗的致病力及其遗传

研究了苎麻疫霉抗甲霜灵(Mt^r)突变株对棉苗的致病力及其遗传,结果显示,抗性突变株对棉苗的致病力与其野生型亲本间无显著差异。而Mt^r突变株XC-6-2对棉苗的致病力在其无性第1代(ZG₁)单孢株间及其与亲本间均存在极显著差异,且XC-6-2对棉苗的致病力性状在单游动孢子第2代(ZG₂)持续发生分离;XC-6-2单卵孢第1代(OG₁)对棉苗的致病力间亦存在极显著差异。上述结果提示,抗甲霜灵突变株对棉苗的致病力在无性单孢和单卵孢子后代间均不能稳定遗传。对比研究发现,野生型亲本菌株XC-6对棉苗的致病力在单游动孢子和单卵孢后代有相似的遗传规律,且单游动孢子群体对棉苗致病力的变异系数较其单卵孢株间的变异系数大。     

马铃薯晚疫病菌抗甲霜灵的SCAR标记

建立马铃薯晚疫病菌抗甲霜灵SCAR(sequenced characterized amplified region,序列特征扩增区域)标记,以马铃薯晚疫病菌对甲霜灵高抗菌株HD01-3和对甲霜灵高感菌株DK98-1为亲本,通过无性单游动孢子分离和有性杂交获得菌株HD01-3无性后代群体、菌株DK98-1无性后代群体以及F1代分离群体,以此为试验材料,利用BSA法(bulked segregant analysis,分离群体分组分析法)构建抗感基因池对后代菌系的甲霜灵抗性进行RAPD(random amplified polymorphic DNA,随机扩增多态性DNA)分析。从178条RAPD随机引物中找到一条特异性引物S2054,其可以扩增出一个与晚疫病菌对甲霜灵抗性相关的遗传标记,将该特异条带回收、克隆、测序,发现此标记大小为457bp,根据测序结果设计特异PCR引物,用于扩增抗感基因池,成功地将特异RAPD标记转化为SCAR标记。初步建立了马铃薯晚疫病菌抗甲霜灵SCAR标记,辅助监测晚疫病菌对甲霜灵的抗性。     

恶疫霉有性生殖交配行为的研究

 对同宗配合疫霉种——恶疫霉(Phytophthora cactorum Schroter)抗甲霜灵突变株和抗地茂散突变株的抗药性在游动孢子和卵孢子后代的遗传分析,筛选到抗性分别由细胞核纯合显性单基因控制的抗甲霜灵突变株和由细胞质可稳定遗传的线粒体基因控制的抗地茂散突变株。将携带抗甲霜灵对地茂散敏感(即Mt^rCn^s)标记的亲本与携带抗地茂散对甲霜灵敏感(即Mt^sCn^r)标记的亲本共同培养,在200个有性生殖单卵孢后代中检测到携带3种不同抗药性标记的单孢株,其中32株携带Mt^rCn^s标记,165株携带Mt^sCn^r标记,另有3株同时携带双亲的标记性状即MtrCnr。结果表明:恶疫霉种内不同菌株共同培养时,有性生殖以配对亲本的各自自交为主,同时配对亲本间也可发生一定频率的杂交。     

大豆疫霉菌单孢分离物生物学性状的遗传变异研究

 本文研究了大豆疫霉菌单游动孢子无性分离物和自交单卵孢子分离物的菌丝生长速率、菌落形态、同宗配合性状、产孢量以及对甲霜灵敏感性的遗传变异。结果表明:菌落形态、生长速率和同宗配合性状在单游动孢子后代和自交后代可稳定遗传,控制上述性状的遗传因子是纯合的;大豆疫霉菌的游动孢子产生能力和对甲霜灵的敏感性在单游动孢子后代和自交后代中均发生连续性变异,表明这两种性状可能是数量遗传性状,也可能控制这两种性状的基因为杂合基因或细胞质遗传因子。     

玉蜀黍赤霉有性重组体的诱导及其生物学特性

 本研究以硝酸盐营养缺陷型突变体(nit)和多菌灵抗性为遗传标记,在5个所选菌株中设计了3个玉蜀黍赤霉病菌Gibberella zeae的杂交组合,使各菌株之间进行杂交,从而诱导有性重组体。从各杂交组合的后代中任意挑选出3个有性重组体,比较了这些有性重组体与其亲本在无性和有性阶段的主要生物学性状。结果表明,玉蜀黍赤霉中的nit基因及对杀菌剂多菌灵的抗药性基因可以通过有性杂交的方式重组,即发生了有性重组。有性重组体与其亲本在菌落生长、培养性状和致病性方面没有显著差异;但某些有性重组体中,产孢量和产子囊壳能力方面存在一定差异。总体看来,有性重组体仍然保持了较高的适合度。因此,可以认为有性重组在玉蜀黍赤霉群体对多菌灵抗药性发生发展以及群体遗传进化中可能起着重要的作用。     

速克灵抗性灰霉病菌菌株性质的研究

研究结果表明,灰霉菌对二甲酰亚胺类与苯并咪唑类药剂之间不存在交互抗药性,与传统的保护性杀菌剂之间也不存在交互抗药性。杀菌剂福美双、克菌丹、百菌清对二甲酰亚胺类抗性菌抑菌效果较好。高抗二甲酰亚胺类药剂的某些菌株产孢能力大于敏感菌株。抗性菌单孢分离后代仍保留抗性。大棚草莓连续9次喷撒速克灵,每次间隔10—14天,未发现速克灵抗性菌株的产生。盆栽草莓试验表明,无药条件下,抗性菌株的致病力明显低于敏感菌;正常用药条件下,速克灵对抗性菌株的防治效果明显低于敏感菌株。     

江西省番茄绵疫病菌的交配型及对甲霜灵敏感性测定

为明确江西番茄绵疫病菌的类别和抗药性水平, 采用单孢菌落直接配对法和菌落生长量法, 测定2018年-2019年江西番茄主产区绵疫病菌的交配型及其对甲霜灵的敏感性。结果表明, 江西番茄绵疫病菌存在A2和A0交配型, 发生频率分别为86.2%和13.8%, 未检测到其他交配型, 且A2交配型在各个产区均有分布。58株番茄绵疫病菌株对甲霜灵的EC50范围为0.602 9~3.313 3 μg/mL, 平均EC50为1.372 7 μg/mL, 最不敏感菌株EC50值为最敏感菌株的5.5倍, 差异较为明显。3个菌株对甲霜灵达到中抗水平, 占供试菌株的5.17%, 病原菌对甲霜灵的抗性水平上升, 亟须加强药剂的轮换或替代。     

辣椒疫霉产毒缺陷与抗药性突变体筛选及其遗传特性

采用MNNG诱变与自交（S1－S3）纯合的方法,从辣椒疫霉的游动孢子群体中,筛选出1株带有抗霜脲氰标记的产毒缺陷突变体、2株抗甲霜灵的产毒突变体。产毒缺陷菌体和产毒菌株均对茄门甜椒致病,但产毒缺陷菌株的致病力降低一半,证明辣椒疫霉的致病显症过程与毒素作用有关,病菌毒素是重要的致病因子。此外,突变菌株的抗药性和产毒缺陷表型在无性游动孢子后代和有性孵孢子后代群体中均可稳定遗传。在姐妹配对F2代卵孢子群体中,辣椒疫霉毒素产生和对甲霜灵的抗性为不完全显性基因所控制,而对霜脲氰的抗性为完全显性基因所控制。     

稻瘟菌致病力变异初探

 从保存的4个单孢分离菌后代再分离若干单胞菌株,接种子国内7个鉴别品种上,又可以分成不同的小种。从6个单孢分离菌再分离的后代单孢菌株,分别对13个抗病品种作致病力测定,各后代单孢菌株仅对少数品种的致病力是一致的。从27个品种的小病斑或个别大病斑上分离的病菌,回接原品种后,有11个分离菌对原品种不致病。这些结果说明大多数分离菌对许多品种的致病力是易变的,只对少数品种的致病力保持相对稳定性。作者认为进行抗性遗传和抗性机制研究需要的致病力稳定分离菌,只能根据其对供试品种致病力的稳定性来选择,而不能按一组鉴别品种来选择。     

掘氏疫霉抗土菌灵突变体的诱导及其特性研究

掘氏疫霉Phytophthora drechsleri是引起黄瓜疫病的主要病原菌。采用紫外线诱导获得了20株掘氏疫霉抗土菌灵的突变体,并对其生物学特性进行了研究。结果表明,掘氏疫霉对土菌灵 的抗性突变频率高于对甲霜灵的抗性突变频率,但其抗性水平低于抗甲霜灵突变体,在1.3~4.3倍 之间;掘氏疫霉对土菌灵的抗性在继代培养和常温保存过程中稳定,突变体的生长速率与亲本敏感 菌株相比发生了不同程度的变化;此外,掘氏疫霉抗土菌灵突变体对甲霜灵和烯酰吗啉无交互抗性。     

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...     

Laboratory studies to assess the risk of development of resistance to zoxamide

Laboratory studies were conducted to evaluate the risk of developing field resistance to zoxamide, a new Oomycete fungicide which acts on microtubules. Zoxamide, metalaxyl and dimethomorph were compared with respect to the ease with which fungicide‐resistant mutants could be isolated and their level of resistance. Attempts to generate mutants of Phytophthora capsici and P infestans with resistance to zoxamide by mycelial adaptation on fungicide‐amended medium were unsuccessful. Similarly, changes in sensitivity to zoxamide were small (resistance factors ≤2.2) in mutants of P capsici isolated by chemical mutagenesis of zoospore cysts. In parallel experiments with metalaxyl, highly resistant mutants were obtained using both adaptation (P capsici or P infestans) and chemical mutagenesis (P capsici). For dimethomorph, chemical mutagenesis (P capsici) yielded moderately resistant mutants (maximum resistance factor = 20.9), and adaptation (P capsici or P infestans) did not induce resistance. It is proposed that failure to isolate mutants resistant to zoxamide results from the diploid nature of Oomycete fungi and the likelihood that target‐site mutations would produce a recessive phenotype. Our studies suggest that the risk of a highly resistant pathogen population developing rapidly in the field is much lower for zoxamide than for metalaxyl. However, as with any site‐specific fungicide, appropriate precautions against resistance development should be taken. © 2001 Society of Chemical Industry     

Sensitivity of Phytophthora infestans to flumorph: in vitro determination of baseline sensitivity and the risk of resistance

The sensitivity of 127 Phytophthora infestans isolates to flumorph was determined in 2003 and 2004. The isolates originated from two geographical regions and showed similar levels of sensitivity in both years. Baseline sensitivities were distributed as a unimodal curve with EC₅₀ values for growth of mycelia ranging from 0·1016 to 0·3228  µ g mL⁻¹, with a mean of 0·1813 (± 0·0405) µ g mL⁻¹. There was no cross-resistance between flumorph and metalaxyl. Laboratory studies were conducted to evaluate the risk of P. infestans developing resistance to flumorph. Mutants resistant to metalaxyl or flumorph were obtained by treating mycelium of wild-type isolates with ultraviolet radiation. Metalaxyl-resistant mutants were obtained with a high frequency and exhibited resistance factor values (EC₅₀ resistant/EC₅₀ sensitive phenotypes) of more than 100, while flumorph-resistant mutants were obtained at much lower frequencies and had very small resistance factors (1·5–3·2). There was cross-resistance between flumorph and dimethomorph, but not with azoxystrobin or cymoxanil. Most flumorph-resistant mutants showed decreases in hyphal growth in vitro and in sporulation both in vitro and on detached leaf tissues. These studies suggested that the risk of resistance developing was much lower for flumorph than metalaxyl. However, as P. infestans is a high-risk pathogen, appropriate precautions against resistance development should be taken.     

疫霉菌对霜脲氰抗性遗传及对霜脲氰和甲霜灵的交互抗性

就疫霉菌对霜脲氰抗性的产生和遗传，以及疫霉菌对霜脲氰和甲霜灵的交互抗性进行了研究。结果表明，苎麻疫霉容易对霜脲氰产生抗药性突变，但抗性在连续３代单游动孢子分离过程中逐渐丧失。大雄疫霉对霜脲氰不易产生抗性突变，恶疫霉、大雄疫霉和苎麻疫霉对甲霜灵和霜脲氰不存在交互抗性     

黄瓜霜霉病菌对双炔酰菌胺的敏感基线及其抗性突变体生物学性状研究

为建立黄瓜霜霉病菌对双炔酰菌胺的敏感基线及评估其抗性风险,采用叶盘漂浮法测定了采自河北、山东未使用过羧酸酰胺类(CAAs)药剂地区的69株黄瓜霜霉病菌(Pseudoperonospora cubensis)对双炔酰菌胺的敏感性,并对黄瓜霜霉病菌抗双炔酰菌胺突变体的获得方法及突变体的生物学性状进行了研究。结果表明:69株黄瓜霜霉病菌对双炔酰菌胺的平均EC50值为(0.358±0.144)μg/mL,不同敏感性菌株的频率呈连续单峰曲线分布,未发现敏感性下降的亚群体,因此可将其作为黄瓜霜霉病菌对双炔酰菌胺的敏感基线;通过药剂驯化的方法未获得黄瓜霜霉病菌抗双炔酰菌胺的突变体;而通过紫外诱导的方法获得了6个抗双炔酰菌胺的突变体,其抗性水平介于5.74~22.96倍之间,突变频率为1.09×10-7,适合度显著低于其亲本菌株,且抗药性不能稳定遗传;双炔酰菌胺与甲霜灵、嘧菌酯、霜脲氰、氟吡菌胺之间无交互抗性关系,与烯酰吗啉之间有交互抗性关系。据此推测黄瓜霜霉病菌对双炔酰菌胺的抗性风险为低到中等。     

马铃薯晚疫病菌对烯酰吗啉的敏感性基线及其室内抗药突变体的研究

 2003-2004年在内蒙古和黑龙江省共分离获得100株马铃薯晚疫病菌,并测定了这些菌株对烯酰吗啉和甲霜灵的敏感性。结果表明,不同地区、不同年份的马铃薯晚疫病菌对烯酰吗啉的敏感性无显著差异,其敏感性呈连续的单峰曲线分布,EC₅₀在0.0631-0.2206μg/mL之间,最不敏感菌株是最敏感菌株的3.1倍,平均为(0.1153±0.0308)μg/mL,未出现敏感性下降的抗药性亚群体,因此可作为马铃薯晚疫病菌对烯酰吗啉的敏感性基线;64%的供试菌株对甲霜灵的敏感性下降,甲霜灵与烯酰吗啉之间不存在交互抗药性。采用紫外线诱变菌丝的方法仅从3个菌株中的1个菌株获得了3%突变频率的抗烯酰吗啉突变体,抗性倍数为1.5-1.9,没有获得高抗药水平的突变体。这些突变体与氟吗啉之间具有正交互抗药性,而与霜脲氰或嘧菌酯之间无交互抗药性;与野生亲本菌株相比,抗药突变体的生长速率和产孢子囊能力显著下降,不利于抗药群体的发展。但由于马铃薯晚疫病菌被杀菌剂抗性行动委员会认为是高抗药性风险病原菌,建议生产上应将烯酰吗啉与其它无交互抗性的药剂交替使用或混合使用,以避免或延缓抗药性的产生。     

Selection and characterization of Phytophthora parasitica mutants with ultraviolet-induced resistance to dimethomorph or metalaxyl

Mutants of Phytophthora parasitica Dast. more or less resistant to dimethomorph or metalaxyl were obtained by treating mycelium with ultraviolet radiation. Some metalaxyl-resistant mutants exhibited levels of resistance (i.e. the ratio between the EC₅₀ values for the mutant and the wild-type strain) greater than 100, but those observed in the dimethomorph-resistant mutants never exceeded 25. Most mutants retained their resistance after sub-culturing on unamended agar medium. Single zoospore clones derived from a metalaxyl-resistant and a dimethomorph-resistant mutant, selected as amongst the most resistant, had levels of resistance similar to those of the parental strains. The metalaxyl-resistant mutants were also resistant to the related phenylamide fungicides, furalaxyl and benalaxyl, but remained sensitive to dimethomorph. The dimethomorph-resistant mutants were not resistant to phenylamide fungicides. The pathogenicity of some metalaxyl- or dimethomorph-resistant mutants on tobacco leaves was similar to that of the wild-type strains.     

黑龙江省和吉林省马铃薯晚疫病菌对氟啶胺和甲霜灵的敏感性变化

2006-2008年在黑龙江省和吉林省共分离获得61株马铃薯晚疫病菌,测定了这些菌株对交配型、氟啶胺和甲霜灵的敏感性。交配型测定结果显示所有菌株都显示A1交配型。甲霜灵测定结果显示10个菌株(16.4%)显示为敏感性,11个菌株(18.0%)显示为中抗,40个菌株(65.6%)显示为抗性。这说明吉林省和黑龙江省发生的晚疫病菌已对甲霜灵产生抗性。氟啶胺敏感性测定结果表明,不同地区、不同年份分离的马铃薯晚疫病菌对氟啶胺的敏感性无显著差异, 其敏感性呈连续的单峰曲线分布, EC50在0.203 3~0.783 7 μg/mL之间, 最不敏感菌株是最敏感菌株的3.85倍, 平均值为(0.4781±0.0163)μg/mL, 未出现敏感性下降的抗药性群体, 因此可作为马铃薯晚疫病菌对氟啶胺的敏感性基线;83.6%的供试菌株对甲霜灵的敏感性下降, 甲霜灵与氟啶胺之间不存在交互抗药性。测定氟啶胺对马铃薯晚疫病的盆栽防治试验结果表明,氟啶胺可湿性粉剂、氟啶胺悬浮剂和烯酰吗啉能很好地控制晚疫病,防治效果分别为85.71%、87.00%、82.19%,而甲霜灵的为23.92%。