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Keywords:

  • AFLP fingerprinting;
  • epidemic parameters;
  • epidemiology;
  • potato late blight;
  • race structure

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The oomycete Phytophthora infestans, the cause of late blight, is one of the most important pathogens of potato worldwide. The pathogen affects leaves, stems and tubers, leading to serious yield losses. To date, the vast majority of potato cultivars commonly grown in Western Europe (Colon et al., 1995b) and North America (Platt & Tai, 1998) are susceptible to late blight. The use of numerous applications of both protective and curative fungicides is common practice to control potato late blight (Schepers & Spits, 2006). The recent displacement of the old clonal population of P. infestans by an aggressive (Day & Shattock, 1997; Flier & Turkensteen, 1999) and more diverse population of P. infestans in Western Europe (Spielman et al., 1991) might have a negative impact on the efficacy of late blight management tools. Evidence is accumulating that P. infestans is sexually reproducing in many countries in Western Europe (Spielman et al., 1991; Drenth et al., 1994; Sujkowski et al., 1994; Andersson et al., 1998; Brurberg et al., 1999; Turkensteen et al., 2000). Sexual reproduction results in variation in the pathogen and might lead to increased and more rapid evolution of the pathogen.

In the European Union (EU), organic growers apply a wide range of measures aimed at controlling late blight in potato crops, including the use of copper-based fungicides, early cropping systems, various cultural practices and cultivar resistance (Tamm et al., 2004). Protective copper-based fungicides, which are currently used to control late blight in most organic production systems, are estimated to slow down an epidemic by approximately 10 to 30 days, depending on weather conditions and cultivar resistance. The additional growth period provided by copper-based fungicide protection is estimated to increase the income of EU-organic potato growers by between 15 and 45 million euro(€) per annum (Zarb et al., 2002). In an agro-economic study, Varis et al. (1996) reported that late blight was a serious problem in cv. Bintje in both integrated and organic systems in Finland. Total yields were 10% and 36% lower, respectively, compared to the conventional cropping system. Similar late blight problems have been reported in organic potato production in the Netherlands (Lammerts van Bueren, personal Communication, Louis Bolk Instituut, Hoofdstraat 24, 3972 LA Driebergen, the Netherlands).

Maximum annual copper use in organic agriculture has been continually reduced, and from 1st January 2006 was limited to 6 kg ha−1 per year (Council Regulation (EEC) No. 2092/91). This regulation on copper-based fungicides may interfere with EU policy aiming to support the expansion of organic production in Europe. Therefore, new disease management tools are urgently needed to replace copper containing fungicides in organic potato production, while securing economic profitability and durability of organic production systems. A shift towards a more widespread use of resistant potato cultivars in organic potato production systems may contribute to a significant reduction of fungicide use, or even facilitate potato production without fungicide inputs, while maintaining an economically acceptable yield and product quality (Inglis et al., 1996).

A potential drawback of the widespread use of more resistant potato cultivars is the increased instability of host resistance in potato against P. infestans that may be associated with the displacement of the old clonal population of P. infestans with a more aggressive and variable population of the pathogen in Europe (Flier et al., 2001, 2003a). This finding is supported by reports in recent years from commercial potato growers who repeatedly suffer severe late blight outbreaks in cultivars with high levels of partial resistance, and high tuber blight incidences, leading to serious yield losses. However, no EU-wide monitoring of these incidences is currently in place. R-gene containing potato cultivars like Escort and Santé, which are widely grown in organic farming in the Netherlands, have shown considerable levels of foliar and tuber blight in recent years, resulting in serious yield losses. The observation of increased yield losses due to late blight in organic crops has raised concern about the possible increase in pathogenicity of P. infestans to more resistant potato cultivars. Information on the pathogenicity of P. infestans populations in organic crops may help to assess the risk of increasing virulence and aggressiveness following a large scale replacement of susceptible potato cultivars by those with higher levels of host resistance.

The aim of the present study was (i) to compare populations of P. infestans collected from organic potato crops grown in France, Norway, Switzerland and the UK in terms of pathogenicity (virulence phenotype and aggressiveness) and genetic marker variation; and (ii) to compare levels of genetic variation and mean pathogenicity of isolates collected from the susceptible cv. Bintje with isolates sampled from the moderately resistant cv. Santé. Finally, the likelihood of a potential shift towards increased levels of pathogenicity in P. infestans populations following a large scale introduction of more resistant potato varieties is discussed. This study was part of the EU funded project ‘Blight-Mop’ which aimed to develop a systems approach for late blight management in organic farming.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Collection and isolation of isolates

Potato leaves naturally infected by P. infestans were collected from experimental organic plots of cv. Bintje and Santé in France, Norway, Switzerland and the UK in 2001 (Table 1). Additional samples were collected from similar experiments in Switzerland in 2002. At each site the experimental plots consisted of nine cultivars in a randomized block design with four replicates, each subplot 3 m wide consisting of four rows, 0·75 m apart and 15 m long (Speiser et al., 2006). Sampling started from one week after the first lesions appeared and continued until 50% of leaf area was infected in the four replicate plots of each cultivar. Isolations were made by trapping P. infestans from leaves with single lesions onto potato tuber slices, followed by culturing on a selective rye agar (RA) (Grünwald et al., 2001). All isolates were subsequently maintained on pea agar at 18°C and sent to the Netherlands within 3 months of collection, where stock cultures of 229 isolates were permanently stored in liquid nitrogen (Flier & Turkensteen, 1999) at Plant Research International, Wageningen.

Table 1.  Sample sites, year of collection, cultivars and number of Phytophthora infestans samples used in subsequent phenotypic and genotypic analyses
Population codeNo. isolates characterized for:
CountryRegionLocationCultivarYear of collectionMating type, haplotype & pathogenicityAFLP
UKS2001United KingdomNorthern EnglandNewcastleSanté20012422
UKB2001United KingdomNorthern EnglandNewcastleBintje20012827
NS2001NorwayRogalandSaerheimSanté20013027
NB2001NorwayRogalandSaerheimBintje20013028
FS2001FranceNorthern BrittanySuscinioSanté20013027
FB2001FranceNorthern BrittanySuscinioBintje20013034
CHS2001SwitzerlandZurichReckenholzSanté20011414
CHB2001SwitzerlandZurichReckenholzBintje2001 4 4
CHS2002SwitzerlandZurichReckenholzSanté2002 9 3
CHB2002SwitzerlandZurichReckenholzBintje20023024

Pea agar was prepared by autoclaving 120 g of frozen peas in 1 L of water. The peas were removed by filtering through cheesecloth and the broth was autoclaved again after adding 15 g L−1 agar. In subsequent analyses of virulence and epidemic parameters, isolates from Switzerland collected in 2001 and 2002 were pooled due to the low numbers of isolates collected from Bintje in 2001 and Santé in 2002.

Culturing and inoculum preparation

All genetic diversity and pathogenicity studies were performed at Plant Research International. Isolates taken from liquid nitrogen storage were first inoculated on tuber slices of the susceptible potato cv. Bintje and incubated in the dark at 15°C for 5 to 7 days. When sporangia were present, small tufts of mycelium were placed in a drop of water on the abaxial epidermis of leaflets of cv. Bintje placed in 9 cm Petri dishes containing 10 mL 2% water agar. The inoculated leaflets were incubated for seven days in a climate chamber at 15°C with a 16 h light period (Philips fluorescence tubes type 33, intensity of 12 Wm−2). Sporangial inoculum was prepared by washing leaflets showing abundant sporulation in 15–20 mL tap water followed by concentration adjustment using a flowcytometer (Beckman Coulter BV). Sporangial suspensions were kept at 18°C and used within 30 minutes of preparation.

Race and mating type determination

The resistance gene (R-gene) differential set of potato clones for race identification consisted of: R1, R2, R3, R4, R5, R6, R7, R10, R11 (Black et al., 1953; Malcolmson & Black, 1966) together with the universal suscept, cv. Bintje. Virulence for R8 and R9 was not assessed as the corresponding R-gene differentials were excluded from the set due to virus infection. Tubers of each differential clone were planted in 12 L plastic pots containing loam-based compost. Plants were grown under greenhouse conditions with at least 16 h light a day, supported by Philips Son-T Agro illumination. The greenhouse temperature was kept at 20°C with 80% relative humidity.

For race determination, detached leaflets of 6 to 10-week-old differential plants were placed abaxial face up in 9 cm diameter plastic Petri dishes containing 10 mL 2% water agar. A sporangial suspension (104 sporangia mL−1) was sprayed to runoff onto the leaflets with a spraying nozzle operated at a pressure of 0·5 kg m−2. Inoculated leaflets were incubated in a growth chamber at 15°C in the dark for 24 h. Subsequently the remaining water on the surface leaf was allowed to evaporate by placing the Petri dishes, without lids, in a laminar flow cabinet for 30 minutes. Incubation was continued in a climate chamber at 15°C with a photoperiod of 16 h provided by fluorescence tubes type 33 (Philips) at an intensity of 12 W m−2. Disease symptoms were recorded seven days after inoculation. Two leaflets per R-gene differential were inoculated in each test, and the experiment was repeated. The interaction between R-gene and virulence factor was considered compatible when sporulation was clearly visible on infected leaflets on at least one of the replicate leaflets in both independent experiments.

Mating type was determined by in vitro crosses with known A1 and A2 tester strains (IPO98014 and IPO82001, respectively) according to Grünwald et al. (2001).

Epidemic parameters

The epidemic parameters infection efficiency (IE), lesion growth rate (LGR) and sporulation density (SPOR) were measured in detached leaflet assays as described by Flier et al. (2003a) with minor modifications. Bintje and Santé plants were grown in the greenhouse from certified seed. Fully-grown lateral leaflets of cv. Bintje or Santé were collected and placed in 9 cm diameter Petri dishes filled with 10 mL of 1·5% water agar. For inoculation, either 10 drops, each of 10 µL, of a sporangium suspension 1·0 × 104 spores mL−1 (IE), or a single drop of 10 µL of a sporangium suspension 5·0 × 104 spores mL−1 (LGR, SPOR) were placed on the lower side of each leaflet. Two replicate leaflets of each plant genotype were inoculated with each isolate. The whole experiment was repeated. The Petri dishes containing the inoculated leaflets were wrapped in transparent polythene bags and incubated in a climate chamber for one week at 15°C with a light intensity of 12 Wm−2, 16 h light per day. Infection was recorded 8 days after inoculation. Infection frequency (IF), based on the fraction of lesions showing sporulation, was calculated and transformed into an estimate for IE according to Colon et al. (1995a) using the formula IE = 1 –H1/k, where H is the fraction of unsuccessful inoculations and k the average number of sporangia per inoculum droplet. The developing lesions were measured three times at three, four and five days after inoculation, using an electronic calliper. Length and width of each lesion were measured and the average diameter, lesion area (LA) and lesion growth rate (LGR) were calculated. Sporulation density (SPOR) was calculated after 8 days of incubation. Lesion area (mm2) was estimated by image analysis using the MINIMOP image analyzer (Kontron). Sporangia from each individual lesion were collected into a single vial containing 10 mL ISOTON 2 solution (Beckman Coulter BV) and counted using a flow cytometer (Beckman Coulter BV). Spore density (SPOR, sporangia per mm2 lesion area) was calculated from the average of two counts of 0·5 mL each.

DNA extraction

Isolates were grown for 10 to 14 days at 20°C in pea broth supplemented with 200 mg L−1 ampicillin. The mycelium was harvested, lyophilized and stored at −80°C. Lyophilized mycelium (10 to 20 mg) was ground in microcentrifuge tubes with a pestle and sterile sand. Total DNA was extracted using the Puregene kit (Gentra/Biozym) according to manufacturer's instructions. DNA was suspended in 100 µL of TE (10 mm Tris-HCl [pH 8·0], 1 mm EDTA [pH 8·0]) and stored at −20°C.

Mitochondrial haplotypes

The P1 (1118 bp), P2 (1070 bp), P3 (1308 bp) and P4 (964 bp) regions of the mitochondrial genome were amplified using primers and methods described by Griffith & Shaw (1998). Reactions were performed in a PTC200 thermocycler (MJ Research). PCR products were digested with restriction enzymes CfoI, MspI and EcoRI resulting in restriction fragment band patterns that could be classified into four different mtDNA haplotypes: Ia, Ib, IIa and IIb.

Fluorescent amplified fragment length polymorphisms (AFLPs)

DNA (250 ng) was digested in a 50 µL reaction volume with EcoRI (10 U) and MseI (10 U) for 6 hours at 37°C in restriction ligation buffer (10 mm Tris/Ac [pH 7·5], 10 mm MgAc, 50 mm KAc, 5 mm DTT, 50 ng µL−1 BSA). Digestion was confirmed on agarose gels. Restriction fragments were ligated to MseI adapters (5′ GACGATGAGTCCTGAG/TACTCAGGACTCAT 5′) and EcoRI adapters (5′ CTCGTAGACTGCGTACC/CTGACGCATGGTTAA 5′) using 0·1 µm EcoRI adapter, 1·0 µm MseI adapter, 0·2 mm ATP and 2·4 Weiss-U T4 DNA ligase (Amersham Pharmacia Biotech). Ligation was performed overnight at 10–12°C and the ligation products were diluted 10 times with filtered ultra pure water. Non-selective PCR amplification was performed using primers Eco00 (5′ GACTGCGTACCAATTC) and Mse00 (5′ GATGAGTCCTGAGTAA). Non-selective PCR amplifications were performed in a PTC200 thermocycler (MJ Research) as described previously by Flier et al. (2003b). The amplified restriction fragment products were checked on 1·0% agarose gels. Selective PCR was performed in a 20 µL reaction volume with 5 µL of 20 × diluted amplification products as described previously (Flier et al., 2003b) using either the primers Eco19 and Mse 16 or Eco21 and Mse 16. Products were loaded on Sequagel (Biozym) polyacrylamide gels and run on an ALFexpress automatic sequencer (Amersham Pharmacia Biotech). Conditions were 1500 V, 60 mA, 35 W, and 55°C. Thirty six samples were loaded on each gel, together with flanking Cy5-labelled fluorescent 50 bp ladders (Amersham Pharmacia Biotech) and two reference isolates (PIC99016 and NL-VK6C).

Data analysis

All statistical tests for the epidemic parameter data were performed using the statistical software Genstat version 6·1 (Payne et al., 1993). Restriction fragments of isolates were visualized on agarose gels using ethidium bromide under UV illumination, and classified according to mtDNA haplotype. AFLP patterns were analysed using Imagemaster ID software (Amersham Pharmacia Biotech), manually correcting for faint bands and exclusion of controversial bands. A total of 137 distinct and reproducible AFLP bands were identified using the primers either Eco19 or Eco21 in combination with Mse16. Bands were treated as putative single AFLP loci and a binary matrix containing the presence or absence of these reproducible bands was constructed and used for further analysis. Statistical analyses were conducted using TFPGA (Tools for Population Genetic Analyses, version 1·3). Each AFLP band was assumed to represent the dominant genotype at a single locus, while the absence of that same band represents the alternative homozygous recessive genotype.

Genotypic diversity analysis was used to determine the distribution of diversity among populations (France, Norway, Switzerland and the UK) and among subpopulations of P. infestans collected from Bintje or Santé. Genotypic diversity was calculated using Shannon's information index (Magguran, 1988) and an unbiased Shannon's index (Abu-El Samen et al., 2003). Pair-wise measures of Roger's modified genetic distance and population differentiation was estimated using an exact test (Raymond & Rousset, 1995) in order to assess the significance of the different statistics for the null hypothesis of no differentiation at the corresponding hierarchical level. Permutation and re-sampling tests (jackknifing and bootstrapping) were carried out to calculate estimates for standard errors. Cluster analysis of AFLP genotypes was based on allele frequencies observed for each sub-population. A phenogram was constructed using the neighbour joining method algorithm from a Rogers’ modified genetic distance matrix. Bootstrap sampling (1000 replicates) was performed for parsimony analysis of the constructed tree.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mating types and mtDNA haplotypes

Both the A1 and the A2 mating type were detected in isolates collected from organic potato crops in Europe in 2001. Only A1 mating type isolates were detected in pathogen population samples from the UK, France and Switzerland (2001 sampling only). The A2 mating type was predominant in isolates collected on cv. Bintje from Switzerland in 2002. Mixed A1 and A2 mating type populations were present in Norway, on both cvs. Bintje and Santé, and in Switzerland on Santé in 2002 (Table 2). Isolates collected from cv. Bintje in Norway were mainly A2 (28 out of 30), while mating types in isolates from cv. Santé were more evenly distributed (15 A1 and 15 A2 strains).

Table 2.  Isolate sample sizes, mating type distributions, mtDNA haplotypes, AFLP genotypes and genotypical diversity of Phytophthora infestans populations collected from the susceptible potato cv. Bintje and the moderately resistant cv. Santé organically grown in France, Norway, Switzerland and the United Kingdom in 2001 and 2002
 Mating typemtDNA haplotypeAFLP genotypesGenetic diversity
Population codeA1A2n.d.aIaIbIIan.d.HsbHsrc
  • a

    Not determined.

  • b

    Genetic diversity within populations.

  • c

    Relative genetic diversity within populations (corrected for sample size).

UKS200124  23  1 51·050·34
UKB200128   6 22  71·340·41
NS20011515 27 3  152·480·75
NB2001 228 1119   81·670·50
FS200129 119 11 112·040·62
FB200130  14 16 132·150·61
CHS200114  14    20·900·34
CHB2001 4       30·560·41
CHS2002 1 8  1 8  30·640·58
CHB2002 30   291 20·460·14

Mitochondrial DNA haplotypes Ia and IIa, both representative for isolates associated with recent migrations of the late blight pathogen, were detected in P. infestans populations from the UK, France and Switzerland (Table 2). In the Norwegian population both the Ia and Ib mtDNA haplotypes were detected. In isolates collected from cv. Bintje, 19 out of 30 isolates were of the Ib haplotype. No statistically significant association between the Ib haplotype and the A1 mating type was observed (data not shown).

Virulence tests

With the exception of virulence for resistance genes R8 and R9, which were not included in the differential set, all nine virulence factors were detected in isolates collected from France, Norway, Switzerland and the UK. The most common virulence phenotype was race 1,3,7,11 with an overall frequency of 42·4%, followed by races 1,2,3,7,11 and 1,7, both with a frequency of 9·3% (Table 3). The most complex race (1,2,3,4,6,7,10,11) was found exclusively in isolates collected from cv. Santé in Switzerland. Race 0 strains were present in all four P. infestans populations sampled. In total, 23 different races were detected in 229 isolates. Virulence complexity, measured as the average number of virulence factors per isolate, ranged from 2·03 for isolates collected from Norway on cv. Bintje to 4·78 for isolates collected from cv. Santé from Switzerland (Fig. 1). Isolates collected from the moderately resistant cv. Santé showed a significantly higher virulence complexity than isolates from cv. Bintje, with the exception of the P. infestans population from France (Fig. 1).

Table 3.  Incidence (in percentage) of physiological races of Phytophthora infestans in isolate samples collected from the susceptible potato cv. Bintje and the moderately resistant cv. Santé organically grown in France, Norway, Switzerland and the United Kingdom in 2001 and 2002
RaceaCountry
FranceNorwaySwitzerlandUKAverage
BintjeSantéBintjeSantéBintjeSantéBintjeSanté
  • a

    Numbers refer to the described R-genes in the differential host plants that each race is capable of infecting.

00·06·76·713·30·04·37·10·04·8
10·00·040·03·30·04·37·10·06·9
30·00·00·03·30·00·00·00·00·4
1,30·00·00·00·00·00·03·60·00·4
1,70·00·016·713·341·20·03·60·09·3
1,100·00·010·00·00·00·00·00·01·3
1,110·00·00·00·00·00·03·60·00·4
1,2,70·00·00·00·035·30·00·00·04·4
1,3,70·00·03·30·00·00·00·04·20·9
1,3,113·33·30·03·30·00·032·14·25·8
1,2,3,70·00·00·00·02·90·00·00·00·4
1,2,7,100·00·00·00·02·90·00·00·00·4
1,3,7,1140·083·320·053·35·921·735·779·242·4
1,3,10,116·70·00·00·00·00·00·00·00·8
1,2,3,7,103·30·00·00·00·00·00·00·00·4
1,2,3,7,1120·03·30·06·72·921·77·112·59·3
1,2,3,10,116·70·00·00·00·00·00·00·00·8
1,3,4,7,110·03·30·00·00·00·00·00·00·4
1,3,7,10,1113·30·00·00·08·821·70·00·05·5
1,2,3,4,7,113·30·00·00·00·00·00·00·00·4
1,2,3,5,7,110·00·00·03·30·00·00·00·00·4
1,2,3,7,10,113·30·03·30·00·013·00·00·02·5
1,2,3,4,6,7,10,110·00·00·00·00·013·00·00·01·6
image

Figure 1. Average number of virulence factors for each Phytophthora infestans population collected from the susceptible potato cv. Bintje and the moderately resistant cv. Santé organically grown in France, Norway, the UK (2001) and Switzerland (2001 and 2002). Bars indicate SED of the mean.

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Epidemic parameters

Considerable variation for the epidemic parameters infection efficiency (IE), lesion area (LA) and sporulation density (SPOR) was present in P. infestans populations sampled from cvs. Bintje and Santé in the four European countries.

There was no consistent evidence across countries for cultivar-specific isolates. However, in many cases the tendency was for more aggressiveness among isolates from cv. Bintje. Infection efficiency ranged from 0·0005 for Swiss isolates from cv. Santé to 0·0079 for Norwegian isolates from cv. Bintje (Table 4). Isolates collected from cv. Bintje showed a higher average IE compared to isolates from cv. Santé (0·0030 and 0·0016, respectively). However, no statistically significant effect for cultivar or cultivar by country was detected (Table 4).

Table 4.  Average restricted maximal likelihood (REML) estimates for epidemiological parameters of Phytophthora infestans isolates collected from the susceptible potato cv. Bintje and the moderately resistant cv. Santé organically grown in France, Norway, Switzerland and the UK in 2001 and 2002
Country of originEpidemiological componentsa
IELASPOR10LOG(SPORCAP)
BintjeSantéBintjeSantéBintjeSantéBintjeSanté
  • a

    IE = infection efficiency, LA = lesion area, SPOR = sporulation density, 10LOG(SPORCAP) = 10Log(LA × SPOR).

Switzerland0·00060·00054262434662465·24·7
France0·00190·001067654927629255·1
Norway0·00790·00133634792812424·75
UK0·00150·00355796774252645·15·2
Average0·00300·00165114873622615·05·0
χ2Cultivar0·409 0·666 <0·001 0·81 
χ2Cultivar × Country0·242 <0·001 <0·001 <0·001 
LSD(0·95)Cultivar0·0061 188 88 0·2 
LSD(0·95)Country0·0063 186 88 0·2 
LSD(0·95)Cultivar × Country0·0064 200 94 0·2 

Lesion area ranged from 243 mm2 to 677 mm2 for isolates collected on cv. Santé from Switzerland and the UK, respectively. Average LA for isolates collected on Bintje was 511 mm2, compared to 487 mm2 for isolates from Santé. No significant difference was found for average LA values for both cultivars, but a highly significant differential interaction for cultivar by country was detected (P > 0·001). Sporulation density varied considerably for P. infestans populations collected from cv. Bintje, but not for populations sampled from cv. Santé (Table 4).

Average SPOR for isolates collected on cv. Bintje varied from 276 to 466 sporangia mm−2 for French and Swiss isolates, respectively. Sporulation density for isolates originating from cv. Santé varied from 242 to 292 sporangia mm−2 for Norwegian and French isolates, respectively. Significantly higher (P > 0·001) average SPOR values were measured on isolates collected from cv. Bintje compared to cv. Santé (362 versus 261 sporangia mm−2). No significant cultivar effect was detected for sporulation capacity estimated as 10LOG(SPOR*LA) (Table 4). A highly significant differential interaction for cultivar by country was detected (P > 0·001) for both SPOR values and 10Log (SPORCAP).

Population structure

A total of 69 AFLP genotypes were detected in 210 isolates using 137 AFLP loci amplified with two primer combinations. A neighbour-joining tree showing the genetic similarities between AFLP genotypes is presented in Fig. 2. No AFLP genotype was detected in more than one national P. infestans population, and no isolates with AFLP genotypes associated with the US-1 clonal lineage, typical of the old population of P. infestans, were detected (Fig. 2) (data not shown). A total of 38 AFLP genotypes were detected only once, and the most frequent AFLP genotype (CHB04) was found in 21 isolates. The normalized Shannon index of genetic diversity varied from 0·14 for Swiss isolates collected from cv. Bintje in 2002 to 0·75 for Norwegian isolates collected from cv. Santé (Table 2).

image

Figure 2. Dendrogram of genotypes of Phytophthora infestans collected from the susceptible potato cv. Bintje and the moderately resistant cv. Santé (numbers of identical isolates in parentheses) organically grown in France, Norway, Switzerland and the UK in 2001 and 2002. Neighbour joining tree was constructed using AFLP markers. The scale shown is the genetic distance calculated using Roger's modified genetic distance coefficients. Numbers at the nodes represent bootstrap values supporting the branches, generated from 1000 random permutations. Isolate VK6C, an A1 isolate collected in the Netherlands in 1958, (Flier et al., 2001) was included as an outgroup.

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Modified Roger's genetic distance coefficients for cultivar specific subpopulations of P. infestans indicate considerable differences between P. infestans populations sampled from organic potato crops from France, Norway, Switzerland and the UK (Table 5, Fig. 3). Cultivar specific subpopulations of isolates from each country are without exception most closely related to each other (Fig. 3). The French pathogen population is most closely related to the Swiss population sampled in 2001, although a significant difference (P > 0·001) was detected between the French population on cv. Bintje and the Swiss population from cv. Santé, based on an exact test for population differentiation (Table 5). The two Norwegian P. infestans subpopulations were significantly different from other subpopulations, except the Norwegian subpopulation on Santé, which was not different from the Swiss subpopulation on Bintje in 2001 (Table 5). Based on AFLP marker data, both Swiss P. infestans subpopulations collected in 2001 differ from the 2002 samples collected on cv. Santé, but not from the 2002 samples from cv. Bintje. The pathogen population from the UK showed some similarity to the Swiss population collected in 2001 but not to other P. infestans populations (Fig. 3, Table 5).

Table 5.  Matrix of genetic distance and population differentiation for Phytophthora infestans populations collected from the susceptible potato cv. Bintje and the moderately resistant cv. Santé organically grown in France, Norway, Switzerland and the UK in 2001 and 2002. Values above the diagonal are probabilitiesa for each pair wise comparison using an exact test (Raymond & Rousset, 1995), values below the diagonal are Wright's modified Roger's distance
 NB2001NS2001FB2001FS2001UKB2001UKS2001CHB2001CHS2001CHB2002CHS2002
  • a

    Probability level considered significant if <0·05.

NB2001 0·0000·0000·0000·0000·0000·0000·0000·0000·000
NS20010·228 0·0000·0000·0000·0000·6070·0000·0000·000
FB20010·3110·267 1·0000·0000·0001·0000·0000·0000·000
FS20010·3400·2830·155 0·0000·0001·0000·4000·0000·000
UKB20010·3620·3260·2740·264 0·0060·9950·0000·0000·000
UKS20010·3690·3120·2800·2730·231 0·9990·0180·0000·000
CHB20010·3800·3290·2600·2380·2860·281 1·0000·0001·000
CHS20010·3630·3230·2600·2300·2790·2730·184 0·0000·168
CHB20020·4380·3960·4020·3870·3920·3840·4260·402 0·000
CHS20020·3900·3610·3470·3260·3400·3200·3550·3210·370 
image

Figure 3. Cluster analysis of 10 subpopulations of Phytophthora infestans collected from the susceptible potato cv. Bintje and the moderately resistant cv. Santé organically grown in France, Norway, Switzerland and the UK in 2001 and 2002. Dendrogram is based on UPMGA clustering using Roger's modified genetic distance coefficients.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Genetic variation in populations of P. infestans collected from organic potato crops from France, Norway, Switzerland and the UK were compared in terms of virulence phenotype, aggressiveness and genetic marker variation. Using the same traits, isolates collected from the susceptible cv. Bintje were compared with isolates sampled from the moderately resistant cv. Santé. The genetic structure of the four P. infestans populations sampled varied from mainly clonal populations showing restricted levels of genetic diversity to mixed A1 and A2 mating type populations showing high levels of genetic diversity, suggesting periodic sexual reproduction.

Based on mating type distribution and mtDNA haplotyping, all four European P. infestans populations sampled belong to the newly established pathogen population that was introduced around 1976 (Drenth et al., 1994). In the UK and France, only the A1 mating type was recovered and all isolates were of the Ia or IIa mtDNA haplotype, both associated with the recently introduced European P. infestans population. The absence of A2 mating type strains in the sample from the UK population is in line with the report by Day & Shattock (1997) in which the A2 frequency in England and Wales from 1993 to 1995 was estimated as 0–3%. Also, the predominance of A1 isolates from Brittany is similar to previous findings by Lebreton et al. (1998), who reported A2 strains only from tomatoes, sampled in north-western France from 1996 to 1997. In Norway, a late blight population comprising both A1 and A2 mating type strains was present at the site of the experimental field in 2001, with the majority of the A1 isolates (88%) collected from cv. Santé. In a multiple year sampling (1993–1996) of 128 isolates of P. infestans collected from south-west Norway, the A1:A2 ratio was calculated as 7:3 (Hermansen et al. 2000), which is quite different from the more limited 2001 data presented here.

The Ib haplotype, associated with the old clonal P. infestans populations present in Europe during most of the 20th Century (Spielman et al., 1991), was found in 37% of the Norwegian isolates but was not absolutely associated with the A1 mating type strains, as was the case in the old clonal population. These results suggest that meiotic recombination has broken the association between the A1 mating type and the Ib haplotype, and therefore supports the view that sexual reproduction occurs in the Norwegian late blight population (Brurberg et al. 1999). The Swiss pathogen population was the only one that was sampled over two years. Evidence exists that oospores play a role in late blight epidemics in Switzerland (Knapova & Gisi, 2002). In 2001, only A1 mating type isolates were recovered, which were all of the Ia haplotype. In 2002, a strong shift towards A2 mating type and IIa haplotype was observed. Only one A1 isolate was recovered, which had the Ia haplotype. The observed shifts in both mating type and mtDNA haplotype frequencies in the Swiss isolate collection indicate that migration and genetic drift at the end of each growing season may strongly influence population structure and diversity in regional P. infestans populations.

Race complexity varied considerably between the regional P. infestans populations sampled. In all pathogen samples but the French, isolates collected from cv. Santé were more complex than isolates collected from cv. Bintje. This may be related to the presence of the R1 and R10 resistance genes in cv. Santé (Flier et al., unpublished data), although race 0 isolates were recovered from cv. Santé at three sampling locations. The collection of isolates without specific virulence factors from an R-gene containing cultivar may be explained by experimental errors in virulence testing, or physiological aging of the host leading to a lower expression of R-gene induced defence mechanisms (Stewart, 1990). Virulence for R5 was only detected in isolates from Norway, while virulence for R6 was only found in the Swiss population. Specific virulence for R4 was not detected in Norwegian samples, and UK isolates were characterized by a lack of virulence to R10. In a previous study of virulence diversity in Norwegian P. infestans isolates collected in 1996 (Hermansen et al., 2000), it was found that virulence to R5 and R6 was rare while virulence for R4 was common. In that study, about 10% of the isolates came from the same region as isolates from the present study, and no regional pattern of the virulence data was found. There appear to be significant differences in virulence spectrum that may reflect random shifts in race composition and differences in R-gene deployment in popular potato cultivars in each country.

AFLP fingerprinting supported the idea of moderate genetic substructuring between the four P. infestans populations sampled. No AFLP based P. infestans genotype was detected in more than one sampling location and many of the AFLP genotypes were only detected once. It is concluded that high levels of genetic variation are being maintained in both putatively sexually reproducing local populations (e.g. Norway) and asexual reproducing populations (e.g. France). Given the very limited sampling scheme which comprised a collection of approximately 30 isolates from two cultivars, it is remarkable that values up to 0·75 for the normalized Shannon index of diversity are found, indicating high levels of genetic diversity. These findings are in line with an earlier report on genetic diversity of isolates from Norway and Finland (Brurberg et al., 1999). Diversity in isolate samples also differed from year to year. For the Swiss sampling site, where two years of data were available, no AFLP genotype detected in the 2001sampling was recovered in the following year and a strong shift in mating type distribution was observed. The results presented here are in line with other studies reporting strong year-to-year effects on pathogen diversity in the Netherlands and England and Wales (UK) (Zwankhuizen et al., 2000; Day et al., 2004).

Recent work by Carlisle et al. (2002) has shown that differences in aggressiveness between P. infestans isolates can be detected with improved resolution when moderately resistant cultivars are used as tester cultivars. In the current experiments, significant differences were detected in epidemic parameters despite the fact that the susceptible cv. Bintje was used. The detection of significant cultivar by country differential interactions for the epidemic parameters LGR, LA and SPOR is in line with the view that the four late blight populations that were sampled show some level of genetic differentiation.

No evidence has been found to support the hypothesis that P. infestans strains collected from the moderately resistant cv. Santé show increased levels of pathogenicity compared to isolates collected from the susceptible cv. Bintje. On the contrary, isolates collected from cv. Bintje were (for the epidemic parameters tested) on average more aggressive than, or comparable to, isolates from cv. Santé. This result is consistent with earlier findings by Pilet (2003) and Montarry et al. (2006), who found that isolates from partially resistant potato cultivars showed lower aggressiveness levels than isolates collected from susceptible cultivars. However, it should be noted that the observed differences in aggressiveness for isolates sampled from cvs Bintje and Santé were not consistently observed for all countries and that significant country by cultivar differential interactions existed.

In this work, the possible confounding effect of adaptation to cv. Bintje cannot be excluded, since all sporangial suspensions were produced and tested on cv. Bintje. However, previous results (Flier et al., unpublished data) indicate this effect to be of limited importance in aggressiveness testing.

Although the sample sizes were small, no evidence was found to suggest a directional selection towards increased aggressiveness to the more resistant cv. Santé and therefore such shifts are unlikely to occur under field conditions. However, a complicating factor in making general predictions on stability of resistance to late blight is the presence of considerable differences in structure and diversity of regional P. infestans populations. Recent international and intercontinental collaborative studies have explored the stability and evaluation of cultivar resistance exposed to indigenous late blight isolates or natural inoculum (Hansen et al., 2005; Forbes et al., 2005).

It is recommended, therefore, that together with a large scale introduction of more resistant potato cultivars, a monitoring programme is set-up to evaluate the stability of the resistance of regionally grown cultivars. Such a monitoring programme could serve as an early warning system, valuable in resistance management in both organic and non-organic potato production systems in Europe. The Concerted Action EUCABLIGHT, a consortium of European blight researchers and breeders, has agreed on improved methods for assessment of resistant germplasm in field trials and in the laboratory (http://www.eucablight.org, ‘Protocols’ section). The inclusion of common standards with a range of late blight resistance will allow researchers to compare the expression of resistance in clones grown in different climates and exposed to different strains and populations of P. infestans (Colon et al. 2005).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This research was supported by the European Commission (EU-Framework Five project QLK5-CT-2000- 01 065 BLIGHT MOP) and the Netherlands Ministry of Agriculture, Nature Management and Food Safety (LNV-DWK research programmes 342 and 397). We thank all those that were involved in providing isolates and we are very grateful for all the stimulating discussions with the members of the BLIGHT MOP community.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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