Phenotypic and genotypic diversity in Phytophthora infestans on potato in Great Britain, 1995–98

A total of 2691 single-lesion isolates of Phytophthora infestans was established from samples of late-blight disease from 354 commercial and garden/allotment sites in Scotland, England and Wales over four growing seasons, 1995–98. The A2 mating type was rare (3·0% of isolates) and was detected at only 34 sites. In vitro tests of sensitivity to the phenylamide fungicide metalaxyl showed that 316 sites yielded isolates with some insensitivity (resistant and/or intermediate); these were more often commercial sites than garden/allotment sites. Over the four seasons, the frequency of isolates with intermediate fungicide sensitivity increased, while the frequency of resistant phenotypes decreased. Resistant isolates were always of A1 mating type. A subset of 1459 isolates from 326 sites was analysed for molecular diversity. Mitochondrial DNA (mtDNA) haplotype Ia predominated (91·0% of isolates); haplotype IIa was present at 54 sites and both haplotypes at 33 sites. The multilocus RFLP probe RG57 detected 30 fingerprints. Four fingerprints were particularly common (RF002, RF006, RF039 and RF040) and 10 were unique to a single site in a single year. The three commonest fingerprints (RF039 > RF002 > RF006) were of A1 mating type and the fourth (RF040) was A2. RF002 isolates were resistant to the phenylamide metalaxyl and were more common in Scotland than in England and Wales. Small sample sizes limited the usefulness of estimates of diversity. Although approximately half of all sites appeared to be colonized by RF039 genotypes, some sites (both commercial and garden/allotment) showed a higher diversity, having both common and unique genotypes. The genotypic diversity within isolates collected from commercial sites and those from garden/allotment sites were similar. The contributions of sexual reproduction and alternatives to sex to the generation of variation are discussed.     

DNA fingerprinting uncovers a new sexually reproducing population ofPhytophthora infestans in the Netherlands

The oomycetous fungusPhytophthora infestans (Mont.) de Bary, which causes late blight disease in potatoes, is heterothallic with two known mating types, A1 and A2. From 1845 until 1980 only A1 mating type isolates were found in Europe. In 1980, the A2 mating type appeared permitting sexual reproduction. Here we show that virulence properties and DNA fingerprint patterns of isolates collected in the Netherlands before and after the appearance of A2 mating type isolates are different. Before 1980, eight different races were found in which virulence factors 1, 2, 3, 4 and 10 were most common. After 1980, new virulence factors (i.e. 5, 6, 7, 8 and 11) showed up and the diversity for virulence increased tremendously: 73 different races were detected among 253 isolates analyzed. DNA fingerprint analyses of isolates collected before 1980 revealed that, for at least two decades, only one RG-57 fingerprint genotype was present in Europe. Among 179 isolates collected after 1980 134 distinct RG-57 fingerprint genotypes were identified. The dramatic increase in genetic diversity strongly suggests that theP. infestans population in the Netherlands is now propagating sexually. The change from asexual to sexual reproduction, and the resulting increased adaptability and ability to survive outside the host, may interfere drastically with the regular disease control methods.     

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.     

The role of aggressiveness and competition in the selection of Phytophthora infestans populations

Selection within populations of Phytophthora infestans was investigated by comparing the aggressiveness of single‐lesion isolates on detached leaflets of four potato cultivars with differing levels of race‐nonspecific resistance to P. infestans. The isolates included 23 representative of Northern Ireland genotypes from the early 2000s, used to inoculate previously reported field trials on competitive selection (2003–2005), plus 12 isolates recovered from the 2003 trial. The cultivars were those planted in the previous trials: Atlantic (blight‐susceptible) and Santé, Milagro and Stirling (partially resistant). Very highly significant variation for latent period, infection frequency and lesion area was found between genotypes and cultivars; differences between genotypes were more marked on the more resistant cultivars, but no one genotype was the most aggressive across all. Detached leaflets were also inoculated with mixtures of isolates from each genotype group at three sporangial concentrations: differences in aggressiveness between genotypes were more apparent at lower concentrations and on the more resistant cultivars. Genotype groups that were the most aggressive on the more resistant cultivars tended to be those selected by the same cultivars in the field. A mixture of all isolates of all genotypes was used to inoculate detached leaflets of the same cultivars. With one exception, single spore isolates recovered from any one leaflet belonged to a single genotype, but different genotypes were recovered from different cultivars. Phytophthora infestans isolates from Northern Ireland showed significant variation for foliar aggressiveness, and pathogen genotypes exhibited differential aggressiveness to partially resistant cultivars and interacted competitively in genotype selection.     

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.     

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.     

Inoculum Sources and Genotypic Diversity of Phytophthora infestans in Southern Flevoland,The Netherlands

Genotypic changes in populations of Phytophthora infestans in Southern Flevoland (150km²) were analysed by characterising isolates from potato refuse piles, conventional and organic potato fields, and potatoes and tomatoes in allotment gardens for mating type (1712 isolates) and DNA fingerprint pattern using probe RG57 (1048 isolates). The overall percentages of genotypes (and of isolates) that were A2 varied from 32 (4) in 1994 to 45 (56) in 1996. Among the 1048 isolates 170 different genotypes were identified, of which 138 (81%) were rare (i.e., detected in only one sampling site in the research area during 1993–1996). Many rare genotypes were encountered in organic potato fields and in allotment gardens. In 1994 and 1995, four genotypes were abundant. The highest percentages of isolates with these common genotypes were encountered in refuse piles and conventional potato fields. The common genotypes were nearly absent in 1996, suggesting that the population may have passed through a bottleneck at the transition from 1995 to 1996. The Shannon index of genotypic diversity was high in allotment gardens and in organic potato fields. For the total populations the normalised Shannon index of genotypic diversity increased from 0.34 in 1994, with weather favourable to late blight, to 0.61 in 1996, with unfavourable weather. The high numbers of rare genotypes detected every year indicate that oospores may act as an infection source in commercial potato fields. However, refuse piles were identified as the most important infection sources for commercial fields in 1994 and 1995. In 1996 disease in commercial organic fields was probably initiated by a few genotypes originating from seed tubers. In allotment gardens oospores were probably the most important infection source.     

Tracking Phytophthora infestans with SSR markers within and between seasons – a field study in Sweden

The dynamics of a late blight epidemic and sexual reproduction in Phytophthora infestans were studied in an experimental field in mid‐Sweden. The field was inoculated with six isolates of P. infestans taken from another potato field where sexual reproduction of the pathogen was suspected. Three weeks after inoculation single‐lesion leaflets were sampled and the resulting isolates characterized using microsatellites (SSRs) and mating type as markers. Among the 151 isolates analysed, the inoculum genotypes constituted more than 80% of the genotypes found, with three other genotypes making up the remainder. The following year, P. infestans obtained from soil samples taken from this field were analysed, and six novel genotypes were identified. Genotypes from the previous summer’s population were not detected. Analysis of the genotypes recovered was consistent with them being recombinants, with the previous summer’s population acting as parents. These findings are consistent with the hypothesis that oospores produced during a summer epidemic in Sweden can overwinter and cause infection the next year.     

Development of a species-specific and sensitive detection assay for Phytophthora infestans and its application for monitoring of inoculum in tubers and soil

A specific and sensitive PCR assay for the detection of Phytophthora infestans , the cause of late blight of potato, in soil and plant tissues was developed. A P. infestans -specific primer pair (INF FW2 and INF REV) was designed by comparing the aligned sequences of rDNA internal transcribed spacer regions of most of the known Phytophthora species. PCR amplification of P. infestans DNA with primers INF FW2 and INF REV generated a 613 bp product, and species specificity was demonstrated against DNA from nine other Phytophthora species and seven potato-blemish pathogens. In a single-round PCR assay, 0·5 pg pure P. infestans DNA was detectable. Sensitivity was increased to 5 fg DNA in a nested PCR assay using Peronsporales-specific-primers in the first round. As few as two sporangia or four zoospores of P. infestans could be detected using the nested assay. Procedures are described for detection of P. infestans in leaves, stem and seed potato tubers before expression of symptoms. A soil assay in which 10 oospores per 0·5 g soil were detectable was developed and validated using samples of field soil. The PCR assay was used to examine the long-term survival of sexual (oospores) and asexual (sporangia and mycelium) inoculum of P. infestans in leaf material buried in a replicated experiment under natural field conditions. Oospores were consistently detected using the PCR assay up to 24 months (total length of the study) after burial in soil, whereas the sporangial inoculum was detected for only 12 months after burial. Sporangial inoculum was shown to be nonviable using a baiting assay, whereas leaf material containing oospores remained viable up to 24 months after burial.     

Characterization of some Asian isolates of Phytophthora infestans

A total of 401 isolates of Phytophthora infestans were collected from eight Asian regions (Korea, India, Taiwan, Indonesia, Thailand, Nepal, China and Japan) between 1992 and 2000 – 318 from potato and 83 from tomato. The isolates were analysed for mating type, metalaxyl resistance, RG57 fingerprinting, mitochondrial DNA (mtDNA) haplotype and the polymorphism of three allozyme loci, i.e. glucose-6-phosphate isomerase ( Gpi ), peptidase ( Pep ) and malic enzyme ( Me ). The isolates were multilocus-genotyped based on RFLP (RG57) fingerprint, dilocus allozyme genotype, mtDNA haplotype and mating type. Twenty multilocus genotypes were identified among 125 isolates. Of these genotypes, 14 had not been previously reported. Some of the multilocus genotypes were common to isolates from several geographical regions, suggesting migration. The metalaxyl-resistant isolates belonged to the multilocus genotypes JP-1, JP-2, and JP-3. Multilocus genotypes coexisting in a single field were found in following regions: Thailand (1994), central China (1996), Nepal (1997) and Japan (1998 and 2000). The possible origins of certain genotypes are discussed, including the possibility of sexual recombination within the P. infestans populations in Nepal and perhaps Thailand.     

Phytophthora infestans in a single field in southwest Sweden early in spring: symptoms, spatial distribution and genotypic variation

Sixty-eight isolates of Phytophthora infestans were collected from an early planted, fleece-protected potato field in southwest Sweden in May 2001. No infection was observed in the neighbouring fields. In the field investigated, infected plants were found in six discrete foci, with symptoms almost exclusively observed in the lower part of the canopy and numerous infections found on stems and on leaves touching the ground. The structure of the population was analysed based on mating type, mitochondrial haplotype and microsatellites (SSRs) as markers. Both mating types were present and haplotypes Ia and IIa were detected. Among 61 isolates analysed with microsatellite markers, 14 multilocus genotypes were distinguished based on six polymorphic loci. Out of the six foci, three included 3–5 genotypes each. There were unique genotypes in all foci, except two closely situated to each other. These findings strongly suggest that soilborne oosporic inoculum contributed significantly to initiate the late-blight epidemic in the investigated field.     

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.     

Changes in specific virulence in Polish populations ofPhytophthora infestans: 1985–1991

Ninety-five isolates ofPhytophthora infestans collected throughout Poland during 1985–1991 and characterized for multilocus genotypes based on mating type, allozymes and DNA fingerprint, were analyzed for specific virulence to differential potato cultivars carrying ten major resistance genes. The multilocus analysis led to three groupings. The first group contained 22 isolates of a clonal lineage (PO-1) that is postulated to have been present in Europe during most of the twentieth century, but PO-1 isolates were recovered in Poland only during 1985–1988. This group contained, on average, virulence to 5.5 specific resistance genes per isolate. The second group consisted of 30 isolates in a clonal lineage (PO-4) that had not been detected before 1988. PO-4 isolates had virulence to a mean of 6.5 resistance genes per isolate. The third group was composed of 43 isolates representing 38 multilocus genotypes also not detected before 1988. These diverse genotypes had virulence to an average of 6.7 specific resistance genes per isolate. More than half (53%) of the PO-4 isolates shared a single pathotype. The group of 43 isolates was dominated by two pathotypes: the most common one (47% of the isolates) was the same pathotype that dominated PO-4 isolates; the next most common one (21%) differed from the most common one by the absence of virulence to resistance gene R5. The recent immigrant isolates (not detected before 1988) generally had virulence to a greater number of specific resistance genes than did isolates in the previous population [detected before 1988 (PO-1)]. Recent immigrant populations were dominated by one or two pathotypes, so their pathotypic diversity values were somewhat less than that of the previous population.     

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).     

Diversity of Phytophthora infestans from Poland

A collection of 96 Polish isolates of Phytophthora infestans sampled in the years 2006, 2008 and 2009 were analysed using phenotypic and genotypic markers. Mating type, virulence, resistance to metalaxyl, mitochondrial haplotype and polymorphism at 12 simple sequence repeat (SSR) loci were determined. The majority of isolates were of the A1 mating type, mitochondrial haplotype Ia and sensitive to metalaxyl. Virulence factors against potato R genes R1, R3, R4, R7, R10 and R11 were present in most isolates. Genotyping using SSR markers revealed high genetic diversity within the Polish P. infestans population. Amongst the 96 isolates 66 unique genotypes were identified, 49 of which were observed only in single isolates. Eight isolates of the genotype 13_A2 lineage that has been reported in other parts of Europe were also found in Poland. The implications of these results are discussed.     

Genetic structure and pathogenicity of populations of Phytophthora infestans from organic potato crops in France, Norway, Switzerland and the United Kingdom

Genetic variation and pathogenicity of Phytophthora infestans isolates collected from organic potato crops of the susceptible cv. Bintje and the moderately resistant cv. Santé were assessed in France, Norway, and the United Kingdom in 2001 and in Switzerland in 2001 and 2002. Population structures differed considerably between the four P. infestans populations. Those from France, Switzerland and the UK were mainly clonal populations showing restricted levels of genetic diversity, whilst those from Norway were mixed A1 and A2 mating type populations with high levels of genetic diversity, suggesting periodical sexual reproduction. Isolates collected from cv. Bintje were on average more aggressive than or comparable to isolates from cv. Santé. Race complexity varied considerably between the regional P. infestans populations, with isolates from France and Switzerland showing the highest number of virulence factors. In all pathogen samples but the French, isolates collected from cv. Santé were more complex than isolates collected from cv. Bintje. No directional selection towards increased aggressiveness towards the more resistant cultivar Santé was observed. This suggests that there is no shift towards increased levels of pathogenicity in P. infestans populations following the large-scale introduction of more resistant potato varieties in organic production systems in Europe.     

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.     

Novel microsatellite markers for the analysis of Phytophthora infestans populations

Co-dominant microsatellite molecular markers for Phytophthora infestans were developed and their potential for monitoring the genetic variation in populations was demonstrated in the UK, across Europe and worldwide. Markers were developed according to two strategies. First, several thousand P. infestans expressed sequence tag (EST) and bacterial artificial chromosome (BAC) sequences were screened for the presence of simple sequence repeat (SSR) motifs, and, of these, 100 candidate loci were selected for further investigation. Primer pairs developed to these loci were tested against a panel of 10 P. infestans isolates and approximately 10% were shown to be polymorphic and therefore appropriate for further testing. Secondly, the construction and screening of a partial genomic library resulted in the development of one additional polymorphic marker. The resulting 12 SSR markers were converted to higher-throughput fluorescence-based assays and used in combination with two previously published markers to characterize a wider collection of 90 P. infestans isolates from the UK and six other countries. Several isolates from the closely related species P. mirabilis , P. ipomoea and P. phaseoli collected from around the world were also genotyped using these markers. Amongst the 90 isolates of P. infestans examined, considerable SSR diversity was observed, with 68 different genotypes and an average of 3·9 (range 2–9) alleles per locus. When other Phytophthora species were genotyped, all loci were successfully amplified and the majority were polymorphic, indicating their transferability for the potential study of other closely related taxa.     

Displacement of US‐1 clonal lineage by a new lineage of Phytophthora infestans on potato in Kenya and Uganda

Phytophthora infestans, the pathogen responsible for causing late blight in potato and tomato, is a rapidly evolving and highly adaptable pathogen. Following the appearance of a new clonal genotype in Kenya (KE‐1 lineage) in 2007, there was a need to document possible displacement of the US‐1 genotype by the newer lineage. Samples from Kenya (260) collected on potato and from Uganda collected on potato (134) and tomato (32) were genotyped with simple sequence repeat (SSR) and mitochondrial DNA haplotype (mtDNA) markers. Results show that, four years after its discovery, the KE‐1 lineage has completely displaced the US‐1 lineage on potato in Kenya. The KE‐1 lineage has also migrated into Uganda through the eastern part of the country that borders western Kenya. All potato samples from eastern Uganda were of the KE‐1 lineage. Moreover, samples from eight fields in western Uganda were also of the KE‐1 lineage, which is in agreement with an ongoing westward displacement. However, the new lineage has not displaced the old lineage on tomato, as all samples from tomato were US‐1. Principal coordinate analysis (PCA) scatter plots revealed clustering that indicated separation between the lineages and also between the subpopulations of the US‐1 lineage on potato and tomato.