Phenotypic and Genotypic Characterization of Phytophthora infestans Isolates from China

A total of 288 (202 from potato and 86 from tomato) isolates of Phytophthora infestans were collected from 1998 to 2007 in China. The isolates were characterized based on mating type, in vitro metalaxyl sensitivity, virulence on potato differentials, allozymes of glucose-6-phosphate isomerase ( Gpi ), peptidase ( Pep ), and mitochondrial DNA (mtDNA) haplotype and examined by DNA-based simple sequence repeat (SSR) and random amplified polymorphic DNA (RAPD) fingerprinting. The majority (283 of 288) of the isolates were of the A1 mating type, the other three were the A2 mating type and two were the A1A2 mating type. Resistance to metalaxyl was frequently observed, with 248 (86.1%) resistant, 21 (7.3%) intermediate and 19 (6.6%) sensitive isolates identified. Virulence was assessed for 125 isolates on a set of 11 potato differentials and 61 races were detected. Most isolates were virulent on the differential genotype with gene R3, and all known virulence genes were found, with race 3.4.7.11 being the most common. This pattern did not appear to be associated with geographic origin, sample type, mating type or metalaxyl sensitivity. The dominant banding patterns for Gpi were 100/100/111 (176 isolates) and 100/100 (109 isolates), but genotypes 86/100 and 100/111 were also identified. All isolates tested were homozygous (100/100) at the Pep locus. The majority (205 of 288) of isolates tested was of mtDNA haplotype IIb, 76 were haplotype IIa and seven were the rare Ib haplotype. The genetic diversity of 60 representative isolates from China was assayed by two types of molecular markers, RAPD and SSR. A high level of polymorphism was found. The results demonstrated the diverse phenotypic and genotypic structure of the current populations of P. infestans in China.     

Phenotypic and Genotypic Changes in the Phytophthora infestans Population in Taiwan – 1991 to 2006

Late blight, caused by Phytophthora infestans , is one of the most destructive diseases of tomato in Taiwan. A total of 655 isolates of P. infestans , including 29 isolates from potato, was collected from major tomato and potato production areas of Taiwan during 1991 to 2006. Isolates were characterized for their pathogenicity, mating type, in vitro metalaxyl sensitivity and molecular genotype (including allozyme pattern, mitochondrial genomic haplotype and DNA fingerprint) to monitor population changes in P. infestans . The population of P. infestans in Taiwan underwent a dramatic genetic shift in the 1997–1998 cool growing season. Isolates collected from tomato before 1997 were aggressive to tomato but not potato; most isolates obtained after 1998, were aggressive to both hosts. Metalaxyl sensitivity of isolates changed from sensitive/intermediate before 1997 to resistant since 1998. Similarly, the isolates obtained before 1997 were all US-1 clonal lineage (including variants US-1.1, US-1.2, US-1.3 and US-1.4). During the 1997–1998 cool growing season, the US-11 clonal lineage and the TW-1 genotype appeared, possibly introduced on imported table potatoes. The US-11 lineage spread rapidly and since 1999 has almost completely displaced the old population in Taiwan. Mating type determined by pairing with A1 and A2 reference isolages of P. parasitica , showed all isolates were of the A1 mating type, suggesting that the A2 mating type had not become established in Taiwan. The increasing percentage (up to 42.3% in 2006) of the US-11 variants (including US-11.l, US-11.2, US-11.3 and US-11.4) implied that genomic diversity of the pathogen is changing quickly. Therefore, it is important to continuously monitor the population changes of P. infestans and develop an integrated management strategy for this disease.     

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.     

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。     

Genetic and Physical Variability at the Mating Type Locus of the Oomycete, Phytophthora Infestans

Mating type in the oomyceteous fungus, Phytophthora infestans, is determined by a single locus. In a previous study of a few isolates, the locus segregated in a manner genetically consistent with its linkage to a system of balanced lethal loci. To determine the prevalence of this phenomenon within P. infestans, genetic analyses were performed using isolates representative of the diversity within the species that had been selected by DNA fingerprinting using probes linked to mating type. Non-Mendelian segregation of the mating type locus was observed in crosses performed with each isolate. An unusual group of isolates was identified in which the mating type determinants had been rearranged within the genome; these strains also produced an aberrantly large number of self-fertile progeny. Curiously, in all isolates, markers linked to the mating type locus appeared prone to duplication, transposition, deletion, or other rearrangement. This was not observed for loci unlinked to mating type. Data from the crosses and analyses of marker variation were used to erect models to explain the bases of mating type determination and of the unusual segregation of the chromosomal region containing the mating type locus.     

Phytophthora infestans in Poland from 1987–1989; Nuclear DNA Content, Mating Type Distribution and Response to Metalaxyl

Isolates of Phytophthora infestans were collected from all potato growing regions of Poland during the blight seasons of 1987—1989. All 1987 isolates were of Al mating type and were sensitive to metalaxyl. In 1988 and 1989, 46.5 % and 55.3 % of the isolates were sensitive to metalaxyl, respectively. The percentage of highly resistant (R) isolates increased from 25.6 % in 1988 to 39.5 % in 1989; however the percentage of intermediately resistant (I) isolates decreased during that period from 27.9 % to 5.3 %. A significant association was observed between the A1 compatibility type and metalaxyl resistance. The A2 mating type first appeared in 1988, and its frequency increased from 4.7 % of the population in 1988 to 47.6 % in 1989. Coincident with this change in mating type frequency, changes in ploidy levels of isolates were observed. Whereas 3 % of the 1988 isolates were diploid, 90 % of the 1989 A2 isolates and 28.6 % of the 1989 Al isolates were diploid. The approximate 1:1 ratio of the two mating types encountered in 1989, and the predominance of diploidy, indicates that the Polish population of P. infestans has the potential to become sexual.     

Genetic analysis of Phytophthora infestans populations in the Nordic European countries reveals high genetic variability

Late blight, caused by the oomycete Phytophthora infestans, is the most important disease of potato (Solanum tuberosum). The pathogen is highly adaptable and to get an overview of the genetic variation in the Nordic countries, Denmark, Finland, Norway and Sweden we have analyzed 200 isolates from different fields using nine simple-sequence repeat (SSR) markers. Forty-nine alleles were detected among the nine SSR loci and isolates from all four Nordic countries shared the most common alleles across the loci. In total 169 multilocus genotypes (based on seven loci) were identified among 191 isolates. The genotypic diversities, quantified by a normalized Shannon's diversity index (H(s)), were 0.95 for the four Nordic countries. The low F(ST) value of 0.04 indicates that the majority of variation is found within the four Nordic countries. The large number of genotypes and the frequency distribution of mating types (60% A1) support the hypothesis that sexual reproduction is contributing notably to the genetic variation of P. infestans in the Nordic countries.     

Genotypic Analysis of Russian Isolates of Phytophthora infestans from the Moscow Region, Siberia and Far East

Phytophthora infestans samples were collected during 1997 and 1998 at multiple sites in Russia from Sakhalin Island in the Far East across Siberia (nine sites, 160 isolates) to the Moscow region (four sites, 325 isolates). In addition, 12 isolates that were obtained previously were included. All isolates were analysed for mating type, and sensitivity to metalaxyl. Isolates from within any of the nine sites outside of the Moscow region were monomorphic for mating type and nearly monomorphic for metalaxyl resistance. In contrast, both A1 and A2 isolates were detected in the Moscow region, and these isolates were also polymorphic for metalaxyl resistance. In two sites in Siberia only A2 mating type strains were detected, in the other six sites in Siberia and in Sakahlin Island, only A1 mating types were detected. A subset of isolates ( n =191) was also analysed for pathotype (virulence to 10 R-genes, each in a distinct differential genotype). All isolates were highly complex (mean number of virulences approximately 8.4 of a maximum number of 10). All isolates ( n =43) from Sakahlin Island were virulent to all 10 of the R-genes tested. A further subset of isolates ( n =70, including 12 isolates collected before 1997) was analysed for genotype at the Glucose- 6-phosphate isomerase and Peptidase loci, mtDNA haplotypes, and RFLP pattern using the RG57 probe. The US-1 clonal lineage (previously dominant) was not detected in the 1997–98 sample. The populations of P. infestans near Moscow in 1997 and 1998 was highly diverse with 15 unique genotypes (including both mating types) among a sample of 18 isolates. In contrast, the populations of P. infestans in Siberia had limited diversity, with only three multilocus genotypes detected and most populations were dominated by the SIB-1 clonal lineage. This lineage accounted for 31 of the 39 strains collected in Siberia that were assayed for multilocus genotype.     

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。     

中国部分地区马铃薯寄主上致病疫霉SSR基因型分析

利用两对SSR引物对两个基因座Pi4B和Pi4G进行了PCR扩增,测定了中国部分地区66个致病疫霉Phyophthora infestans(马铃薯晚疫病菌)菌株和2个参考菌株的SSR基因型,并对菌株的基因型进行了鉴定和命名.在被测定的66个致病疫霉菌株中,共产生了7种SSR基因型D-03,D-05,D-06,G-02,H-01,F-01和F-06,其中F-06为本研究新命名的基因型.F-01基因型菌株53个,占总菌株数目的80.3%,该基因型为中国致病疫霉的优势基因型.在对两个基因座Pi4B和Pi4G产生的等位基因统计分析发现基因座Pi4B产生的多样性比Pi4G高.对SSR数据揭示的河北、黑龙江和云南3个不同省份致病疫霉遗传多样性的比较发现,河北省和黑龙江省致病疫霉遗传多样性几乎相同,然而与云南省致病疫霉有较大的遗传差异.此外,发现致病疫霉SSR基因型与其对甲霜灵抗性无相关性.     

中国番茄晚疫病菌交配型及其分布研究

本文对中国番茄晚疫病菌交配型的发生和分布及病菌的生物学特性进行了研究,结果表明,我国主要番茄产区晚疫病菌以A1交配型为主,占总菌株数的96.02%;1999~2001年间从全国18个省市采集的201个番茄晚疫病菌株中,仅发现8个A2交配型菌株,分布于广西、云南、河北和福建;被测菌株在10%V8培养基上菌落生长均受到一定的限制,而在黑麦培养基上菌落的生长速度、产孢量有明显的差异,但菌落形态与交配型类型不相关。     

中国马铃薯晚疫病菌AFLP遗传多样性分析

应用AFLP分子标记检测了我国部分马铃薯主要产区马铃薯晚疫病菌的遗传多样性及不同地区菌株间的亲缘关系。在200对引物组合中,利用6个菌株筛选出12对多态性好、带型清晰的引物组合。利用这12对引物组合对1997-2002年间采自我国黑龙江、河北、四川和云南4省的50株菌株进行了PCR扩增,共扩增出922条谱带,其中多态性标记530条,占57.5%。利用NTSYSpc软件中UPGMA算法构建了我国马铃薯晚疫病菌的亲缘关系树状图,聚类分析结果表明我国马铃薯晚疫病菌的遗传多样性与病原菌的地理来源有一定的相关性,而与交配型、生理小种和对甲霜灵的抗性无明显的相关性。用POPGENE软件计算了各群体间的遗传多样性参数,结果表明我国马铃薯晚疫病菌的遗传多样性程度不高,不同地区种群间分化不明显。     

Chromosomal heteromorphism and an apparent translocation detected using a BAC contig spanning the mating type locus of Phytophthora infestans

Genetic and physical irregularities associated with the mating type locus of the oomycete, Phytophthora infestans, were revealed by analyzing a contig spanning the locus that was constructed using a bacterial artificial chromosome library. Contigs from both homologs of an A1 strain A/a genotype at mating type locus) had chromosome-specific differences, flanked by regions of similarity. Such heteromorphism was detected within multiple isolates. The mating type locus was narrowed to a 60-70kb interval by genetic mapping of candidate genes, identified using a cDNA library. During these analyses, an unusual isolate of P. infestans was identified in which the mating type determinant had apparently translocated from its location in typical strains. Comparative mapping of the cDNAs between P. infestans and P. parasitica revealed partial synteny between the species however; substantial rearrangements existed and no cDNA was tightly linked to mating type in P. parasitica. These findings add to previous observations of unusual genetic behavior involving mating type in Phytophthora.     

Inhibition of fungal and bacterial plant pathogens by synthetic peptides: in vitro growth inhibition, interaction between peptides and inhibition of disease progression

Four synthetic cationic peptides, pep6, pep7, pep11 and pep20, were tested alone and in combinations for their antimicrobial activities against economically important plant pathogenic fungi (Phytophthora infestans and Alternaria solani) and bacteria (Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica). In in vitro studies, P. infestans and A. solani were inhibited by all four peptides, while E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica were inhibited only by pep11 and pep20. All peptides completely inhibited P. infestans and A. solani on potato leaves and P. infestans on tubers at concentrations comparable to the in vitro IC50 (effective concentration for 50% growth inhibition) values, suggesting that these peptides are more potent in preventing infection than in inhibiting hyphal growth in vitro. Microscopic observations of P. infestans and A. solani when treated with these peptides revealed hyphal anomalies. In tuber-infectivity assays, pep11 and pep20 reduced bacterial softrot symptoms by 50% at 2.0 to 2.30 microM and by 100% at 20 microM. In assays involving two-way combinations of these peptides, growth inhibitions of fungi and bacteria by the combinations were no more than the sum of growth inhibitions by each peptide when used alone, indicating that they act additively. pep11 and pep20 are not phytotoxic to potato plants at 200 microM. With strong and broad-spectrum antimicrobial activities of pep11 and pep20 against fungi and bacteria, and with no antagonistic activities, the expression of these peptides in transgenic potato plants could lead to enhanced disease resistance against these pathogens.     

Purification of an isoform of patatin with antimicrobial activity against Phytophthora infestans.

Phytophthora infestans (Mont.) de Bary is infamous as the causal agent of the late blight epidemic contributing to the Irish potato famine of the mid 19th century and remains agriculture's most destructive disease as new mutations and migrations confound control measures. In efforts to develop resistant varieties, a somatic hybrid (the Wisconsin J series) between potato (Solanum tuberosum) and a wild relative (Solanum bulbocastanum) has been found to convey durable resistance against the pathogen. We screened the total protein (100 microg ml(-1)) of somatic hybrid varieties J138, J138A12, J101K12, J103K12, and J101K9 for in vitro spore germination inhibition of P. infestans. Since J138 exhibited maximum inhibition at 150 microg ml(-1) in comparison to other varieties, we purified a 40 kD protein from J138 tubers by assaying its ability to inhibit spore germination in P. infestans spores. The highly purified protein was able to inhibit P. infestans spore germination by 70% at the 2.5 microg ml(-1) concentration. The N-terminal sequence of this protein was found to have exact amino acid homology to patatin, the major storage protein of potato tubers. The inhibitory protein has the same molecular weight as patatin and cross-reacts with patatin antibodies. The infection of J138 plants with spores of P. infestans under greenhouse conditions showed that patatin is expressed in stem tissue 72 h after the plant is inoculated with field isolates of P. infestans (US8). In this communication, we report the purification, characterization and antifungal activity against spores of P. infestans of patatin-J from potato tubers.     

马铃薯晚疫病生防木霉菌的筛选及鉴定

采用马铃薯活体筛选法从268株木霉菌中筛选获得两株对致病疫霉有较强抑菌活性的木霉菌株R-5和T-15。这两株木霉菌代谢液可抑制病原菌生长及孢子囊萌发。温室防病试验发现,接种两株木霉菌可以减轻晚疫病的发生。田间试验进一步证明,两株木霉菌对晚疫病具有良好的田间防治效果,防效分别达到了72.4%和70.0%。经分子生物学方法鉴定,两株木霉菌分别为拟康氏木霉和棘孢木霉。实验构建的以活体筛选为基础的生防木霉菌筛选方法是一种可行高效的生防木霉菌筛选方式。     

Genetic Mapping and Non-Mendelian Segregation of Mating Type Loci in the Oomycete, Phytophthora Infestans

DNA markers linked to the determinants of mating type in the oomycete, Phytophthora infestans, were identified and used to address the genetic basis of heterothallism in this normally diploid fungus. Thirteen loci linked to the A1 and A2 mating types were initially identified by bulked segregant analysis using random amplified polymorphic DNA markers (RAPDs) and subsequently scored in three crosses as RAPD markers, restriction fragment length polymorphisms (RFLPs), single-strand conformational polymorphisms (SSCP), cleaved amplified polymorphisms (CAPS), or allele-specific polymerase chain reaction markers (AS-PCR). All DNA markers mapped to a single region, consistent with a single locus determining both mating types. Long-range restriction mapping also demonstrated the linkage of the markers to one region and delimited the mating type locus to a 100-kb region. The interval containing the mating type locus displayed non-Mendelian segregation as only two of the four expected genotypes were detected in progeny. This is consistent with a system of balanced lethal loci near the mating type locus. A model for mating type determination is presented in which the balanced lethals exclude from progeny those with potentially conflicting combinations of mating type alleles, such as those simultaneously expressing A1 and A2 functions.     

Phytophthora infestans isolates from Northern China show high virulence diversity but low genotypic diversity

Phenotypic and genotypic characteristics of 48 Phytophthora infestans isolates , collected in five provinces in Northern China between 1997 and 2003, were determined and compared with reference isolates. Characterisation included mating type, virulence, mitochondrial DNA (mtDNA) haplotype and DNA fingerprinting patterns based on simple sequence repeats (SSR) and amplified fragment length polymorphisms (AFLP). All isolates had the A1 mating type, mtDNA haplotype IIa and an identical SSR genotype (designated as SG-01-01) that differed from SSR genotypes found in the reference isolates, including those representing the 'old' US-1 lineage that dominated the P. infestans population worldwide prior to 1980. In contrast, the virulence spectra were highly variable and virulence to all resistance genes present in the standard differential set ( R1 to R11 ) was found. AFLP analysis revealed some diversity; eight different AFLP genotypes were found that could be grouped into two major clusters. This study shows that there is very little genotypic diversity in the P. infestans population in Northern China. The occurrence of many different races within this rather uniform population is discussed in the framework of recent insights into the molecular determinants of avirulence in potato– P. infestans 'gene-for-gene' interactions.     

Genetic diversity and differentiation of Indian isolates of Phytophthora infestans as revealed by RAPD analysis

Sixty-seven isolates of Phytophthora infestans collected from Himalayan hill regions and subtropical planes of India were characterized by RAPD markers to assess diversity and differentiation based on location of origin. Ten random decamer primers generated 161 polymorphic fragments. Association of P. infestans isolates on the dendrogram and PCO plot revealed two clear grouping based on geographical location of origin-hill isolates and plane isolates. Quantification of diversity by Shannon index of diversity analysis demonstrated that most of the diversity was present with a particular population (hill or plane) of P. infestans isolates, with 85% variation being within and 15% being between hill and plane isolates. Subtropical plane isolates of P. infestans exhibited higher variability compared to hill isolates and they were more dispersed on the PCO plot. No clear differentiation of isolates based on mating type was reflected on the dendrogram and PCO plot.     

利用马铃薯晚疫病菌粗毒素离体筛选马铃薯抗晚疫病无性系

以3个马铃薯品种叶片外植体的愈伤组织为材料、马铃薯晚疫病病菌培养粗毒素为筛选压力进行马铃薯抗晚疫病细胞变异无性系筛选。结果表明,不同品种的叶片、愈伤组织对毒素有不同的忍耐力。“费乌瑞它”最大忍耐的粗毒素浓度为40％,“东农303”、“早大白”则为50％。马铃薯晚疫病病原菌粗毒素对愈伤组织的诱导、生长、不定芽的分化及再生苗生根有显著的抑制作用,而且浓度越大,抑制作用越强。筛选结果抗性鉴定表明,经过粗毒素筛选的细胞无性系及其再生植株对高浓度粗毒素的抗性能力显著高于对照。