Evidence for presence of the founder Ia mtDNA haplotype of Phytophthora infestans in 19th century potato tubers from the Rothamsted archives

Late blight remained a significant disease for potato growers in Europe long after the famine of the 1840s. Of the four mitochondrial haplotypes of Phytophthora infestans, only the Ia mitochondrial DNA (mtDNA) haplotype has been identified previously in infected potato leaves from famine‐era herbarium specimens collected in England, Ireland and Europe in the 19th century. Long‐term soil fertility experiments were conducted on potato between 1876 and 1901 in Rothamsted to investigate effects of combinations of organic manures and mineral fertilizers on disease and yield. This report identifies for the first time the same Ia mtDNA haplotype of P. infestans in three diseased tubers from 1877 from the long‐term Rothamsted trials, thus providing the earliest evidence of the presence of the founder Ia mtDNA haplotype of P. infestans in potato tubers in England. Soil amendments had a significant impact on disease and yield. A real‐time PCR assay was used to detect and quantify P. infestans in tubers. The level of pathogen DNA was greatest in tubers from highest yielding plots that received combinations of inorganic nitrogenous and mineral fertilizers and least in tubers from plots with organic farmyard manures or non‐nitrogenous mineral fertilizers. The Ia mtDNA haplotype was also confirmed from diseased potato leaves during the same time period. Thus, the founder Ia mtDNA haplotype survived in potato tubers after 1846 and was present over 30 years later in the UK.     

Phenotypic variation in Nordic populations of Phytophthora infestans in 2003

A total of 743 single-lesion isolates of Phytophthora infestans were collected in summer 2003 from Denmark, Finland, Norway and Sweden. Most of the isolates were tested for mating type, and subsets were tested for sensitivity to fungicides and virulence (host specific pathogenicity). Approximately 60% of the isolates were A1 mating type in each country. Both mating types were present in 40% of the fields where more than one isolate was tested, indicating strong potential for sexual reproduction. The proportion of metalaxyl-resistant isolates dropped to under 15% from the 60% observed in the early 1990s in Norway and Finland, possibly due to lower selection pressure because of decreased use of metalaxyl. Propamocarb-HCl sensitivity remained unchanged in the Nordic countries compared to the situation in 1997–2000 in Finland. Four isolates collected from Finland and Sweden were able to sporulate in the presence of this fungicide at a concentration of 1000 mg L⁻¹. In Norway and Finland the frequencies of virulence factors and pathotypes remained nearly unchanged since the 1990s, but the mean number of virulence factors per isolate increased from 5·6 to 6·3. In Denmark and Sweden virulence factors 2 and especially 6 were more common than in Norway and Finland. In addition, in the Swedish population the frequencies of pathotypes were quite even while in other countries pathotype 1,3,4,7,10,11 was most prevalent.     

Severe outbreaks of late blight on potato and tomato in South India caused by recent changes in the Phytophthora infestans population

Late blight, caused by Phytophthora infestans, has emerged as the most destructive disease of potato and tomato in South India since 2008. One hundred and fifty‐seven isolates of Phytophthora infestans, 63 from potato and 94 from tomato, were collected from major potato and tomato production areas of South India between 2010 and 2012. Their phenotypic and genotypic characteristics were determined and compared with reference isolates. Isolates were characterized based on mating type, in vitro metalaxyl sensitivity, mitochondrial DNA haplotype, RG57 DNA fingerprinting patterns, SSR markers and aggressiveness on potato and tomato, in order to monitor population changes in P. infestans. All isolates were A2 mating type, metalaxyl resistant, mtDNA haplotype Ia and had RG57 and SSR fingerprints almost identical to the 13_A2 clonal lineage reported in Europe. Variation at the D13 and SSR4 loci allowed discrimination of minor variants, designated as 13_A2_3, 13_A2_3b, 13_A2_3c and 13_A2_1. A comparison of the lesion diameters caused by 157 isolates on detached leaflets of three potato and tomato cultivars showed all isolates to be equally aggressive, confirming that the same clonal population is infecting both hosts. This study demonstrates that the 13_A2 lineage was responsible for severe late blight outbreaks on potato and tomato in South India and has replaced the prior population represented by the US‐1 and other genotypes. Revised management strategies will be required to combat this destructive 13_A2 clonal lineage and monitoring of the population across other potato‐ and tomato‐growing regions of India is warranted.     

Genetic variation among asexual progeny of Phytophthora infestans detected with RAPD and AFLP markers

Genotypic variation among 32 single-zoospore isolates (SZI) of Phytophthora infestans , derived asexually from two hyphal-tip parental isolates (PI-105 and PI-1) of the US-8 genotype, was assessed with 80 random amplified polymorphic DNA (RAPD) primers and 18 amplified fragment length polymorphic DNA (AFLP) primer pairs. In previous investigations, the SZIs from parental isolate PI-105 showed high levels of virulence variability and were differentiated into 14 races, whereas the SZIs from PI-1 showed identical virulence to the parent. The purpose of this investigation was to determine if phenotypic variation observed among SZIs of P. infestans could be detected at the DNA level in these isolates. Polymorphism was detected with 51 RAPD primers and with all 18 AFLP primer pairs in PI-105 SZIs. In SZIs from PI-1, polymorphism was also detected with 25 RAPD primers and 17 AFLP primer pairs. Cluster analysis using the unweighted pair-group method with arithmetic averages (UPGMA) separated the SZIs from parent PI-105 into six virulence groups, 11 RAPD groups and three AFLP groups. Cluster analysis of PI-1 SZIs, which all belong to the same virulence group, differentiated them into four RAPD groups and six AFLP groups. No close correlation among RAPD, AFLP and virulence groups could be established within the two progenies of SZIs. Results of this study suggest that there is a considerable level of inherent genetic variability among SZIs derived asexually from the same parental isolate. The possible mechanisms and implications of this genetic variation are discussed.     

Phenotypic and genotypic variation in Michigan populations of Phytophthora infestans from 2008 to 2010

Severe potato and tomato late blight epidemics in Michigan since 2008 prompted characterization of Phytophthora infestans isolates from the region. From eight counties in Michigan, 124 isolates were collected from infected potato and tomato plants from 2008 to 2010 and characterized using ‘classical’ markers and microsatellites. The classical markers included mating type, Gpi allozyme, mitochondrial DNA haplotype, sensitivity to metalaxyl‐M and tuber pathogenicity. All isolates from 2008 to 2010 were A2 mating type and Ia mtDNA haplotype (124 isolates), 105 isolates had Gpi profile 100/122, 17 isolates had the profile 100/100/111 and the remaining two isolates had 100/111/122. Sensitivity to metalaxyl‐M, expressed as EC₅₀ for mycelial growth in vitro, ranged from <0·1 to 91 μg mL^?1, where 95 and 96% of isolates were classified as either sensitive or intermediate in 2008–2009 and 2010 respectively. The metalaxyl‐M sensitivity and dominant Gpi profile were typical of clonal lineage US‐22, first isolated in 2008 in North America from tomato plants. Tuber pathogenicity, characterized as severity of tuber late blight, was also variable among isolates; however, isolates were less aggressive than previous genotypes present in Michigan, such as US‐8. Microsatellites (simple sequence repeats; SSRs) revealed a shift in the population, characterized by two clusters differentiated over time. These results suggested displacement of the US‐8 genotype by US‐22 from 2008 to 2010 in Michigan. Continuous tracking of changes within P. infestans populations provides evidence of genetic shifts due to migration, prompting modification of management strategies based on the phenotypic characteristics of causal genotypes.     

Genetic analysis of Phytophthora sojae populations in Fujian, China

Phytophthora sojae is a destructive soilborne pathogen causing seedling damping‐off and root rot of soybean (Glycine max). The goal of this study was to determine the genetic structure of P. sojae populations in Fujian, China. Nine microsatellite markers were used to investigate the genetic variation in 19 P. sojae populations, sampled from Fujian Province and northeastern China (Jilin and Heilongjiang Provinces) between 2002 and 2013. Overall, a low genetic diversity, Hardy–Weinberg disequilibrium, and an index (an index of association) that was significantly different from zero were detected in populations; these results were consistent with self‐fertilization and clonal modes of reproduction for this pathogen. However, using Bayesian Markov chain Monte Carlo approach, principal component analysis and neighbour joining (NJ) algorithm, the Fujian P. sojae populations clustered into three distinct groups, one of which included most isolates of the northeast populations. What is more, significant estimates of pairwise fixation indices (F_ST) were detected between most populations, especially in different clusters. It is hypothesized that the cropping system used, the limited dispersal ability, and human‐mediated gene flow may account for the observed genetic structure of P. sojae populations in Fujian, China. In addition, a high virulence frequency of the pathogen on different cultivars carrying known major R genes for resistance, and a rapid increase in virulence frequency, indicated that these major R genes should not be used to manage seedling damping‐off and root rot diseases of soybean (Glycine max).     

Variation in populations of Phytophthora infestans in Finland and Norway: mating type, metalaxyl resistance and virulence phenotype

Phytophthora infestans was isolated from potato leaves and tubers collected from different parts of Finland in 1990–96 and Norway in 1993–96. Isolates were assessed for mating type, resistance to metalaxyl and virulence phenotype. In Finland 15% of 200 isolates tested and in Norway 25% of 642 tested were A2 mating type. Differences in the A1/A2 ratio were evident among regions and A2 was not found in northern areas. In Finland the frequency of metalaxyl-resistant isolates, among 1834 tested, decreased from 59% in 1990–95 to 2% in 1996. In Norway 59% of 491 isolates tested were resistant to metalaxyl in 1996. Among 269 Finnish and 105 Norwegian isolates, all known virulence genes were found in both countries and race 1.3.4.7.10.11 was the most common (resistance gene R9 was not included in the differential set). Oospores were observed in potato leaves from three locations in the southern part of Norway. The implications of the 'new' populations in the Nordic countries are discussed.     

SSR assessment of Phytophthora infestans populations on tomato and potato in British gardens demonstrates high diversity but no evidence for host specialization

Phytophthora infestans populations can differ in composition as a result of host specialization on tomato and potato hosts. In Great Britain many amateur gardeners grow outdoor tomatoes but there is little or no commercial tomato production outdoors. This study analysed isolates of P. infestans from British gardens with 12 multiplexed simple sequence repeat markers that are used to monitor the disease on commercial potato crops. Samples of P. infestans from tomato hosts were collected in 3 years and from potato in 1 year from across Great Britain. Seven previously unreported clonal lineages were detected in garden populations and higher frequencies of unique clonal lineages (28–40%) were present compared with populations from British commercial potato crops reported elsewhere. Garden populations had a lower proportion (11–48% less) of the most common lineages (13_A2 and 6_A1) that together made up at least 86% of the commercial potato populations during the sampling period. Host species accounted for only 2·0% of molecular variance detected between garden potato‐ and tomato‐hosted samples. No significant difference in clonal lineage composition was found between host species in Great Britain and this could be due to the whole P. infestans population overwintering on potato. British garden populations on both hosts were much more diverse than those on commercial potato crops; this finding may be influenced by less frequent fungicide use by gardeners and a higher diversity of unsprayed susceptible potato cultivars, enabling metalaxyl‐sensitive and less aggressive genotypes to survive in gardens.     

Population genetics of Phytophthora infestans in Denmark reveals dominantly clonal populations and specific alleles linked to metalaxyl‐M resistance

Control of the potato late blight pathogen Phytophthora infestans relies heavily on chemicals. The fungicide metalaxyl‐M (Mefenoxam) has played an important role in controlling the disease, but insensitivity to the fungicide in certain isolates is now of major concern. A genetic basis for resistance to metalaxyl suggests the possibility for linking resistance phenotypes to specific population genetic markers, but in order to do this, the population genetic structure and mode of reproduction in a population must first be well described. The dynamics of metalaxyl‐M resistance in the Danish population of P. infestans was characterized over the course of the 2013 growing season, as was the population genetic structure, using simple sequence repeat (SSR) genotypes and single nucleotide polymorphism (SNP)‐based mitochondrial haplotyping of over 80 isolates. Both mating types A1 and A2 were present in most fields, but tests for recombination showed that clonal reproduction dominates in Danish populations. Genotype was not linked to haplotype and no differentiation was observed at the haplotype level, but rather between fields. Resistance phenotypes were linked to specific SSR alleles, demonstrating the potential for a more precise SNP‐based marker system for predicting resistance to metalaxyl‐M.     

中国不同地区致病疫霉遗传多样性的RAPD分析

 本文应用RAPD技术检测了我国主要马铃薯产区致病疫霉的遗传分化情况及不同地区菌株间的亲缘关系。用筛选出的10个随机引物对1997-2001年间采自我国9省市的82株及3株来自日本的致病疫霉DNA进行了PCR扩增,获得了79条谱带,其中多态性标记75条,占95%。根据扩增结果,运用UPGMA分析,获得了表现菌株间亲缘关系的树状图。菌株间的最大遗传距离为0.5,以距离0.3为阈值,可将供试菌株划分为10个组(RG1-10)。结果发现:A1交配型菌株群体内的差异大于A1和A2菌株群体之间的;RAPD分组与菌株的地理来源、交配型及对甲霜灵的敏感性无明显相关性。研究结果显示,来自中国北方甘肃、内蒙、吉林、黑龙江地区的菌株与一些来自云南、四川等西南地区的菌株亲缘关系相近。病原菌随种薯的迁移可能是导致这种现象的原因之一。     

Population genetic analysis of Phytophthora infestans in northwestern China

Late blight caused by Phytophthora infestans is the most devastating disease of potato worldwide. To understand the P. infestans population structure and dynamics in northwestern China, 959 single‐lesion isolates were purified in three consecutive years (2009–2011) and were characterized for mating type, pathotype, mtDNA haplotype and molecular variation at eight SSR loci. The results showed that the distribution of mating types changed significantly over years, with self‐fertile isolates dominant in 2010 and 2011. SSR genotyping distinguished 959 isolates into 151 genotypes, and association analysis indicated that P. infestans populations in 2010 and 2011 were strictly asexual while in 2009 they showed signs of sexual reproduction. Population analysis showed that the majority of genetic variation was within P. infestans populations. Isolates sharing identical SSR genotypes were detected in distant regions, indicating that migration of P. infestans could have occurred between regions. Pathogenicity assays on a set of potato differential lines containing R1 to R11 resistance genes detected four pathotypes from 74 selected isolates, with the pathotype virulent against all 11 R genes being dominant. Three mtDNA haplotypes (Ia, IIa, IIb) were detected with Ia being dominant among 507 isolates examined. Phylogenetic analysis indicated that P. infestans populations in northwestern China are distant from European lineages including 13‐A2 (blue‐13) at the time of this survey. The results have implications for the trade of healthy seed tubers as a means of managing late blight.     

Implication of peroxynitrite in defence responses of potato to Phytophthora infestans

In this study peroxynitrite (ONOO^?) is proposed as an important player in defence responses during the interaction of potato (Solanum tuberosum) and the oomycete pathogen Phytophthora infestans. The potato–avr P. infestans model system exhibited a transient programme of boosted ONOO^? formation correlated in time with the burst of nitric oxide (NO) and superoxide during the first 6 h post‐inoculation (hpi). The early ONOO^? over‐accumulation was not accompanied by TPx gene expression. In contrast, the compatible interaction revealed a 24 h delay of ONOO^? formation; however, an enhanced level of NO and superoxide correlated with TPx up‐regulation was recorded within the earlier stages of pathogen infection. Peroxynitrite over‐accumulation in the susceptible potato coincided with an enhanced level of protein tyrosine nitration starting from 24 hpi. Surprisingly, the nitroproteome profile of the resistant potato did not show any visible difference after inoculation, apart from one band containing subtilisin‐like protease‐like proteins, which appeared 48 h after pathogen attack. An additional pharmacological approach showed that treatment of the susceptible genotype with ONOO^? followed by inoculation with P. infestans contributed to slowing down of the colonization of host tissues by the pathogen via a faster and stronger up‐regulation of the key defence markers, including the PR‐1 gene. Taken together, the results obtained indicate that a precise control of emitted NO and superoxide in cooperation with thioredoxin‐dependent redox sensors in sites of pathogen ingress could generate a sufficient threshold of ONOO^?, triggering defence responses.     

Diversity of Avr‐vnt1 and AvrSmira1 effector genes in Polish and Norwegian populations of Phytophthora infestans

The oomycete Phytophthora infestans, the cause of late blight, is one of the most important potato pathogens. During infection, it secretes effector proteins that manipulate host cell function, thus contributing to pathogenicity. This study examines sequence differentiation of two P. infestans effectors from 91 isolates collected in Poland and Norway and five reference isolates. A gene encoding the Avr‐vnt1 effector, recognized by the potato Rpi‐phu1 resistance gene product, is conserved. In contrast, the second effector, AvrSmira1 recognized by Rpi‐Smira1, is highly diverse. Both effectors contain positively selected amino acids. A majority of the polymorphisms and all selected sites are located in the effector C‐terminal region, which is responsible for their function inside host cells. Hence it is concluded that they are associated with a response to diversified target protein or recognition avoidance. Diversification of the AvrSmira1 effector sequences, which existed prior to the large‐scale cultivation of plants containing the Rpi‐Smira1 gene, may reduce the predicted durability of resistance provided by this gene. Although no isolates virulent to plants with the Rpi‐phu1 gene were found, the corresponding Avr‐vnt1 effector has undergone selection, providing evidence for an ongoing ‘arms race’ between the host and pathogen. Both genes remain valuable components for resistance gene pyramiding.     

French isolates of Phytophthora infestans from potato and tomato differ in phenotype and genotype

Prior to 1996, the A2 mating type of Phytophthora infestans was not detected on potato in France, but was found at one site on tomato in 1995. This finding lead to the question of the extent of differences and relationships existing between the populations of P. infestans present on each host. A collection of 76 isolates collected in France, mainly in 1996, from potato and tomato was characterised for mating type, allozyme genotype at the Gpi and Pep loci, and mitochondrial DNA haplotype; 74 of these isolates were also characterised for multilocus RFLP fingerprint, and 62 for virulence. All isolates except four showed allozyme genotypes (Gpi 90/100 or 100/100, Pep 83/100 or 100/100) and mtDNA haplotypes (Ia or IIa) characteristic of the populations introduced into Europe in the late 1970s. The four exceptions were isolates collected from tomato in Southern France in 1988-1991, which showed some characteristics of the former European populations (Gpi 86/100, Pep 92/100, mtDNA Ib). Both mating types were present among the collections from both hosts, but isolates with the A2 mating type were found on potato only in one garden crop, adjacent to tomato. Nine different RG57 fingerprints were observed, with a greater diversity among tomato isolates. Furthermore, tomato and potato collections differed markedly in the frequencies of genotypes present. Finally, tomato isolates generally had a lower virulence complexity than potato isolates. These data suggest that P. infestans populations on tomato and potato are largely separated, despite the occurrence of limited gene flow.     

Diversity of populations of Phytophthora infestans in relation to patterns of potato crop management in Latvia and Lithuania

Potato crop losses can be substantial when conditions for late blight (Phytophthora infestans) development and spread are favourable. In this study, drivers of differences between the P. infestans population structures in Latvia and Lithuania, two neighbouring countries with similar potato-growing traditions, were investigated. Genotypes of P. infestans and population genetic diversity were analysed using a 12-plex simple sequence repeat (SSR) marker assay. High genetic diversity was demonstrated in both populations, with population diversity being higher in Latvia. It would appear that local populations established from soilborne oospores early in the season are well adapted to the conditions in the region. However, somewhat greater spread and survival of local clones was detected in Lithuania, suggesting that potato cropping there is more vulnerable to clonal invasion than in Latvia. For effective disease management, current strategies should be adjusted according to the specific pathogen populations in the region, considering the reproduction and survival of the pathogen. Potato growers should implement late blight preventive measures such as longer field rotation to prevent oospore infections, especially in Latvia, and should use more disease resistant cultivars and high-quality seed potatoes.     

云南省马铃薯晚疫病菌交配型及生物学特性研究

 作者对1998~2000年间采自云南省13个县、23个地点的马铃薯晚疫病菌的交配型、菌落形态、燕麦培养基上生长情况、生长速度和产孢量进行了测定。结果显示,采自云南13个县、23个地点的共157个菌株全部为A1交配型,表明云南马铃薯主产区的晚疫病菌以A1交配型为主,同时,被测的代表菌株在生长速度和产孢量上存在显著差异,表明这一地区的晚疫病菌种群内存在丰富的遗传多样性。此外,结果还显示,晚疫病菌菌株在燕麦培养基上的生长情况与其菌落形态和交配型不相关。     

Markers, old and new, for examining Phytophthora infestans diversity

Late blight, caused by Phytophthora infestans , is an ongoing threat to potato and tomato crop production worldwide and considerable fundamental and applied research is conducted with the long-term aim of improved disease control. Understanding the mechanisms, processes and rates of P. infestans evolution is an important factor in predicting the effectiveness and durability of new management practices. A range of phenotypic and genotypic tests has been applied to achieve this goal, but each has limitations and new methods are sought. Recent progress in P. infestans genomics is providing the raw data for such methods and new high-throughput codominant biomolecular markers are currently being developed that have tremendous potential in the study of P. infestans population biology, epidemiology, ecology, genetics and evolution. This paper reviews some key applications, recommends some changes in approach and reports on the status and potential of new and existing methods for probing P. infestans genetic diversity.     

Stability of partial resistance in potato cultivars exposed to aggressive strains of Phytophthora infestans

Potato cultivars were evaluated for their resistance responses to aggressive strains of Phytophthora infestans in field and laboratory experiments. Analysis of variance revealed differential cultivar-by-isolate interactions for both foliar and tuber blight resistance. Differential responses occur as revealed by specific susceptibilities of cultivars to certain pathogen genotypes and changing rank order. In general, severity of late blight epidemics as observed in the haulms did not correlate well with foliar blight resistance ratings as presented in the National List of Recommended Potato Varieties. No significant correlation was found between tuber blight incidence under field conditions and the tuber blight rating in the National List. Also, there was no relation between the field and laboratory tuber blight resistance assessments. A significant association was demonstrated between late blight infection in the foliage and tuber blight incidence under field conditions. The presence of differential interaction, independent of R-gene-based resistance, indicates some adaptation of P. infestans to partial resistance and consequently adverse effects on the stability and durability of partial resistance to potato late blight.     

Influence of competition and host plant resistance on selection in Phytophthora infestans populations in Michigan, USA and in Northern Ireland

Competition between genotypes of Phytophthora infestans was studied by inoculating potato cultivars with differing susceptibility to late blight in field experiments over three years in Northern Ireland, UK, and Michigan, USA. Multiple isolates of six genotype groups of P. infestans were chosen from the local populations in both N. Ireland and Michigan for inoculation of separate field trials planted in 2003, 2004 and 2005. Four cultivars were used in each trial; two (susceptible cv. Atlantic and the partially resistant cv. Stirling) were common to both locations, whereas the two additional cultivars (with partial resistance to late blight) were cvs Santé and Milagro in N. Ireland and cvs Pike and Jacqueline Lee in Michigan. Single-lesion isolates of P. infestans were obtained from leaves at 1% level of infection, characterized using pre-assigned markers and re-assigned to their respective genotype groups. Extreme selection occurred within the population of genotypes of P. infestans in N. Ireland in each year, with different genotype groups dominating the infection of different cultivars. Selection was observed on all cultivars tested, but was greatest on the more resistant cultivars. Over the 3 years, all of the 114 isolates obtained from cv. Milagro belonged to a single group, whereas among the 118 isolates from cv. Atlantic all six groups were represented. By contrast, in Michigan, the US-8 genotype dominated infection in all cultivars in each year; only 12 of 374 isolates characterized belonged to other genotypes (11 US-14 and a single US-10 isolate).