三种致病疫霉交配型分子检测方法的比较

致病疫霉Phytophthora infestans属于异宗配合卵菌,当A1、A2两种交配型同时存在时,可以进行有性生殖,产生卵孢子。检测疫霉菌交配型的传统方法是采用对峙培养,这种方法耗时长并且需要标准的A1、A2交配型菌株作为参照。因此,人们希望开发出更加简便和快捷的可直接基于核苷酸序列差异的分子检测方法。目前,已报道了3个与致病疫霉交配型紧密连锁的分子标记可用于交配型的检测。本研究用64株致病疫霉菌比较了3种基于交配型分子标记的检测方法与传统方法检测的结果。结果显示,依据分子标记的3种分子检测方法与传统对峙培养方法测定的交配型结果一致率为61%-73%,而且3种分子检测方法都不能检测出自育菌株。因此,致病疫霉交配型的分子检测方法还有待进一步研究。     

河北省番茄上致病疫霉Phytophthora infestans群体结构的分析

番茄晚疫病是河北省番茄生产上最具毁灭性的病害之一,对引起该病害的致病疫霉群体结构进行分析有利于病害的防治。利用对峙培养法和菌落直径法对2007-2008年采自河北省保定、沧州和唐山分离自番茄的49个致病疫霉菌株进行了交配型和甲霜灵抗性的表型测定,结果表明该群体所有菌株均为A1交配型,以甲霜灵敏感菌株为主,抗性菌株仅7株。利用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)、简单序列重复(SSR)和扩增片段长度多态性(AFLP)等分子技术对该群体的基因型进行了分析,结果表明供试菌株线粒体基因型均为Ia型,共鉴定出了Ⅰ、Ⅱ和Ⅲ3种SSR基因型,AFLP聚类分析在相似系数0.87时可以形成α、β和γ等3个不同的分支。河北省所有番茄上致病疫霉菌株均分布在α分支上,该分支又可进一步分为7个亚分支。AFLP亚分支与甲霜灵抗性和地理来源均无明显相关性,但Ⅱ型SSR与甲霜灵抗性和地理来源有明显的相关性。综合表型和基因型数据说明河北省番茄上致病疫霉群体结构比较单一,遗传多样性程度较低。     

疫霉蛋白质的聚丙烯酰胺凝胶盘状电泳

对北京,河北、青海、山东、湖北、广东等地不周寄主上收集的,属于 Phytophthoracapsici,P．Colocasiae,P．Heveac,P．Infestans, P．Melonis P．Nicotianac var. nicotianae 和 P．Nicotianae var. parasitica 7个种和变种的17株菌的蛋白质,进行了聚丙烯酰胺凝胶盘状电泳的分析和比较,经多批次试验,7个种和变种各自恒定地呈现典型的蚤白质图谱,显示出种间的差异。其中 P. infestans 占11株,它们虽采自不同地区和寄主,又分属5个生理小种,经多次电泳,电泳图谱基本相同,但不同菌株存在一些差异。同一株菌,培养条件对电泳图谱影响不大,不论是培养在含苹果酸的培养基上,还是培养在延胡索酸培养基上,电泳图谱基本一致,只是电泳材料必须新鲜。     

番茄幼苗对接种晚疫病菌的生理响应

以番茄晚疫病抗病品系CLN2037E、感病品系5号自交系及其杂种一代F1为材料,人工接种番茄晚疫病菌生理小种T1、T2、T3,研究番茄苗期抵抗晚疫病的生理响应.结果显示：（1）感病品系的膜脂过氧化产物丙二醛（MDA）与过氧化氢（H2O2）的含量增加幅度较大,抗病品系的含量则相对稳定,F1代的变化趋势接近感病品系;（2）抗病品系和感病品系的多酚氧化酶（PPO）、过氧化物酶（POD）和超氧化物歧化酶（SOD）活性变化都呈先升高后下降,抗病品系的酶活性高峰出现早且峰值小于感病品系.研究发现,抗病番茄品系幼苗的多项生理指标在接种晚疫病菌后变化幅度小而稳定,并能在短时间内基本恢复到原来的正常状态,从而表现出较强的抗病性.     

华南地区瓜类疫霉对甲霜灵的田间抗药性

【目的】了解华南地区瓜类疫霉(Phytophthora melonis)对甲霜灵的田间抗药性。【方法】2007-2010年从广西、广东两省(区)9个市冬瓜和黄瓜产区采集疫病样品,分离纯化瓜类疫霉,分别采用菌落生长速率法和叶盘漂浮法测定瓜类疫霉对甲霜灵的敏感性,并用药剂驯化方法从敏感性菌株诱导瓜类疫霉抗甲霜灵突变体。【结果】从9个市24个样点共分离纯化获得193株瓜类疫霉,抗药性检测结果表明,敏感菌株、中等抗性菌株和抗性菌株分别占测试菌株的29.0%、18.1%和52.8%;不同地区、不同寄主分离的菌株的抗性频率和抗性水平差异较大,来源于广东的菌株抗性频率和抗性水平一般高于来源广西的菌株,分离自黄瓜的菌株高于分离自冬瓜的菌株,大部分样点抗性菌株占据优势群体,个别菌株的抗性指数高达4226.9,叶盘漂浮法测定结果和菌落生长速率法相似;在含药平板上对敏感菌株进行甲霜灵抗性诱导结果表明,从60%的敏感菌株中成功诱导出对甲霜灵抗性稳定的突变体,突变体的抗性水平为敏感性亲本的189-407倍;9株来源于未施用过甲霜灵等苯基酰胺类杀菌剂样点的菌株均为敏感性菌株,其EC50值为0.0429-0.5461μg/mL,将它们EC50的平均值0.3200±0.1617μg/mL确定为华南地区瓜类疫霉对甲霜灵的敏感性基线;对两个样点的监测结果表明,瓜类疫霉抗甲霜灵菌株的频率及抗性指数有逐年增高趋势。【结论】华南广西和广东两省(区)瓜类疫霉对甲霜灵抗性普遍发生,瓜类疫霉对甲霜灵抗药性产生与其和药剂的接触密切相关。瓜类疫霉敏感性基线的建立,可为今后瓜类疫霉抗甲霜灵的评价和进一步监测提供科学依据。     

致病疫霉有性生殖诱导及F1代遗传多样性

致病疫霉Phytophthora infestans为马铃薯晚疫病的重要病原菌。通过从昆明市寻甸县采集110P和H-6两株致病疫霉,明确其染色体倍性、交配型、线粒体单倍型、毒性和甲霜灵敏感性,经对峙培养,利用改良的卵孢子萌发方法获得有性生殖F1代群体POP1（60株）,并对POP1进行表型和基因型测定。结果表明：冷冻处理24h为最佳条件,卵孢子萌发率达5.09%±0.15%;POP1的交配型、毒性和甲霜灵敏感性均发生了分离,其中交配型分离比为A1:A2:A1A2:自育型（SF）=16:5:17:22,毒性分离比为抗性（R）:敏感性（S）=11:49,甲霜灵敏感性分离比为抗性（R）:敏感性（S）=2:58;3个表型的分离均偏离孟德尔单基因显性遗传特点。基于8对SSR多态性引物对POP1基因型分析表明,遗传相似系数为0.98时,可将所有菌株分为14个基因型;遗传相似系数为0.95时,可将POP1分为6个分支,其中优势群体为S1,占分离群体的61.67%。关联分析进一步表明,8对SSR所代表的基因型和几个重要表型有显著相关性（R2=0.6667）。本研究建立了高效的致病疫霉卵孢子萌发体系,解析了有性生殖后代群体遗传结构特点,为深入探索致病疫霉的变异规律及病害流行趋势提供了理论基础。     

致病疫霉超级生理小种遗传多样性分析

通过交配型和甲霜灵抗性以及线粒体DNA单倍型、SSR和AFLP基因型分析对40个超级生理小种菌株进行了遗传多样性分析。在被测菌株中发现了A1、A2和自育3种不同类型的交配型。其中,A1和自育型菌株数量多,分别为21株和14株,而A2交配型仅5株。甲霜灵抗性测定检测出高抗菌株26株,敏感菌株14株。线粒体DNA单倍型测定出Ia型和IIa型两种,比例接近1:1。基于5个基因座被测40个超级生理小种菌株共鉴定出了7种SSR基因型。利用6对荧光引物共检测到258条AFLP谱带,其中多态性谱带204条,多态性为79.1%。将供试的40个菌株划分为38个基因型,几乎每个菌株都为1个特有基因型。而且,我国南方和北方超级生理小种群体存在着明显的遗传差异。结果表明我国致病疫霉超级生理小种具有丰富的遗传多样性,可以推断致病疫霉中的任何小种都可在多个抗病基因的强大选择压力下,在短时间内通过与之对应的无毒基因快速突变而成为超级生理小种。当前对致病疫霉生理小种的鉴定及监测对生产上利用抗病品种防控晚疫病的指导意义不大。     

紫外光诱导苎麻疫霉抗甲霜灵突变及对苎麻疫霉生物学特性的影响

作者研究了紫外光对苎麻疫霉的抗甲霜灵诱变效应及对苎麻疫霉生物学特性的影响。结果表明,供试6个苎麻疫霉野生型菌株经菌丝块紫外光药剂诱变和菌丝块药剂驯化3周后,均获得抗甲霜灵突变体,且紫外光药剂诱变处理角变区出现频率明显高于药剂驯化处理,说明紫外光对苎麻疫霉抗甲霜灵突变有一定促进效应。紫外光显著地抑制苎麻疫霉游动孢子的萌发和菌丝的生长,在一定范围内,处理时间愈长,抑制率愈高。苎麻疫霉耐紫外光菌株(经亚致死剂量的紫外光照射处理后存活的游动孢子所形成的菌株)与野生型亲本相比,对温度和pH的敏感性大致相同,但菌落形态有一定变异,菌丝生长速率、卵孢子产生量均显著下降,表明紫外光对苎麻疫霉的菌丝生长和卵孢子产生量有明显的抑制作用。苎麻疫霉耐紫外光菌株EC50值比野生型亲本菌株EC50值提高了23.21%-56.70%,即耐紫外光菌株对甲霜灵敏感性比野生亲本菌株显著下降,这与紫外光诱变试验结果是相一致的。     

掘氏疫霉遗传学研究II．交配型遗传与变异的研究

本研究以掘氏疫霉P“yl口外埔o,o dr~cksleri丁ucke r野生型菌株的单游动孢子无性系为亲本,测定了自交、杂交后代交配型的遗传,经Kmno·处理引起的交配型变异以及Ft代可自孕单卵孢株有性生殖后代交配型的遗传与变异。聚碳膜间隔配对诱导A,和^,菌株自交产生的卵孢子经H zot处理索0激萌发(萌发率1 z～16％)获得单卵孢株。交配型测定结果表明,^t和A,亲本自交st代单卵孢株均保持与亲本一致的交配型。用KMnO．处理上述卵孢子导致A-和^z亲本的部分s一代单卵孢株出现自孕现象,少数A,亲本自交后代改变为A：交配型;从A。交配型亲本的s-代可自孕菌株产生的卵孢子萌发所建立的单卵孢株中,同时获得扎和^。交配型菌株。上述结果不支持San somc关于疫霉菌AhA,交配型分别由纯合、杂合基因控制的假说,进一步证明了Ko关于交配型抑制因子控制交配型表达的假说的合理性。 掘氏疫霉种内菌株直接配对产生的卵孢子用HtO,刺激萌发获得Ft代单卵孢拣。测定结果表明,在F。代出现A。A,、A,A;、A口4种交配型的单卵孢株,其比例因不同亲本组合而有较大差异oF。代出现的^。A,菌株自孕产生的卵孢子经HtO,处理刺激萌发建立自交系,观察到^。^：交配型在有性生殖后代发生分离,各交配型比例因亲本不同而异o A·At菌株的自孕能力在单游动孢子无性系后代可稳定遗传,认为F-代出现的AJAt个体来自亲本的杂交。AtA-单卵孢株保藏4～6个月后大多仍具自孕能力,少数改变为Az交配型。     

Potato late blight in Morocco: characterization of Phytophthora infestans populations (virulence and mating type)

Late blight caused by Phytophthora infestans, is the most important disease of potato in Morocco. Use of partially resistant cultivars should be an essential component of a sustainable management strategy of potato late blight, provided the durability of this form of resistance. It is therefore important to determine the nature of P. infestans Moroccan populations. Mating types were determined for 91 strains of P. infestans collected in the northern (Larache-northern plain), north western (Kénitra) and north eastern (Méknès, Middle Atlas) potato cropping areas of Morocco in 1999-2000, 2000-2001 and 2003-2004. They showed a clear regional structure of these populations, with the presence of both mating types (A1 and A2). Of all isolates collected since 1999, A2 mating type constituted 56% (54 isolates), following by A1 mating type (40.7%, 31 isolates) and A1-A2 (self-fertile) mating type (3.30%, 3 isolates). Populations from Méknès and Kénitra consisted mainly of A2 mating type, whereas populations from Larache predominantly included A1 mating type. Physiological race study revealed the presence of 19 races of P. infestans in the first collection of 25 isolates tested between 1999 and 2001. All known virulence genes were detected in western and northern Moroccan isolates, except virulence for resistance genes R2, R5, and R6 which were absent. All isolates were able to overcome two or more R genes except one isolate (5-1) corresponding to race 1.     

马铃薯晚疫病菌DK98-1和HD01-3无性后代生物学特性的研究

在已明确我国存在甲霜灵抗性菌株的基础上,通过单游动孢子分离技术获得马铃薯晚疫病菌DZO1和HZO1无性后代菌系。研究了无性单游动孢子后代菌系的生物学特性及甲霜灵抗性,主要结果如下:对无性后代菌系的菌落形态、生长量和产孢能力进行测定,发现被测的DZO1和HZO1无性后代菌系大部分菌株的生物学性状与亲本菌株相比没有发生明显的变异,个别菌株的生物学性状与亲本菌株有显著差异,说明晚疫病菌的无性繁殖未引起较大的变异;对后代菌系的甲霜灵抗性水平进行测定,结果表明DZO1和HZO1无性后代菌系的甲霜灵抗性水平与其对应的亲本菌株基本相同。     

Mating type and sensitivity of Phytophthora nicotianae from tobacco to metalaxyl and dimethomorph in Henan province,China

Phytophthora nicotianae causes black shank, one of the most important diseases of tobacco worldwide. Metalaxyl and dimethomorph are two fungicides which have been used widely for control of this disease in Henan province, China. A study was conducted to determine the level of metalaxyl and dimethomorph sensitivity in isolates of P. nicotianae from tobacco in Henan province and mating type structure of the pathogen population. A total of 32 isolates were isolated from 11 cities in Henan province. Sensitivity of all isolates to metalaxyl and dimethomorph was tested in vitro, and mating types of all isolates were determined by pairing known A1 and A2 testers. For metalaxyl, EC₅₀ values of 32 P. nicotianae isolates ranged from 0.08 to 2.82 mg/L. Sixteen isolates were sensitive, and the rest were intermediate to metalaxyl. None were classified as resistant isolates. For dimethomorph, EC₅₀ values of 32 P. nicotianae isolates ranged from 0.07 to 0.59 mg/L. All isolates were sensitive to dimethomorph. Thirty‐one isolates were A2 mating type, and one isolate was A0 mating type. No isolate was identified as A1 mating type. These results suggested that the P. nicotianae population in Henan province has already exhibited intermediate resistance to metalaxyl and was still sensitive to dimethomorph, and asexual reproduction was the major form of reproduction for the P. nicotianae population.     

Dominance and recessiveness at loci for virulence against potato and tomato in Phytophthora infestans

Summary In this study we investigated the genetic control of virulence in the diploid fungal pathogen, Phytophthora infestans, against host resistance genes R1, R2, R3, and R4 (potato) and Ph1 (tomato). For four of these virulence traits, the presence or absence of segregation indicated conclusively which phenotype was dominant. We observed a 31 (virulentavirulent) segregation on R2 in the progeny of parents which were both virulent, suggesting that virulence is dominant and both parents are heterozygous. In a cross in which one parent was virulent and the other avirulent on potato gene R3, all progeny tested were avirulent, so avirulence against R3 is dominant. The same virulent parent crossed with a different avirulent parent produced virulent and avirulent progeny in a 13 ratio, indicating that a second locus may be involved. The progeny of two parents virulent on R4 segregated for virulence and avirulence, so virulence against R4 is dominant. For Ph1, a 13 segregation in the progeny of two avirulent parents showed that the avirulent phenotype is dominant, and a 31 ration in a second cross suggested the involvement of a second locus. The segregations for virulence against R1 did not indicate which phenotype was dominant, but did suggest singlelocus control.     

甲霜灵对大豆疫霉野生菌株及突变菌株生长发育影响的细胞学研究

本文利用电子显微镜技术研究了内吸性杀菌剂甲霜灵(Metalaxyl)对大豆疫霉Phytophthoras ojae野生菌株和突变菌株的形态学及超微结构的影响。结果表明:不同浓度甲霜灵处理后可导致野生菌株和突变菌株发生一系列不同的变化。低浓度(1μg/mL)处理后,野生菌株在培养基上的生长即可受到抑制,菌丝呈现不规则的肿胀、过度分枝;菌丝细胞壁不规则加厚,菌丝细胞内液泡增加,脂肪粒累积,细胞器排列紊乱,原生质最终坏死。随浓度的升高,野生菌株立即停止生长,菌丝干瘪坏死。而突变菌株只在高浓度(10μg/mL)甲霜灵处理后顶端菌丝出现少量较小的分枝,菌丝细胞壁无增厚现象,但细胞内脂肪粒大量积累,明显高于敏感性菌株;突变菌株在高浓度甲霜灵压力下仍继续生长。     

Occurrence and management of fungicide resistance in Botrytis cinerea on tomato from greenhouses in Hebei,China

Grey mould, caused by the fungal pathogen Botrytis cinerea, is one of the most devastating tomato diseases, and the control of this disease is mainly by the application of chemicals. In this study, 512 isolates of B. cinerea were collected from tomato grown in greenhouses at 10 locations in 10 cities of Hebei Province from 2011 to 2016 and tested for their sensitivities to carbendazim (Car), diethofencarb (Die), iprodione (Ipr) and pyrimethanil (Pyr). Of these tested isolates, 95.7%, 95.2%, 31.6% and 89.4% were resistant to Car, Die, Ipr and Pyr, respectively. There were nine fungicide‐resistant phenotypes in the tested isolates. Car^RPyr^RDie^RIPR^S and Car^RPyr^RDie^RIPR^R were the most common phenotypes, accounting for 59.6%, and 31.1% of the tested isolates, respectively. The field trials showed that the control efficacies (CE) of carbendazim + diethofencarb (WP, 25% + 25%), pyrimethanil (EC, 40%) and iprodione (WP, 50%) at the recommended doses were 22.75%–29.23%, 58.44%–64.19% and 61.02%–65.17%, respectively, significantly lower than those of boscalid (WG, 50%) and pyrisoxazole (EC, 25%). The resistance management trial conducted from 2015 to 2017 indicated that the CE of tomato grey mould in the experimental fields was higher than 90% and the sensitivity to carbendazim, diethofencarb and pyrimethanil of B. cinerea isolates from the experimental fields increased on a yearly basis. These results showed that the frequency of resistance to Car, Die, Ipr and Pyr was high, and these four fungicides could not effectively control tomato grey mould. Tomato grey mould could be controlled by using biopesticides and newly synthesized fungicides with different modes of action. Our findings would be useful in designing and implementing fungicide resistance management spray programmes for the control of tomato grey mould.