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.     

云南番茄致病疫霉的交配型、甲霜灵敏感性及毒力类型

对2003~2004年云南省番茄致病疫霉的交配型、甲霜灵敏感型、毒性因子及毒力类型进行了系统的测定和分析。结果表明:云南番茄致病疫霉只存在A1交配型,未发现A2交配型或自育型。甲霜灵抗性和中抗菌株是主要菌系,抗性、中抗、敏感菌株分别占测定菌株的51.2%、31.7%、17.1%。云南番茄致病疫霉居群对已知的抗性基因R1、R2、R3、R4、R6、R7、R8、R9、R10、R11有毒性,其毒性频率在9.8%~95.1%之间。测定的61个菌株中检测到26种不同的毒力类型,每种类型平均含有5.2个毒性因子,其中毒力类型1.3.4.7.9出现频率最高,为优势的毒力类型,出现频率为44.3%,其次为1.3.4.6.7.9、1.3.7.9,出现频率均为4.9%,其它23种毒力类型出现频率仅在1.6%~3.3%之间。云南番茄致病疫霉居群显示了复杂的表型特征。     

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。     

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.     

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.     

四川省马铃薯晚疫病菌群体表型和遗传变异的分析

致病疫霉Phytophthora infestans引起的晚疫病是马铃薯的一种毁灭性病害。为了对四川省近年马铃薯晚疫病菌进行系统多样性分析,本研究从表现型和基因型两个方面鉴定了四川省马铃薯晚疫病菌的群体多态性。交配型、甲霜灵敏感性和生理小种的鉴定结果发现A1交配型菌株只有2份,A2型有29份,自育型12份;甲霜灵敏感菌株3株,中抗菌株22株,高抗菌株18株;共测定出11个生理小种,其中全毒力小种发生频率最高,占供试菌株的25.58%。基因型鉴定结果表明,43份材料共发现了10种SSR基因型,5个SSR标记在该种群上共产生了16个SSR位点,每个标记平均有3.2个位点,多态性信息含量平均值为0.46,SSR4是多态性最丰富的标记。本试验中A2菌株和自育型的基因型SSR相同,其中全毒力小种是当地的优势致病菌,该研究为今后四川马铃薯晚疫病的有效防控提供理论依据。     

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.     

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。     

The R3 resistance to Phytophthora infestans in potato is conferred by two closely linked R genes with distinct specificities

The R3 locus of potato (Solanum tuberosum L.) confers full resistance to avirulent isolates of Phytophthora infestans, the causal agent of late blight. R3 resides in the distal part of chromosome 11 and segregates in a potato mapping population, from which a well-saturated amplified fragment length polymorphism map is available. Using a population of 1,748 plants, we constructed a high-resolution genetic map at the R3 locus. Using the combination of fine mapping and accurate disease testing with specific P. infestans isolates, we detected that the R3 locus is composed of two genes with distinct specificities. The two genes R3a and R3b are 0.4 cM apart and have both been introgressed from S. demissum, the 'donor' species of most characterized race-specific R genes to P. infestans. A natural recombinant between R3a and R3b was discovered in one accession of S. demissum. The synteny between the R3 locus and the tomato I2 locus is discussed.     

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.     

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。     

Qualitative and quantitative late blight resistance in the potato cultivar Sarpo Mira is determined by the perception of five distinct RXLR effectors

Potato defends against Phytophthora infestans infection by resistance (R)-gene-based qualitative resistance as well as a quantitative field resistance. R genes are renowned to be rapidly overcome by this oomycete, and potato cultivars with a decent and durable resistance to current P. infestans populations are hardly available. However, potato cultivar Sarpo Mira has retained resistance in the field over several years. We dissected the resistance of 'Sarpo Mira' in a segregating population by matching the responses to P. infestans RXLR effectors with race-specific resistance to differential strains. The resistance is based on the combination of four pyramided qualitative R genes and a quantitative R gene that was associated with field resistance. The qualitative R genes include R3a, R3b, R4, and the newly identified Rpi-Smira1. The qualitative resistances matched responses to avirulence (AVR)3a, AVR3b, AVR4, and AVRSmira1 RXLR effectors and were overcome by particular P. infestans strains. The quantitative resistance was determined to be conferred by a novel gene, Rpi-Smira2. It was only detected under field conditions and was associated with responses to the RXLR effector AvrSmira2. We foresee that effector-based resistance breeding will facilitate selecting and combining qualitative and quantitative resistances that may lead to a more durable resistance to late blight.     

The R1 gene for potato resistance to late blight (Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant resistance genes

Late blight caused by the oomycete Phytophthora infestans is the most destructive disease in potato cultivation worldwide. New, more virulent P. infestans strains have evolved which overcome the genetic resistance that has been introgressed by conventional breeding from wild potato species into commercial varieties. R genes (for single-gene resistance) and genes for quantitative resistance to late blight are present in the germplasm of wild and cultivated potato. The molecular basis of single-gene and quantitative resistance to late blight is unknown. We have cloned R1, the first gene for resistance to late blight, by combining positional cloning with a candidate gene approach. The R1 gene is member of a gene family. It encodes a protein of 1293 amino acids with a molecular mass of 149.4 kDa. The R1 gene belongs to the class of plant genes for pathogen resistance that have a leucine zipper motif, a putative nucleotide binding domain and a leucine-rich repeat domain. The most closely related plant resistance gene (36% identity) is the Prf gene for resistance to Pseudomonas syringae of tomato. R1 is located within a hot spot for pathogen resistance on potato chromosome V. In comparison to the susceptibility allele, the resistance allele at the R1 locus represents a large insertion of a functional R gene.     

Effector genomics accelerates discovery and functional profiling of potato disease resistance and phytophthora infestans avirulence genes

Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties.     

Diversity of the Phytophthora infestans population in Flanders, Belgium

A total of 94 isolates of Phytophthora infestans were collected from disease outbreaks in commercial potato crops and private gardens in 2002 and 2003. The isolates were recovered successfully from single lesions of diseased potato foliage. Not from all isolates pure cultures were obtained due to contaminations with Fusarium species and bacteria. The structure of the population was analysed phenotypically. Characteristics of the isolates included in vitro growth rate, mating type, in vitro sensitivity to the phenylamide fungicide metalaxyl-M and allozyme genotype at glucose-6-phosphate isomerase (Gpi) and peptidase (Pep) loci. Significant differences in in vitro growth rate were observed among the 52 isolates by comparing the main radial growth of the isolates after 7 days. Forty seven from the isolates tested were the Al mating type. Only one isolate was characterized as A2 mating type. Isolates with sensitive, intermediate and resistant responses to metalaxyl-M were detected in the populations. Forty isolates had a growth of less then 40 % at 5 ppm metalaxyl-M. Three isolates had a growth of less then 40 % at 100 ppm metalaxyl-M. Eight isolates had a growth of more then 40 % at 5 and 100 ppm metalaxyl-M. Cellulose acetate electrophoresis was used to examine Gpi and Pep banding pattern of the population of P. infestans attacking potato in Flanders. All the isolates tested produced the 100/100 Gpi isozyme electromorph. Five different allozyme genotypes of the Pep loci were identified: 92/92, 96/96, 100/100, 92/100, 83/100.     

Cloning and characterization of r3b; members of the r3 superfamily of late blight resistance genes show sequence and functional divergence

Massive resistance (R) gene stacking is considered to be one of the most promising approaches to provide durable resistance to potato late blight for both conventional and genetically modified breeding strategies. The R3 complex locus on chromosome XI in potato is an example of natural R gene stacking, because it contains two closely linked R genes (R3a and R3b) with distinct resistance specificities to Phytophthora infestans. Here, we report about the positional cloning of R3b. Both transient and stable transformations of susceptible tobacco and potato plants showed that R3b conferred full resistance to incompatible P. infestans isolates. R3b encodes a coiled-coil nucleotide-binding site leucine-rich repeat protein and exhibits 82% nucleotide identity with R3a located in the same R3 cluster. The R3b gene specifically recognizes Avr3b, a newly identified avirulence factor from P. infestans. R3b does not recognize Avr3a, the corresponding avirulence gene for R3a, showing that, despite their high sequence similarity, R3b and R3a have clearly distinct recognition specificities. In addition to the Rpi-mcd1/Rpi-blb3 locus on chromosome IV, the R3 locus on chromosome XI is the second example of an R-gene cluster with multiple genes recognizing different races of P. infestans.     

A genetic analysis of quantitative resistance to late blight in potato: towards marker-assisted selection

Late blight caused by the oomycete Phytophthora infestans is the most important fungal disease in potato cultivation worldwide. Resistance to late blight is controlled by a few major genes (R genes) which can be easily overcome by new races of P. infestans and/or by an unknown number of genes expressing a quantitative type of resistance which may be more durable. Quantitative resistance of foliage to late blight was evaluated in five F₁ hybrid families originating from crosses among seven different diploid potato clones. Tuber resistance was evaluated in four of the families. Two of the families were scored for both foliage maturity and vigour. The five families were genotyped with DNA-based markers and tested for linkage with the traits analysed. QTL (quantitative trait locus) analysis identified at least twelve segments on ten chromosomes of potato having genes that affect reproducibly foliage resistance. Two of those segments also have major R genes for resistance to late blight. The segments are tagged by 21 markers that can be analyzed based on PCR (polymerase chain reaction) with specific oligonucleotide primers. One QTL was detected for tuber resistance and one for foliage vigour. Two QTLs were mapped for foliage maturity. Major QTL effects on foliage and tuber resistance to late blight and on foliage maturity and vigour were all linked with marker GP179 on linkage group V of potato. Plants having alleles at this QTL, which increased foliage resistance, exhibited decreased tuber resistance, later maturity and more vigour.     

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.