Diversity of Phytophthora infestans from Poland

Authors


Abstract

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.

Introduction

Phytophthora infestans is the causative agent of late blight, which is considered to be the most important disease of potato in the world economically. In Europe the total annual cost of late blight of potato and tomato is estimated to be around €1 billion. That amount includes crop losses, as well as the cost of fungicides used for crop protection (Haverkort et al., 2008). In Poland, because of the high cost of fungicide protection and the traditional farming on small plots, a minority of fields (49% in year 2003) are sprayed against late blight (Kapsa, 2004). Phytophthora infestans is widespread in Poland, and its population characteristics should be taken into consideration in management advice for potato cropping.

Phytophthora infestans is a heterothallic, hemibiotrophic oomycete with two mating types (A1 and A2). The first global appearance of the pathogen took place in the 19th century, and had a devastating effect on Ireland. The Great Irish Famine caused by late blight led to the death of 1 million people, and another 1·5 million were forced to emigrate (Govers, 2005). Until recently it was assumed that widespread migration of a single clonal lineage of A1 mating type and mitochondrial haplotype Ib (Ib mtDNA), termed US-1, resulted in its domination of the worldwide population of P. infestans. The A2 mating type existed previously only in central Mexico, which is considered to be the centre of origin of P. infestans (Fry et al., 1993). However, samples of blighted potatoes from the 19th century were studied for mitochondrial haplotype and Ib mtDNA has not been found among those samples (Ristaino, 2002; Ristaino et al., 2013). This clearly demonstrates that US-1 was not the only clonal lineage present outside central Mexico in the 19th century. In the winter of 1976/1977 large shipments of potatoes from Mexico to Europe introduced the A2 mating type (Niederhauser, 1991), and, with potato trade from Europe, it spread across the world. In Poland, the A2 mating type was detected for the first time in 1988 (Sujkowski et al., 1994). The presence of both mating types enables sexual reproduction, which increases genetic diversity in the pathogen population leading to increased adaptability. The second important effect of sexual reproduction is the production of oospores, which are highly tolerant to adverse environmental conditions and can survive in soil between growing seasons (Turkensteen et al., 2000). In recent years the European population of P. infestans has undergone major changes. The ratio of mating types changed from more than 80% of A1 in 2004 to below 30% in 2008 (www.eucablight.org). Genotype 13_A2 was reported in the Netherlands and Germany in 2004 and went on to comprise 80% of the population in Great Britain in 2007 (Cooke et al., 2012).

Over the years a range of phenotypic and genotypic markers have been used for studying the diversity of P. infestans. Phenotypic markers include: isozymes, mating type, virulence, metalaxyl resistance and aggressiveness. Determination of mating type can be also classified as a genotypic marker as PCR-based methods have been developed. Other genotypic markers include: restriction fragment length polymorphism (RFLP), mtDNA haplotype analysis, amplified fragment length polymorphism (AFLP), single nucleotide polymorphism (SNP), sequence analysis and simple sequence repeats (SSRs) (Cooke & Lees, 2004). SSRs are neutral, co-dominant, single-locus markers, and so they are considered to be ideal for population studies (Cooke & Lees, 2004; Lees et al., 2006). The use of standardized, validated SSRs is a reliable method for comparison of P. infestans populations among various regions, and to monitor genetic variability of the P. infestans population. Since the first published study of P. infestans using SSR markers (Pi4B and Pi4G) (Knapova & Gisi, 2002), various other markers have been developed. At present more than 20 markers are available. In a previous investigation, two sets of SSR markers (12 and eight markers) were used to genotype Polish isolates of P. infestans from 2005. This revealed their high genetic variability as among 43 isolates only three genotypes occurred more than once (M. Chmielarz, unpublished data). For the present study, 96 Polish P. infestans isolates from 3 years (2006, 2008, 2009) were tested, using a set of 12 SSR markers (Li et al., 2012a). Mitochondrial haplotype was also assessed, as well as a range of phenotypic traits: mating type, virulence and resistance to metalaxyl. The goal of this study was to detect if recent changes occurring in the P. infestans population in Europe, for example France (Montarry et al., 2010) or the UK (Cooke et al., 2012), are reflected in the population of this pathogen in Poland. In addition, the Polish population of P. infestans was surveyed with regard to traits important for potato breeding and control.

Materials and methods

Collection of P. infestans isolates

Samples of potato leaves and stems, as well as tomato fruits and leaves infected with P. infestans were collected from various locations by inspectors from the Main Inspectorate of Plant Health and Seed Inspection (Fig. 1). From P. infestans isolates collected in 2006, 2008 and 2009, 96 were selected for characterization and SSR genotyping (24, 36 and 36 from each year respectively). Eleven of the isolates collected in 2006 were from tomato plants. Thirty-nine isolates were from sites that were protected against potato late blight and 56 isolates were from unprotected sites, with no such data available for one isolate. The isolates were collected from allotments (nine in 2006), experimental fields (seven in 2006, 13 in 2008 and five in 2009), a foil tunnel (two in 2006), commercial fields (five in 2006, 23 in 2008 and 31 in 2009), and from an unknown field type (one isolate).

Figure 1.

Locations of fields in Poland from which Phytophthora infestans isolates were sampled in 2006 (a), 2008 (b) and 2009 (c).

The number of samples collected from one field varied from one to 11, with a mean of 1·65. Pure cultures of P. infestans were isolated following a modified procedure described by Zarzycka (2001), using agar media with pimaricin and rifamycin (0·001 and 0·003% in media, respectively). Pure cultures were grown on rye A agar media (Griffith et al., 1995) at 16°C in the dark and stored long-term using two methods modified from Dahmen et al. (1983): on agar slopes under mineral oil at 4°C or in liquid nitrogen with 15% DMSO as a cryoprotectant. Isolates from 2006 and 2008 were stored on agar slopes; isolates from 2009 were stored on agar slopes and in liquid nitrogen.

Phenotypic markers

Virulence of the isolates was tested on 12 potato genotypes, susceptible cultivar Craigs Royal and 11 lines with genes R1–R11 from Solanum demissum (Black's differential set obtained from SASA, Edinburgh, UK), using a detached leaflet test, with two replicates of three leaflets each (Colon et al., 2004).

Resistance to metalaxyl was tested on rye A agar media amended with metalaxyl (Metalaxyl PESTANAL®; Sigma-Aldrich) at final concentrations of 5 and 100 mg L−1. All isolates were incubated in the darkness, at 16°C, for 13 days. Isolates were classified as sensitive when diameters of P. infestans cultures on media with both 5 and 100 mg L−1 metalaxyl were smaller than 40% of the culture diameter on the control plates without metalaxyl. Isolates were defined as intermediate when cultures were >40% of control on media with 5 mg L−1 metalaxyl, but smaller than 40% of the control on media with 100 mg L−1 metalaxyl. Isolates were scored as resistant when their cultures were larger than 40% of the control on both types of metalaxyl plates (Bakonyi et al., 2002). The following isolates were used as references: resistant isolate US-8 (US940480), two intermediately resistant Hungarian isolates (3/1/02 and 13/04/02) and two susceptible isolates of genotype US-1 (82.07.7 from Wales and 74001 from the Netherlands) kindly supplied by W. Fry, J. Bakonyi, D. S. Shaw and F. Govers, respectively.

Genotypic markers

To isolate DNA, pure cultures of P. infestans were grown on liquid rye A medium. Mycelia were harvested, washed in sterile distilled water and frozen in liquid nitrogen before placing at −70°C. The mycelia were later freeze-dried and stored at −20°C. Freeze-dried mycelia were ground to a powder in microcentrifuge tubes using micropestles and liquid nitrogen. DNA was isolated using a DNeasy Plant Mini Kit (QIAGEN) according to the manufacturer's instructions.

Mating type was tested using PCR with primers S1a (5′-AGGATTTCAACAA-3′) and S1b (5′-TGCTTCCTAAGG-3′) (Judelson, 1996). Each PCR was conducted in a 20 μL reaction mixture consisting of 2 μL 10× buffer containing MgCl2 (Genoplast Chemicals), 0·1 μm each of dATP, dCTP, dGTP and dTTP, 0·4 μm each primer, 1 U Taq DNA polymerase (Genoplast Chemicals) and 10–50 ng template DNA. Reactions were carried out using the following thermal cycling parameters: initial denaturation for 5 min at 94°C, followed by 35 cycles of: 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C; and a final extension for 4 min at 72°C. PCR products were resolved on a 1·5% agarose gel stained with ethidium bromide (1 mg mL−1) and visualized on a UV transilluminator. Thirty-five imported isolates of known mating types were used to validate the PCR test for mating type determination of P. infestans. The method gave consistent results in 34 out of 35 tested isolates, and therefore was used to determine mating types of P. infestans isolates for this study.

The mitochondrial DNA haplotype was tested using a modified version of the method described by Griffith & Shaw (1998). Two sets of primers were used: P2 (5′-TTCCCTTTGTCCTCTACCGAT-3′ and 5′-TTACGGCGGTTTAGCACATACA-3′) and P4 (5′-TGGTCATCCAGAGGTTTATGTT-3′ and 5′-CCGATACCGATACCAGCACCAA-3′). The PCR reaction for P2 was conducted in a 25 μL reaction mixture consisting of 2·5 μL 10× buffer, 250 μm dNTP, 1·875 μm MgCl2, 2·5 μm of each primer, 1 U Taq DNA polymerase and 10–50 ng template DNA. Reactions were carried out using the following thermal cycling parameters: initial denaturation for 30 s at 94°C, followed by 40 cycles of: 30 s at 94°C, 60 s at 64°C, 60 s at 72°C; and a final extension of 5 min at 72°C. PCR products were digested using restriction enzyme MspI, resolved on a 1·5% agarose gel stained with ethidium bromide (1 mg mL−1) and visualized on a UV transilluminator. The PCR reaction for P4 was conducted similarly to the P2 reaction, but with 0·7 μm MgCl2. Reactions conditions were also similar, but initial denaturation time was 90 s, primer annealing temperature was 60°C, and extension time was 90 s. PCR products were digested using restriction enzyme EcoRI, and visualized as described for the P2 reaction.

Genotyping using SSR markers was performed according to the method described by Li et al. (2012a). A subgroup of 16 isolates was tested by additional SSR markers: Pi16, Pi26, Pi33 (Lees et al., 2006) and Pi4G (Knapova & Gisi, 2002). For automated fragment analysis, one primer of each locus was labelled with a fluorescent dye. The dyes were assigned to loci in such a way that loci with the same dye had non-overlapping ranges of allele size, allowing simultaneous loading of all amplification reactions from one isolate onto the capillary system. PCR reactions were performed in a 10 μL reaction mixture consisting of 1 μL 10× AmpliTaq (Applied Biosystems), 200 μm dNTP, 1·5 mm MgCl2, 10 pmol each primer, 1 U Taq DNA polymerase (Applied Biosystems). The reactions were carried out using the following thermal cycling parameters: initial denaturation for 4 min at 95°C, followed by 35 cycles of: 30 s at 95°C, 30 s at 59°C, 30 s at 72°C; and a final extension for 7 min at 72°C. The fluorescently labelled PCR products were analysed using an ABI3730 DNA Analyzer (Applied Biosystems) with 36 cm capillaries, using Performance Optimized Polymer (POP-7; Applied Biosystems) for 3730 DNA Analyzers. One microlitre of diluted PCR products was added to a loading buffer containing 8·8 μL Hi-Di™ formamide (Applied Biosystems), and 0·2 μL of GeneScan 500 LIZ size standard (Applied Biosystems). Electrophoresis of the samples was carried out at 66°C, at 15 kV, for 20 min. data collection v. 2.0 (Applied Biosystems) was used to collect data, and GeneMapper v. 4.0 (Applied Biosystems) was used to derive the fragment length of the labelled DNA fragments using the known fragment length of the LIZ-labelled marker peaks. The fragment lengths of the detected alleles are presented as a result of the genotyping process.

Data analysis

A principal component analysis (PCA) was performed using the statistical software R v. 2.14.0 (R Foundation for Statistical Computing, 2011). PCA was based on the genetic distance metric of Bruvo et al. (2004) implemented in the R package polysat v. 1.2-1 (Clark & Jasieniuk, 2011) and was followed by cluster analysis using the UPMGA method available in R. The method described by Bruvo et al. (2004) can be used to compute genetic distance, regardless of the ploidy of the individuals. Trisomy may occur in P. infestans (Lees et al., 2006) and therefore, to avoid underestimation of genetic variation, polysat was used and the SimpleFreq function from this package was used to calculate allele frequencies. This estimation method assumes polysomic inheritance. For genotypes with allele copy number ambiguity, all alleles are assumed to have an equal chance of being present in multiple copies (Clark & Jasieniuk, 2011). Diversity for each locus (inline image, where xj is the frequency of the jth allele at the locus) was calculated according to Nei (1978). To measure the amount of genetic differentiation between populations, Wright's pairwise FST values were calculated according to Nei (1978) using the polysat function Calc FST (Clark & Jasieniuk, 2011). Differences in frequencies of mating type, virulence, resistance to metalaxyl and mitochondrial haplotype were tested using two-tailed Fisher's exact test for frequencies.

Results

In total 96 isolates were analysed, 24 from 12 locations in 2006, 36 from 14 locations in 2008 and 36 from 29 locations in 2009.

Both A1 and A2 isolates were found every year (Fig. 2). The A1 mating type prevailed (68 out of 96 isolates). However, among isolates collected in 2006 the A1 mating type was in the minority (eight out of 24). In 2008 only one isolate out of 36 was of mating type A2 and also in 2009 the majority of isolates (25 out of 36) represented the A1 mating type.

Figure 2.

Mating type of 96 isolates of Phytophthora infestans sampled from Poland.

Virulence factors against all R genes from S. demissum were found among the tested P. infestans isolates (Fig. 3). All tested isolates were able to overcome genes R3 and R7. The majority of isolates were virulent against R1, R4, R10 and R11. The virulence against R2, R6 and R8 was moderately frequent, whereas virulence against R5 and R9 was generally rare with the exception of isolates collected in 2006. Data were statistically tested using the two-tailed Fisher's exact test (< 0·05). Virulence against R1, R3, R4, R7, R10 and R11 was not statistically different between years and no statistical difference was found between 2008 and 2009 for virulence against R2, R5, R6, R8 and R9. However, isolates from 2006 were significantly different from isolates from both 2008 and 2009 with respect to virulence against R2, R5, R6, and R9. For virulence against R8, results from 2006 were statistically different from 2009, but not from 2008.

Figure 3.

Results of virulence testing of 96 isolates of Phytophthora infestans from Poland. Each three bar set represents isolates from years 2006, 2008 and 2009 respectively. Arrows mark differentials for which statistically significant differences between years were found.

In all years the majority of tested isolates were sensitive to metalaxyl (14 out of 24, 32 out of 36 and 23 out of 36 for years 2006, 2008 and 2009, respectively). Among tested isolates from 2006 only metalaxyl sensitive or resistant isolates were found, with no intermediate phenotype. Among isolates from 2008 and 2009 all three types of reaction to metalaxyl were found (Fig. 4). A significant increase in the number of intermediate isolates was observed in 2009, compared to previous years (Fisher's exact test, < 0·05). Also a significantly greater proportion of resistant isolates was observed in 2006 (Fisher's exact test, < 0·05). No relationship was found between resistance to metalaxyl and chemical protection of the site from which the sample was collected. Resistant isolates as well as sensitive and intermediate ones originated from both protected and unprotected locations.

Figure 4.

Resistance to metalaxyl of the tested Phytophthora infestans isolates.

The great majority of tested isolates were of the Ia mtDNA haplotype, but IIa haplotypes were also detected (Fig. 5). In 2008 only isolates with Ia haplotype were detected, which means that isolates from 2008 were significantly different from isolates from 2006 and 2009 (two-tailed Fisher's exact test, < 0·05).

Figure 5.

Mitochondrial DNA haplotype of tested Phytophthora infestans isolates.

In total 57 alleles were detected over the 12 SSR loci, with 2–10 alleles per locus, with a mean of 4·75 (Table 1). Some alleles were detected only once among all sampled isolates (frequencies of 0·005 and 0·004, depending on ploidy). A null allele was observed only at the D13 locus. The 96 P. infestans isolates were categorized into 66 unique genotypes, 49 of which were represented by only a single isolate. Ten genotypes were represented by two isolates each, three by three isolates and one by four isolates. The most frequent genotype was represented by 16 isolates.

Table 1. Allele frequencies of SSR markers in the 96 tested isolates of Phytophthora infestans
SSR locusAlleleFrequencySSR locusAlleleFrequency
  1. a

    Locus was not amplified.

Pi022580·037D131180·021
2640·005 1320·010
2660·068 1340·010
2680·789 1360·198
2700·095 1400·021
2720·005 1420·005
Pi4B2050·445 1540·078
2130·152 1580·010
2170·403 2080·005
G111420·052 2100·005
1480·026 nulla0·635
1520·005SSR113310·026
1540·089 3410·827
1560·356 3560·147
1580·037SSR21730·974
1600·058 1750·026
1620·340SSR42850·107
2060·037 2870·017
Pi041600·031 2890·278
1660·455 2910·043
1680·063 2930·239
1700·450 2950·269
Pi632700·260 2970·043
2730·310 2990·004
2790·430SSR62400·270
Pi701920·968 2420·325
1950·032 2440·397
SSR82600·516 2460·004
2660·484 2580·004

Diversity of each locus was calculated using the H value in which 0 means no diversity at that locus and 1 means maximum diversity. The lowest score was for Pi70, which had two alleles and showed almost no diversity. Highest scores were for SSR4 and G11, which had eight and nine alleles, respectively (Table 2).

Table 2. Diversity of SSR loci (according to Nei, 1978)
SSR locus H a
  1. a

    An H value of 0 denotes no diversity at that locus and 1 denotes maximum diversity.

Pi020·362
Pi4B0·616
G110·740
Pi040·585
Pi630·651
Pi700·027
SSR80·499
D130·550
SSR110·293
SSR20·050
SSR40·777
SSR60·664

FST values were calculated for isolates from 2006, 2008 and 2009 (Table 3). The scores were low (0·01–0·05), which indicate that the populations sampled from each year are not genetically differentiated from each other.

Table 3. The amount of genetic differentiation between populations shown by Wright's pairwise FST values
 200620082009
  1. a

    0 means that there is no difference in genetic structure between populations, 1 means a great difference in genetic structure between populations (according to Nei, 1978).

20060·00a0·050·03
20080·050·000·01
20090·030·010·00

Principal component analysis (PCA) based on Bruvo distances of the SSR marker genotyping showed a high variability among the tested isolates. The scatter of the isolates is an indication of the variance of the data and confirms the lack of population structure based on the year of sampling. Isolates from 2008 were slightly more closely grouped than those collected in 2006 or 2009 (Fig. 6).

Figure 6.

PCA analysis using Bruvo distances of tested Phytophthora infestans isolates.

A cluster tree made with the UPMGA method, based on the Bruvo distances calculated from the SSR results (Fig. 7), was used to further investigate differences between years. Isolates from 2006 occur in the upper part of the tree, while isolates from 2008 and 2009 are scattered across the rest of the tree. A group of 16 identical isolates was further analysed using additional markers (Pi16, Pi26, Pi33, Pi4G), which split this group into four different genotypes. Two of them showed no diversity, with alleles 173/176 for Pi16, and 176/180 for Pi26. In the case of marker Pi33, two patterns were found: 203/206 for 15 isolates and 203/203 for one isolate. Marker Pi4G produced three patterns: 160/164 for five isolates, 162/164 for seven isolates and 164/164 for three isolates. It failed to produce any alleles in one isolate. These data were not included in the analysis.

Figure 7.

Cluster tree made with UPMGA method based on SSR results.

Discussion

The Polish population of P. infestans, unlike the French, British or Dutch populations (Montarry et al., 2010; Cooke et al., 2012; Li et al., 2012b), remained diversified, with low frequency of the A2 mating type, rare metalaxyl resistance and no single invasive genotypes spreading. It resembled more closely P. infestans populations in the Nordic countries (Denmark, Finland, Norway and Sweden; Brurberg et al., 2011) and Estonia (Runno-Paurson et al., 2010), with the majority of isolates of mating type A1 and high genetic variation.

The majority (72%) of isolates in this study were sensitive to metalaxyl. In the Czech Republic the majority of P. infestans isolates are sensitive to metalaxyl (95% of isolates collected in 2003–2008;Mazáková et al., 2011). In Estonia 78·8% of isolates were sensitive to metalaxyl in 2007, although in 2005 only 15·2% were sensitive (Runno-Paurson et al., 2010). In many other countries high frequencies of isolates resistant to metalaxyl have been reported. In Belarus only 29% of isolates collected in 2006 and 2007 were classified as sensitive (Pliakhnevich & Ivaniuk, 2008) and in France frequencies of isolates resistant to metalaxyl have been reported to reach up to 80% (Duvauchelle et al., 2009). High sensitivity to metalaxyl, which is characteristic for the Polish population of P. infestans, could be directly explained by the limited use of metalaxyl-based fungicides in Poland compared to western Europe. In fact <50% of fields are chemically protected against late blight (Kapsa, 2004). Mitochondrial haplotype Ia dominated the Polish isolates in this study, but IIa was also present. Domination of Ia mitochondrial haplotype is also common in other European populations of P. infestans, and IIa is widespread, but with a lower frequency. However in Estonia Ia and IIa haplotypes are found in a ratio close to 1:1 (Runno-Paurson et al., 2010).

Using a set of twelve SSR markers developed by Li et al. (2012a), a total of 57 alleles were detected among the tested Polish P. infestans isolates. Most of the markers were useful in differentiation of genotypes of the Polish isolates; only two markers (Pi70 and SSR2) showed almost no diversity. Alleles detected in the Polish P. infestans isolates were also detected in other European populations, but some new, rare alleles were also found, for example allele 208 of marker D13, alleles 264 and 272 of marker Pi02, allele 286 of marker SSR4 and alleles 246 and 258 of marker SSR6.

PCA analysis and low FST values suggest that the tested isolates represent a population with no clear structure. However, some isolates from 2008 and 2009 formed a group of individuals with very similar genotype as shown in the PCA and the lower clade of the cluster tree in Figure 7. The majority of isolates with the same genotype were collected from the same field, and even on the same day. A group of 16 isolates representing the same genotype was collected in different years and different regions: 11 isolates were collected in 2008 from four regions, and five in 2009 from two regions. Another group was formed by four genetically identical isolates, which were collected in two years (one in 2008, three in 2009) from four different regions. This suggests that alleged clonal lineages are able to spread over significant distance, and survive from season to season, probably in seed tubers or in a pile of waste. Variation within a single field occurred as well. Results of genotyping using SSR markers were compared with phenotypic traits and no correlations were found, nor were any regional patterns found.

The 16 isolates of the same genotype represented the same mating type (A1) and mitochondrial haplotype (Ia), yet there were some differences in virulence and resistance to metalaxyl (15 sensitive and one intermediate isolate). Results of the virulence test showed that those isolates represented six different virulence patterns. Although this may suggest that those isolates were in fact different genotypes, it may be that differences in virulence expression occurred. Andrivon et al. (2011) showed that virulence of P. infestans isolates is liable to change; virulence factors may be gained or lost within a single isolate. Furthermore, in the case of virulence against the R2 gene, it was proven that it may depend on the expression pattern of the PiAVR2 effector and not on genetic differences and is therefore vulnerable to change (Gilroy et al., 2011). The 16 isolates with identical SSR genotypes were further tested using additional SSR markers (Pi16, Pi26, Pi33 and Pi4G), and the results revealed that these isolates in fact represented four different, but related, genotypes.

Previously, a considerable genetic diversity in the Polish population of P. infestans was observed, using both the DNA fingerprinting probe RG57 (Sujkowski et al., 1994) and SSR markers (Śliwka et al., 2006; Lebecka et al., 2007). In the Nordic countries (Denmark, Finland, Norway and Sweden), an even higher genotypic variation was observed among P. infestans isolates which were tested with nine polymorphic SSR markers, revealing 169 genotypes in 191 isolates collected in 2003 (Brurberg et al., 2011). PCA analysis showed no pattern of clustering of isolates from a certain country, and low FST values indicates that the majority of variation is found within, and not among countries. It also supports the idea that sexual oospores form an important source of primary inoculum. In France, 220 isolates of P. infestans were collected in 2004 and 2005 and tested using eight SSR markers (Montarry et al., 2010). Among those isolates 70 different genotypes were detected, 48 of which were represented by single isolates. Genotypes were split further into two distinctive clusters by PCA analysis, containing predominantly clonal lineages, as deduced from the results. In their work, Montarry et al. (2010) observed changes occurring in the French population of P. infestans, when one cluster had been replaced by another. It was assumed that this second cluster represented a new, invasive population.

The highly aggressive genotype 13_A2 has been spreading in western Europe; it was first found in 2004 and in 2006–2007 it was dominating in the Netherlands, France, United Kingdom, Switzerland, Germany and Belgium, although not in Denmark (Gisi et al., 2011). This genotype was also found in the present study, represented by eight isolates. Seven of them were collected in 2006, and only one in 2009. The 13_A2 isolates were collected from five different locations; four were collected in northern, three in central-western, and one in southern Poland. They represented five different variants of 13_A2. As most of those isolates were collected in 2006 and only one was found in subsequent years, it can be concluded that this genotype is not spreading. It is unclear why 13_A2 is unsuccessful in some countries; more studies on this subject would be needed to determine the reason. Three factors may be relevant: climate, usage of fungicides and genetic diversity of the P. infestans population present in the invaded country. The average temperature in Poland in the coldest months (December, January, February) is below 0°C and in Denmark it is between −1 and 2°C, whereas in the countries where 13_A2 is common, the average temperature in those months is considerably higher, up to 10°C (http://www.eurometeo.com). Because usage of fungicides in Poland is low, metalaxyl resistant isolates (such as 13_A2) lack an advantage over sensitive isolates, which they would have on intensively sprayed fields. It is harder for a single genotype to invade a highly diversified population, like the one in Poland, than a population with low diversity, dominated by a few genotypes, like the population in the UK in 2004 (Cooke et al., 2012).

The high diversity found in the Polish P. infestans population may be caused by several factors. One of them is the structure of agriculture in Poland. Most fields are small and unprotected against late blight (Kapsa, 2004). As a result, outbreaks of late blight take place in small areas, often surrounded by forests and other obstacles, which limits the spreading of particular genotypes. The low usage of fungicides and cultivation of susceptible cultivars mean that the growth of P. infestans is not limited. In the present study, both mating types have been found in the same field, once in each of the tested years. This is an indication that sexual reproduction and the production of oospores may take place and enhance P. infestans diversity.

Results obtained in this study can be helpful for both potato breeders and farmers. Knowledge of which R genes are effective against Polish P. infestans populations should be used in potato breeding programmes. Currently it is known that the R1 gene is present in late starch cultivars, such as Bzura, Hinga, Jagoda, Jasia, Ślęza and in the medium early cultivar Kuba, besides other unknown R genes from S. demissum. In cultivar Bzura the gene R2 or its homolog is also present (J. Plich, Plant Breeding and Acclimatization Institute - National Research Institute, Młochów, Poland, personal communication). The present study suggests that sexual reproduction of P. infestans may take place, which increases the pathogen's adaptability. Therefore it would be advisable to use multiple resistance genes in a single cultivar, as single gene resistance can be overcome relatively quickly. Genes R5 and R9 would be the most useful, as they provide resistance against most of the tested isolates; genes R2, R6 and R8 could also be used. Use of genes R1, R3, R4, R7, R10 and R11 is not advised as they were inefficient in virulence tests. Farmers should be aware that if P. infestans is present in the field, sexual reproduction will produce oospores, which can survive winter and are a source of primary infection in the next season. Therefore it is important to use crop rotation and remove any volunteer potato plants. Low resistance to metalaxyl of the Polish P. infestans population indicates that this agent could be used for chemical protection against late blight, but with caution, as resistant isolates are present nationwide and widespread usage of fungicides would soon result in the selection of such isolates leading to metalaxyl being ineffective.

Acknowledgements

This research was funded by research programmes PW 3-1-06-0-01 and PW 3-6-00-0-01. The study visit to Norway was funded by the Foundation for the Development of the Education System within the programme Scholarship and Training Fund. The authors would like to thank E. Zimnoch-Guzowska for valuable comments on this paper and the Main Inspectorate of Plant Health and Seed Inspection for their help regarding collection of samples.

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