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Keywords:

  • AFLP;
  • genotypes;
  • late blight;
  • mating type;
  • microsatellites;
  • phenotypes

Abstract

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

A total of 134 isolates of Phytophthora infestans were collected from potato and 42 from tomato fields in Switzerland and France in 1996 and 1997, and compared with isolates from other countries. The structure of the populations was analysed phenotypically and genotypically, and associated to geographical, seasonal and host origin. Phenotype characteristics of the isolates included mating type; sensitivity to phenylamide fungicides; virulence on potato differentials; and pathogenic fitness. Genotypes were assessed for mitochondrial DNA haplotype with restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) as well as amplified fragment length polymorphism (AFLP) and simple sequence repeats or microsatellites (SSR). The majority (96%) of isolates originating from potato were the A1 mating type, whereas half the isolates collected from tomato were A2 mating type. Isolates with sensitive, intermediate and resistant responses to the phenylamide fungicide metalaxyl-M were detected in the populations. Isolates from potato represented races with highly complex virulence spectra, whereas those from tomato belonged to simple races. The pathogenic fitness of isolates was highest on the host of origin, and was significantly reduced for isolates from potato on tomato. One of the four haplotypes, Ia, dominated the population (93% of isolates). Among isolates collected from potato, 15 different SSR genotypes were detected of which two, A-03 and A-06, dominated the population. From tomato, 11 SSR genotypes were found of which four, A-03, B-03, D-03 and F-01, formed the major proportion of the population. Every ninth and fourth isolate from potato and tomato, respectively, represented a different SSR genotype. Four genotypes were isolated from both hosts, whereas 11 and seven genotypes, respectively, originated exclusively from either potato or tomato. The SSR genotype D-02, represented by the ‘old’ Ib haplotype, was still detected in a few isolates in the current population, and in older reference isolates from different countries. The SSR genotype was not associated with mating type or sensitivity to phenylamide fungicides. A total of 40 AFLP genotypes were distinguished among the isolates, every second isolate representing another genotype. The diverse phenotypic and genotypic structure of the current field populations in Europe suggests that they may have evolved from local processes including sexual recombination, host preference and selection rather than through long-distance migration.


Introduction

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

It has been reported repeatedly that the population structure of Phytophthora infestans, the causal agent of late blight of potato and tomato, has undergone major changes in Europe over the past 20 years (Fry et al., 1993; Drenth et al., 1994; Gisi & Cohen, 1996). The predominant ‘old’ population of P. infestans in Europe was apparently displaced by a ‘new’ population, probably introduced in the late 1970s by a potato shipment from central Mexico to Europe (Niederhauser, 1991; Fry et al., 1993). The old population consisted exclusively of A1 mating type and Ib mitochondrial (mt) haplotype isolates, whereas the new population comprised isolates of both A1 and A2 mating types, and Ia and IIa mt haplotypes (Spielman et al., 1991; Day & Shattock, 1997; Lebreton & Andrivon, 1998). A similar displacement by isolates from Mexico was also described for populations of P. infestans in the USA (Fry et al., 1993), and it is assumed that the current populations in the USA and Europe have evolved further independently (Andrivon, 1994). Since 1980, the A2 mating type has been detected in almost all European countries as well as other parts of the world (Gisi & Cohen, 1996): in 1980/81 in former Eastern Germany (Dagget et al., 1993); in 1981/83 in Switzerland (Hohl & Iselin, 1984); in 1981 and following years in the UK (Tantius et al., 1986); and in 1981 in the Netherlands (Frinking et al., 1987). The proportion of A2 mating type isolates collected from commercial potato fields has remained low in the UK, France, Germany and Switzerland (Gisi & Cohen, 1996; Day et al., 2001), whereas in the Netherlands and Scandinavia it has reached over 50% (Hermansen et al., 2000; Turkensteen et al., 2000). High proportions of A2 mating type isolates have also been detected in allotment gardens in the Netherlands (Drenth et al., 1993a), and in certain years in potato fields in Poland (Therrien et al., 1993). In addition, both A1 and A2 isolates were recovered in 1995 and 1996 from tomato in private gardens in France and Switzerland; the frequency of A2 isolates was often higher on tomato than on potato (Lebreton et al., 1996; Duvauchelle et al., 1997; Lebreton & Andrivon, 1998).

The structure of European P. infestans populations changed significantly after new genotypes of both A1 and A2 mating type isolates were introduced in the late 1970s: genetic variation at the molecular level increased (Drenth et al., 1993b), as did race diversity, and additional virulence factors were detected (Drenth et al., 1994). In addition, higher fitness and aggressiveness were reported for certain new genotypes on different cultivars of potato and tomato (Day & Shattock, 1997), as described for populations evolving in the USA (Legard et al., 1995). These observations raise the question of whether new genotypes in the pathogen population may have evolved through sexual reproduction of isolates from potato (although the A1 : A2 ratio may be in disequilibrium), or from potato and tomato. One study reported that P. infestans populations on tomato and potato were largely separated, but limited gene flow did occur (Lebreton & Andrivon, 1998).

The objective of this study was to describe the current structure of the P. infestans population in France and Switzerland, and to compare that population with reference isolates from other countries. Isolates collected in 1996 and 1997 from potato and tomato fields were characterized phenotypically for mating type; sensitivity to the phenylamide fungicide metalaxyl-M; virulence on R-gene differentials 1–11; and pathogenic fitness. In the past, genotypic characterization of P. infestans isolates included allozyme pattern, mitochondrial DNA haplotype and RFLP fingerprints with RG57. In this study, isolates were also characterized genotypically for mitochondrial DNA haplotype with restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). The isolates were also characterized with two additional molecular methods: amplified fragment length polymorphism (AFLP) banding pattern, and simple sequence repeat (SSR) markers, also called microsatellites. Due to the high level of polymorphisms and a random distribution of the markers throughout the genome, the latter two methods are ideal tools for studies on population genetics and genetic identity of individuals. Based on a different molecular design, the two methods complement each other and are not normally linked to single phenotypic traits.

Materials and methods

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

Origin of P. infestans isolates

A total of 176 isolates of P. infestans were collected in Switzerland and France during 1996 and 1997 from areas with natural late blight infections. The isolates from potato were obtained between 23 May and 28 August from commercial fields of the major potato-growing areas of Switzerland (15 cantons) and France (mainly départements 60, Oise, and 62, Pas-de-Calais). The isolates from tomato were collected between 29 June and 8 September from home gardens of the same regions (plus département 68, Haut-Rhin, in France). Each site was visited once per season, and the closest sites were separated by at least several kilometres. Single leaves of tomato plants with freshly sporulating lesions of P. infestans (two replicates per site) were collected and shipped in paper bags to the laboratory. For potato samples, a single diseased leaflet was collected from each disease focus (three replicates per site) and placed between tuber halves held together with rubber bands. These were packed individually in paper bags and placed in cardboard boxes for shipment. Tuber halves were separated in the laboratory and infected leaflets were removed. Tuber halves (infected leaves in the case of tomato samples) were placed in glass Petri dishes containing moist filter paper and incubated for 7 days (2 days for tomato leaves) at 15°C in the dark to promote sporulation. Freshly formed sporangia were washed from infected tissue (bulked from the replicates) and adjusted to 2 × 104 mL−1 in distilled water. The sporangial suspension (bulk sample) was used immediately to infect leaves or leaf discs (of potato and tomato) to assay virulence, pathogenic fitness and sensitivity to metalaxyl-M (see below). The determination of mating type and genotypic characterization required pure cultures of the pathogen, which were produced by placing small pieces of infected tissue (tomato leaf or potato tuber) on V8 agar amended with antibiotics (Ribeiro, 1978) and incubated in the dark for 12 days at 18°C. Agar plugs of pure cultures were stored under liquid nitrogen and used later to grow mycelium in liquid cultures for DNA extraction.

Several isolates of P. infestans collected in different countries between 1971 and 1990 (obtained from the Syngenta strain collection and from Dr Brigitte Mauch-Mani, University of Neuchatel) were used as reference isolates (Table 1).

Table 1.  Origin and characteristics of Phytophthora infestans reference isolates collected in different countries between 1971 and 1990
CountryIsolateHost originYearbMating typemt haplotypeSSR genotype
  • a

    Isolate from trial garden, first record of A2 mating type isolate in Europe (Hohl & Iselin, 1984).

  • b

    Date of isolation.

  • c

    Date when isolate was received from collection, date of isolation unknown.

  • ?, No information.

SwitzerlandCH 12?1971A1IbD-02
 CH 129apotato1981A2IaA-08
 CH 204potato1984A2IaD-05
 CH 219potato1985A1IIaH-02
 CH 220potato1985A2IaD-03
 CH 49?1986A1IbD-02
 CH 237potato1987A1IaH-01
NetherlandsNL 193?1983A1IaH-01
 NL 194?1983A1IaD-03
 NL 88069tomato1988A1IaH-01
IrelandIRL 235potato1980A1IIaH-02
MexicoMEX 143potato1982A2IaB-03
 MEX 144potato1982A1IaD-07
 MEX 618potato1987A2IaD-05
Costa RicaCR 134, 138, 141potato1982A1IaB-05
USAUS 1306tomato1982A1IIbD-05
 US-1potato?A1IbD-02
 US-7tom/pot?A2IaB-02
 US-8tom/pot?A2IaC-01
IsraelIL 6tomato?A1IaF-03
 IL 7tomato?A1IaF-04
JapanJ 314potato1990cA1IbD-02
 J 317potato1990cA2IIaA-01

Phenotypic characterization of isolates

All isolates used in this study were characterized for mating type (A1, A2); sensitivity to the phenylamide fungicide metalaxyl-M; pathogenic fitness on detached leaves of potato and tomato plants; and virulence on detached leaves of potato differentials. Mating type was determined by placing a mycelial plug of the unknown isolate culture on V8 agar plates (Ribeiro, 1978) 2 cm away from a plug of either an A1 or A2 reference isolate. The cultures were incubated in the dark for 15 days at 18°C, then examined microscopically for the presence of oospores in the centre of the plates where the two isolates had grown together (Vega-Sanchez et al., 2000).

To determine the sensitivity of isolates to the phenylamide fungicide metalaxyl-M (formulated as EC 480), the sporangial suspension described above was inoculated on leaf discs of potato (cv. Bintje, five discs per 5 cm dish) floating on a range of five fungicide concentrations (100, 10, 1, 0·1, 0·01 mg a.i. L−1) as described by Sozzi et al. (1992), representing a standard in vivo assay. Leaf discs were floated on distilled water as a control. After incubation for 7 days, sporulation intensity was evaluated on treated and control discs and converted to percentage disease control. The test was repeated three times. EC50 values were calculated from dose–response curves. The isolates were assigned to three groups: sensitive (s, EC50 ≤ 0·1 mg L−1); intermediate (i, 0·1 mg L−1 < EC50 ≤ 10 mg L−1); and resistant (r, EC50 > 10 mg L−1) (Gisi et al., 1997).

The pathogenic fitness of isolates was evaluated separately on leaves of both hosts by using sporangia collected directly from the field samples as described above. Potato plants (cv. Bintje) and tomato plants (cv. Baby) were grown for 4–6 weeks in a greenhouse at 18–20°C in plastic pots (10 cm diameter) containing a peat/sand mixture. Natural sunlight was augmented by illumination with 40 W sodium-vapour lamps (16 h). Leaf discs (18 mm in diameter) cut from the fourth and fifth leaf of potato and tomato plants were placed, upper surface down, in Petri dishes (9 cm diameter) lined with thick filter paper wetted with 4 mL distilled water. Each leaf disc was centrally inoculated with a 10 µL droplet of the original sporangial suspension (≈250 sporangia per disc) of the isolate to be tested. Each isolate was tested on six leaf discs of each host. After inoculation the Petri dishes were incubated for 4 days at 20°C with 12 h light per day. Lesion size on leaf discs was measured 3 and 4 days after inoculation. Sporulation capacity was assessed 4 days after inoculation by collecting the sporangia from all six leaf discs into 20 mL 10% ethanol. Discs were vortexed for 1 min to dislodge and suspend sporangia. The number of sporangia was counted with the aid of a haemocytometer. Sporulation capacity was expressed as sporangia produced per square millimetre of leaf disc area. Latent period was assessed every 12 h by recording the time period between inoculation and appearance of first necrotic symptoms, excluding hypersensitive reactions. In most cases symptoms coincided with appearance of first sporulation (10% sporulating disc area; Flier & Turkensteen, 1999). Pathogenic fitness was estimated by calculating a fitness index using the formula (lesion size × sporulation capacity)/latent period.

An R-gene differential set of 11 potato clones, each carrying a single resistance gene (R1–R11) was used to assess the specific virulence and race structure of P. infestans isolates (Malcolmson & Black, 1966). The potato cultivar Bintje, which has no known R genes, was also included. Three detached leaflets of each differential were placed abaxial side up in a 9 cm Petri dish lined with wet filter paper and inoculated with five 10 µL droplets of a sporangial suspension (20 000 sporangia mL−1) of each isolate. Six days after inoculation (incubation at 18°C and 12 h light), leaflets (three per isolate) were examined microscopically for the presence of sporulation. The interaction between differentials and specific field isolates was rated compatible or incompatible (virulence present or absent) as described by Flier & Turkensteen (1999).

Genotypic characterization of isolates

Three different molecular methods were used for isolate characterization: RFLP-PCR, AFLP and SSR (microsatellites). DNA was extracted from mycelium using the method of Zolan & Pukkila (1986). RFLP-PCR was used as described for P. infestans by Griffith & Shaw (1998); it distinguishes four known polymorphic regions of the mitochondrial genome of P. infestans (haplotypes Ia, Ib, IIa, IIb). Type II differs from type I by a 1·6 kb insert and rearrangement of flanking sequences. Haplotype Ib isolates are associated with the nuclear DNA fingerprint (probe RG57) of US-1, the ‘old’ clonal lineage (Goodwin et al., 1992).

The AFLP method was used as described previously by Van der Lee et al. (1997) for P. infestans, except that instead of radiolabelled primers, the analyses were done with fluorescent-labelled (FAM and JOE) fragments detected on an ABI Prism 377 DNA Sequencer. The selective PCR was performed with primers containing two selective 3′-nucleotides, E + AC/M + CA or E + AA/M + CA (Van der Lee et al., 1997). For AFLP genotyping, a total of 31 AFLP markers were used (Knapova et al., 2001). Of the 176 field isolates, a subset of 74 were analysed, representing all major SSR genotypes. The calculations of genetic similarity and cluster analysis for AFLP and SSR genotypes were performed with the computer package phylip Version 3·573c (Felsenstein, 1995, phylogeny inference package, Department of Genetics, University of Washington, Seattle, USA) using the Jaccard distance coefficient and the unweighted pair group method algorithms (UPGMA).

Microsatellite (SSR) markers were specifically developed for P. infestans using a genomic library of P. infestans enriched with the simple sequence repeat (CT)n (Knapova et al., 2001). After positive clones were sequenced and unique microsatellite loci identified, primers were designed for each locus. Microsatellite PCR amplifications were performed in 25 µL containing 50 ng genomic DNA, 2·5 µL tenfold reaction buffer B (Promega, Southampton, UK), 0·075 mm of each dNTP, 0·5 µm each of forward and reverse primers, and 0·63 U Taq DNA polymerase (Promega). PCR was performed in a Gene Amp PCR System 9600 (Perkin Elmer, Norwalk, CA, USA). The PCR program was 40 s at 94°C, 40 s at 58°C, and 20 s at 72°C, repeated for 33 cycles and then held for 10 min at 72°C. For automated analysis, forward primers were 5′-end-labelled at each locus with a fluorescent phosphoramidite dye (HEX and NED). A quantity of 1·5 µL microsatellite PCR product was added to 5 µL formamide, 1 µL 5% blue dextran and 1 µL GS-500 ROX (Perkin Elmer). Before loading, the mixture was denatured by heating at 94°C for 5 min. Afterwards, 2 µL of this mixture was fractionated by PAGE (4·75%) on an ABI Prism 377 DNA Sequencer and analysed using the genescan software package (PE Applied Biosystems, Foster City, CA, USA). Of the six selected microsatellite markers, three (Pi4B, Pi4G, PiG11) were polymorphic for the tested isolates of P. infestans. For SSR genotype classification of the isolates, a total of 10 different alleles at two loci were chosen (Table 2). Alleles 206, 214, 218 and 226 were at locus 4B, and alleles 157, 159, 161, 163, 165, 177 at locus 4G (Knapova et al., 2001). Genotype definitions are based on banding pattern similarity at the two loci, as indicated by capital letters (A–H), followed by a two-digit number (01–07) (Table 2).

Table 2.  SSR genotype classification of isolates of Phytophthora infestans collected in Switzerland and France in 1996 and 1997, based on allele frequency at loci Pi4B and Pi4G
GenotypePi4BPi4G
206214218226157159161163165177
A-01  X       
A-02  X     X 
A-03  X   X   
A-04  X  X    
A-05  X  X X  
A-06  X  XX   
A-07  X X X   
A-08  X   XX  
B-01 X        
B-02 X       X
B-03 X    X   
B-04 X    XX  
B-05 X   X    
C-01 X X X   X
D-01 XX       
D-02 XX    X  
D-03 XX   X   
D-04 XX   X X 
D-05 XX  X    
D-06 XX  XX   
D-07 XX X     
E-01X     X   
F-01X X   X   
F-02X X  XX   
F-03X X  X    
F-04X X  X X  
F-05X X       
G-01XX        
G-02XX    X   
H-01XXX  X    
H-02XXX  X X  

Results

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

Phenotypic characterization of field isolates

The P. infestans isolates collected from potato (n = 134) differed from those from tomato (n = 42) for mating type distribution and sensitivity to the phenylamide fungicide metalaxyl-M. The majority (96%) of isolates originating from potato were A1 mating type, while only 50% of isolates from tomato were A1 (Table 3). The population from tomato comprised 93, 5 and 2% of isolates with a sensitive (s), intermediate (i) and resistant (r) response to metalaxyl-M, respectively, whereas the population from potato comprised 34, 18 and 48% of s, i and r isolates, respectively. Of the 27 A2 mating type isolates (tomato and potato origin), only one was resistant and two were intermediate. All others were sensitive (Table 3).

Table 3.  Number of Phytophthora infestans isolates collected from potato and tomato fields in Switzerland and France in 1996 and 1997, categorized according to mating type (A1, A2) and sensitivity to the phenylamide fungicide metalaxyl-M
 PotatoTomato
19961997Total19961997Total
A1A2A1A2A1A2A1A2
Switzerland
Sensitive332 42 4131121127
Intermediate121 70 2001 1 0 2
Resistant351170 5300 0 0 0
France
Sensitive 30 20  522 2 612
Intermediate 40 00  400 0 0 0
Resistant 40 70 1100 1 0 1
Total (176)91437213454161742

The virulence spectra differed significantly among isolates. Among isolates from tomato, A1 isolates that infected four and more differentials were more frequent than simple races, whereas A2 isolates generally infected fewer than four differentials (data not shown). The difference in race structure was very pronounced between isolates from tomato and those from potato. Isolates from potato represented highly complex races (most infected six to eight, with an average of 7·6 differentials), whereas isolates from tomato belonged to simple races (most infected one to four, with an average of 3·4 differentials) (Fig. 1). Four simple races with the virulence combination (4) (1.4) (3.4.7) and (1.3.4.7) dominated in P. infestans isolates from tomato (72% of isolates), whereas the four highly complex races (1.3.4.7.10.11) (1.3.4.6.7.10.11) (1.3.4.7.8.10.11) and (1.3.4.6.7.8.10.11) were most frequent on potato (91% of isolates) (Fig. 2). There was some overlap in the two populations. Four races were observed on both hosts, but these made up only a small proportion of the population on either of the two hosts (Fig. 2). Most isolates were virulent on the differential genotype with R gene 4, followed by R genes 1, 3, 7, 10 and 11. Differentials with R genes 2, 6 and 8 were infected by only a limited amount of isolates; virulent isolates on differentials with R genes 5 and 9 were not detected.

image

Figure 1. Percentage of Phytophthora infestans isolates (n = 176) infecting different numbers of potato differentials (each with one of R genes 1–11). Isolates were collected from tomato and potato fields in 1996 and 1997 in Switzerland and France.

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image

Figure 2. Percentage of Phytophthora infestans isolates (n = 176) characterized by race structure (virulent on differentials with one of R genes 1–11). Isolates were collected from tomato and potato fields in 1996 and 1997 in Switzerland and France. Race structure is as follows: 1: (1); 2: (4); 3: (1.4), 4: (2.4); 5: (3.7); 6: (4.11); 7: (1.3.4); 8: (1.4.7); 9: (1.4.11); 10: (1.10.11); 11: (3.4.7); 12: (4.10.11); 13: (1.3.4.7); 14: (1.2.4.7); 15: (3.4.7.11); 16: (1.2.3.4.7); 17: (2.3.4.7.8); 18: (3.4.7.10.11); 19: (1.3.4.7.10.11); 20: (1.2.3.4.7.10.11); 21: (1.3.4.6.7.10.11); 22: (1.3.4.7.8.10.11); 23: (1.2.3.4.6.7.10.11); 24: (1.3.4.6.7.8.10.11).

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A total of 81 P. infestans isolates, 40 from tomato and 41 from potato, were tested for pathogenic fitness (Table 4). Latent period was rather short for all isolates compared to results of other authors (e.g. Flier & Turkensteen, 1999), and did not differ much between the population from potato and that from tomato. Isolates from potato caused significantly smaller lesions and had lower sporulation capacity on tomato than on potato. Isolates from tomato colonized and sporulated about equally well on both hosts. Therefore the fitness index of isolates was highest on the host of their origin, somewhat lower for tomato isolates on potato and significantly lower (by a factor of >20) for potato isolates on tomato (Table 4). Also, A2 isolates from tomato caused smaller lesions and had lower sporulation capacity than A1 isolates when tested on potato, but not when tested on tomato.

Table 4.  Fitness parameters of Phytophthora infestans isolates collected from tomato and potato fields in 1996 and 1997 in Switzerland and France tested on tomato and potato leaf discs
Isolate originLatent period (h)aLesion size (mm2) after 4 daysaSporulation capacitya (sporangia mm−2)Fitness indexb
TomatoPotatoTomatoPotatoTomatoPotatoTomatoPotato
  • a

    Means of six replicates with standard deviation; one-tailed t-test indicates significant difference between means at P = 0·05 if followed by different letters; capital letters are applicable for horizontal comparison of pairs of values (tomato vs potato), small letters for vertical comparison (A1 vs A2).

  • b

    Fitness index = (lesion size × sporulation capacity)/latent period.

  • c

    All isolates are A1 mating type.

Tomato
All (n = 40)52 ± 1252 ± 10164 ± 135 (A)157 ± 136 (A)77 ± 56 (A)61 ± 48 (A)243184
A1 (n = 24)51 ± 1151 ± 10168 ± 149 (a)224 ± 175 (a)75 ± 57 (a)77 ± 57 (a)247332
A2 (n = 16)53 ± 1352 ± 11158 ± 133 (a) 44 ± 24 (b)79 ± 55 (a)35 ± 24 (a)236 30
Potato
All (n = 41)c29 ± 1043 ± 17 64 ± 44 (A)214 ± 116 (B) 5 ± 3 (A)47 ± 20 (B) 11234

Genotypic characterization of field isolates

With RFLP-PCR of mtDNA, one of the four haplotypes, Ia, was identified as dominating (93%) the Swiss and French P. infestans field population in 1996 and 1997. The old clonal haplotype, Ib, was found in only 4% of the isolates (three and four isolates from potato and tomato, respectively) and haplotype IIa in 3% of the isolates (three and two isolates from potato and tomato, respectively) (Table 5). The reference isolates US-1, J 314 and the Swiss isolates CH 12 (collected in 1971) and CH 49 (collected in 1986) were haplotype Ib and A1 mating type, whereas J 317 (A2) and CH 219 (A1) were haplotype IIa. All other reference isolates were haplotype Ia (Tables 1 and 5).

Table 5.  SSR genotype, mt haplotype (HT), origin, host preference and mating type (A1, A2) of Phytophthora infestans isolates collected from potato and tomato fields in Switzerland and France in 1996 and 1997, and of reference isolates
SSR genotypeamt HTOrigin: potato (P)Origin: tomato (T)Host preferenceReference isolates
TotalA1A2TotalA1A2
  • a

    SSR (simple sequence repeat), nomenclature based on definition in Table 2.

A-01IIa202  PJ 317
A-02Ia110  P 
A-03Ia79790725P, T 
A-04Ia  202T 
 IIa  110T 
A-05Ib110  P 
A-06Ia35341  P 
A-07Ia110  P 
A-08Ia     CH 129
B-01Ia  330T 
B-02Ia     US 7
B-03Ia  440TMEX 143
 Ib  101T 
 IIa  101T 
B-04Ib110  P 
B-05Ia      CR 1·34
C-01Ia     US 8
D-01Ia  330T 
D-02Ib101  PCH 12 & 49; US 1; J 314
D-03Ia  514TCH 220; NL 194
D-04Ia110  P 
D-05Ia440312P, TCH 204; MEX 618US 1306
 IIb     
D-06Ia101  P 
D-07Ia     MEX 144
E-01Ia  202T 
F-01Ia330440P, T 
 Ib  303T 
F-02Ia110  P 
 IIa110  P 
F-03Ia     IL 6
F-04Ia     IL 7
F-05Ia  110T 
G-01Ia101  P 
G-02Ia110211P, T 
H-01Ia     CH 237; NL 193 & 88069
H-02IIa     CH 219; IRL 235
        11P, 7T, 4P + T 
3141341286422121  

With the two microsatellites Pi4B and Pi4G, a total of 31 P. infestans genotypes were identified, of which 22 were detected among the 176 isolates collected in Switzerland and France in 1996 and 1997. Three Swiss genotypes, A-08, H-01 and H-02, isolated in 1981, 1985 and 1987, respectively, were no longer present in the 1996/97 population (Table 5). Six additional genotypes were not found in Swiss and French populations, but occurred in other countries: B-02 (clonal lineage US-7, mating type A2); B-05 (isolate CR 134 collected in 1982 in Costa Rica, mating type A1); C-01 (clonal lineage US-8, mating type A2); D-07 (Mexican isolate MEX 144 collected in 1982, mating type A1); F-03 and F-04 (Israeli isolates IL 6 and IL 7 collected from tomato, mating type A1) (Tables 1 and 5). The genotype B-03, represented by the A2 isolate MEX 143, collected in Mexico in 1982, was also found in the Swiss and French population, but only on tomato. On the other hand, the genotype D-02, characteristic of the ‘old lineages’ (CH 12 from 1971, CH 49 from 1986, US-1 and J 314, all A1 mating type) was detected only once in the current field population and as A2 mating type (Table 5).

Among the 134 P. infestans isolates collected from potato, 15 different genotypes were detected (Table 5), of which two, A-03 and A-06, were dominant in the population (84%). Among the 42 isolates collected from tomato, 11 different genotypes were found (Table 5), of which four, A-03, F-01, B-03 and D-03, made up 60% of the population. Four genotypes (A-03, F-01, D-05 and G-02) were isolated from both hosts and formed 64 and 46% of the populations on potato and tomato, respectively, whereas 11 genotypes were specific to potato and seven were specific to tomato (Table 5). This means that approximately every ninth and fourth isolate collected from potato and tomato, respectively, was a unique genotype. All 22 genotypes were isolated in both 1996 and 1997, and 19 of these occurred in both Switzerland and France, although in different proportions. Three genotypes, A-07, F-02 and G-02, were each detected only once from potato in France. The proportion of heterozygous loci for the populations collected from the two hosts was somewhat higher on tomato (45%) than on potato (31%), suggesting that sexual recombination occurs under field conditions more frequently on tomato.

The seasonal distribution of the 12 SSR genotypes collected from potato fields was evaluated for the Swiss isolates (total of 112 isolates) (Fig. 3). The genotypes A-03 (66 isolates) and A-06 (33 isolates) were detected continuously over the entire sampling period from late May to late August (55 days). The five genotypes A-05, B-04, D-05, F-01 and G-01 were found only in the first half of the season, and the four genotypes A-02, D-02, D-04 and D-06 only in the second half of the season (few isolates per genotype; Fig. 3). The A-01 genotype occurred once at the beginning and once at the end of the sampling period. The increase of isolates over time was much faster for the A-03 than for the A-06 genotype (Fig. 3a,b). All isolates of the A-03 genotype were A1 mating type and predominantly resistant to metalaxyl-M (46 of 66 isolates), whereas A-06 isolates were mostly sensitive (25 of 33) and A1 (except for one A2 isolate). Isolates with an intermediate response to metalaxyl-M were detected for both genotypes in similar proportions. Therefore the population consisted of sensitive and resistant as well as A1 and A2 mating type isolates over the entire sampling period. There was no obvious association between mating type, sensitivity to metalaxyl-M, geographical origin and SSR genotype of isolates, as all possible combinations were detected.

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Figure 3. Cumulative number of SSR genotypes A-03 (a) and A-06 (b) of Phytophthora infestans isolates with a sensitive (s), intermediate (i) and resistant (r) response to metalaxyl-M, collected from potato fields in 1996 and 1997 in Switzerland over a sampling period of 80 days. Occurrence of additional genotypes (single isolates) indicated by arrows.

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Among the 176 field and 23 reference isolates, 31 different SSR genotypes were distinguished by microsatellite markers (Table 5). In a dendrogram, they can be differentiated into four major similarity clusters (Fig. 4). Clusters 1 and 2 contained all B, C, E and G genotypes, including the Mexican isolate MEX 143 collected in 1982 (A2 mating type), both the US-7 and US-8 isolates (both A2 mating type), and the A1 mating type isolates collected in 1982 in Costa Rica. Cluster 3 was composed of a few isolates with genotypes A-01 and A-02, whereas cluster 4 contained over 130 isolates of the genotypes A-03, A-06 and A-07, as well as all D genotypes (D-01 to D-07) except D-05. The ‘old’ (haplotype Ib) isolates of genotypes D-01 and D-02 (A1 isolates CH 12, CH 49, US 1, J 314) were closely related to D-07, an A1 isolate collected in Mexico (MEX 144) in 1982. All F and H genotypes and the genotypes A-04, A-05 and D-05 were also in cluster 4 (Fig. 4).

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Figure 4. Genetic similarity and clusters of SSR genotypes of Phytophthora infestans isolates (total number of isolates, Q = 176) collected from one of two hosts (H), potato (P) or tomato (T), or from both hosts (P + T), in 1996 and 1997 in Switzerland and France and of reference isolates (n = 16).

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The majority of A-03 and A-06 isolates (42 and 21 isolates, respectively) from potato were further characterized by measuring important parameters of pathogenic fitness. The average values of the fitness index of A-03 and A-06 isolates were 455 and 345, respectively (significantly different at P = 0·028) (Fig. 5). Although a large number of isolates within a genotype did not differ significantly, there were some A-03 isolates with clearly higher fitness compared to the average of the isolates.

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Figure 5. Pathogenic fitness index (lesion size × sporulation capacity/latent period) of two SSR genotypes, A-03 (n = 42) and A-06 (n = 21), of Phytophthora infestans isolates collected from potato fields in Switzerland in 1996. Each cross represents an individual isolate. Box comprises 50% of population with median (line), mean (dot) and standard error of mean (bar). One-way anova analysis indicated significant difference between means at P = 0·05.

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From the 74 field isolates (the 13 most frequent of 22 SSR genotypes) and six reference isolates analysed with AFLP markers, a total of 40 AFLP genotypes were distinguished – about every second isolate was a unique genotype (Fig. 6). There was no correlation between SSR and AFLP genotypes. Isolates of the two most frequent SSR genotypes A-03 and A-06, and also both mating types, were distributed over the entire AFLP dendrogram (Fig. 6). Nevertheless, the three reference isolates CH 49, US-1 and J 314 (SSR genotype D-02, Fig. 4) were closely related in the AFLP dendrogram (cluster 3, Fig. 6), but were distinct from both the US 8 isolate (cluster 3) and the US-7 isolate (cluster 4). The 40 AFLP genotypes can be grouped as seven clusters (Fig. 6). Isolates originating from tomato occurred only in clusters 5, 6 and 7, whereas isolates from potato were found in all seven clusters. Clustering of AFLP genotypes was not associated with mating type, sensitivity to metalaxyl-M, haplotype, SSR genotype or geographical origin of isolates (data not shown).

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Figure 6. Genetic similarity and clusters of AFLP genotypes of Phytophthora infestans isolates (Nr, number of isolates = 74) collected from the two hosts (H), potato (P) and tomato (T), in 1996 and 1997 in Switzerland and France, and of six reference isolates (US-1, US-7, US-8, CH 49, J 314, IL 7). Additional characteristics are mating type (MT), A1 (1) and A2 (2), and SSR genotypes. Isolates Nr 35 and 64 are haplotype IIa, isolates Nr 55–57 are Ib, all other isolates are Ia.

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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

Since the early 1980s, dramatic changes have been reported repeatedly in the European population of P. infestans, including uneven mating type distribution, a rapidly increasing but now stabilized proportion of isolates resistant to phenylamide fungicides (Gisi & Cohen, 1996), and the substitution of ‘old’ by ‘new’ genotypes (Carter et al., 1991; Drenth et al., 1994). The changes in population structure were mainly based on differences between ‘old’ (mt haplotype Ib) and ‘new’ (mt haplotype Ia) isolates, for Gpi and Pep alleles and RFLP fingerprint with probe RG57 (Spielman et al., 1991; Fry et al., 1993), as well as genotype diversity and race composition (Lebreton et al., 1998). Mitochondrial DNA haplotyping differentiates between only four genotypes. This study aimed to characterize the recent P. infestans populations in Switzerland and France on potato and tomato, using alternative molecular methods such as AFLP and SSR (microsatellites), and to compare these isolates with those of earlier years and other countries, including well characterized isolates representing US-1, US-7 and US-8 lineages. A direct comparison between RG 57 fingerprinting and the AFLP and SSR markers described here has not been done, although other data sets have been compared (Purvis et al., 2001).

When RFLP-PCR of mt DNA was used for characterization of the population of P. infestans, over 90% of the isolates were Ia haplotype. On the other hand, the genotypic structure was more diverse when the population was analysed with SSR markers, producing 15 different SSR genotypes on potato and 11 on tomato. Even more diversity was detected with AFLP, which identified 40 different AFLP genotypes among 80 isolates. Thus it is obvious that the degree of diversity that can be detected in P. infestans populations depends on the molecular method used. Although SSR detected a large number of genotypes, only a few of these dominated the populations studied. Two and four genotypes accounted for more than 80 and 60% of the isolates on potato and tomato, respectively. Furthermore, these frequent SSR genotypes were detected over the entire sampling period and at all locations. This may indicate a fitness advantage for the most frequent genotypes during the entire epidemic.

In the collected populations of P. infestans attacking potato and tomato in France and Switzerland, about every ninth and fourth isolate from potato and tomato, respectively, was a unique SSR genotype, suggesting that the genetic diversity may be higher in the population on tomato. Similar results based on different methods were also reported by Lebreton & Andrivon (1998). Considering this, and the higher heterozygosity for the population on tomato compared to potato, sexual recombination is likely to occur more frequently in the former case. Nevertheless, both mating types, sometimes in similar proportions, have been found in the same potato field in the Netherlands, Scandinavia and the USA, producing oospores in the plant tissue (Drenth et al., 1993a; Andersson et al., 1998; Gavino et al., 2000; Turkensteen et al., 2000), and oospores have been produced easily at rather high frequencies in potato leaf tissue in controlled experiments (Cohen et al., 1997).

This study produced evidence that P. infestans populations on potato and tomato are generally, but not exclusively, host-specific. Both pathogen aggressiveness and specific virulence support general host specificity. The aggressiveness tests demonstrated that isolates, although pathogenic on both hosts, are generally more aggressive on the host of origin. Low aggressiveness was especially pronounced for A1 isolates from potato on tomato. Many A2 isolates appear to prefer tomato over potato. Based on virulence and race structure, the current populations could be differentiated, with highly complex races on potato and simple races on tomato, and little overlap between the two populations. It is not known whether the different race structures affect gene flow between the populations. Potato isolates may carry ‘unnecessary’ virulence factors, and it is an open question whether modification in race structure should be attributed to displacement of genotypes in a population (Andrivon, 1994). In addition, most commercial potato (and tomato) varieties carry only a few major resistance (R) genes, which might not be responsible for race selection. Furthermore, the race structure was determined with the standard set of potato differentials, which probably underestimates the complexity of race structure on tomato.

Although populations on potato and tomato appear to be generally host-specific, evidence for gene flow between them was also found. Several SSR genotypes were associated with only one host, but four SSR genotypes were common to both hosts, accounting for at least half the respective population. Therefore, it appears that there is considerable exchange of isolates between hosts. Although isolates are more aggressive on their host of origin, they apparently also attack the alternative host. In another study, host preference was observed for many isolates, but gene flow occurred between populations of P. infestans attacking tomato and potato (Lebreton & Andrivon, 1998). As sexual recombination may be frequent in the population on tomato studied here, it may serve as a source for new genotypes spreading to potato through local migration in areas where both crops are planted in close vicinity and at similar periods in the season. Nevertheless, isolates from this study originated from commercial potato fields and home gardens, with tomato separated by many kilometres. Results presented here suggest that certain SSR genotypes express a differential host preference. The reasons for this observation are not known and are currently under investigation (Knapova et al., 2002).

The ‘old’ haplotype, Ib, was identified in seven of 176 field isolates, three on potato and four on tomato. The three isolates from potato represented three different SSR genotypes, A-05 (mating type A1), B-04 (mating type A1), and D-02 (mating type A2). The latter genotype, D-02, was associated with several reference isolates from ‘old’ but also more recent populations, which are all A1 mating type (Table 5). Thus ‘old’ Ib haplotypes still exist in the current population, but they are not necessarily A1 mating type and they belong to different SSR genotypes. The four Ib isolates from tomato were all A2 mating type and belonged to SSR genotypes F-01 and B-03. The latter genotype, B-03, was also identified for an isolate collected in Mexico in 1982 from potato (MEX 143), and might support the theory of migration from Mexico to Europe in the late 1970s (Fry et al., 1993). However, MEX 143 is Ia haplotype. If the B-03 genotype has persisted in Europe until today, it apparently emerged through sexual recombination that resulted in a new haplotype. The first A2 mating type isolate detected in Europe in 1981 by Hohl & Iselin (1984), CH 129 (SSR genotype A-08), was not found in the current population. It may have disappeared or does not represent the ‘migrated, new’ population. Also, isolates collected in the early 1980s in Ireland and the Netherlands (Table 5) were ‘new’ haplotypes (Ia and IIa) and A1 mating type, but represented genotypes (SSR H-02 and H-01) not found in the current population of Switzerland and France. The genotypes forming major parts of the current population in the USA, US-7 and US-8 (SSR B-02 and C-01, respectively), were also not present in the European population. Based on the tested isolates and the molecular methods used in this study, it is proposed that local processes such as host adaptation, selection and sexual recombination are important forces affecting the evolution of new European populations. Migration of foreign genotypes may have occurred earlier and probably still occurs through potato shipments within Europe. Genotypes introduced earlier through migration may not have succeeded in persisting within the ‘new’ population in Europe or, alternatively, underwent continuous sexual recombination with pre-existing genotypes giving rise to new, locally produced genotypes which were fitter than the parents. As suggested earlier (Gisi & Cohen, 1996), sexual recombination may have occurred at a certain level in European populations for many years, but was underestimated due to the low proportion of A2 mating type.

SSR markers revealed a close similarity between both US-7 and US-8 isolates and only a few isolates of the current field population, including the B-03 genotype from tomato (clusters I and II), whereas the majority of the other field isolates were not related to these US genotypes (clusters III and IV). With AFLP markers, again the US-7 and US-8 isolates, and also the ‘old’ genotypes (haplotype Ib, SSR D-02) represented by isolates US-1 and CH 49, were closely related to each other (clusters 3 and 4) but were separated from most other field isolates (including B-03 isolates). These results again suggest that European populations evolved independently from those in the USA, and that sexual recombination occurs recurrently in both populations.

The variability detected by SSR and AFLP markers suggests that recent sexual recombination and subsequent migration and selection may have occurred in the field populations investigated. This is further supported by a study in which different SSR genotypes were crossed under laboratory conditions (Knapova et al., 2002). In a cross between an A-01 and a D-03 isolate, new genotypes were generated in the F1 progeny that were also detected in the current field population, including A-03, the most prominent SSR genotype on potato.

For many purposes, mt DNA analyses are of limited value for the characterization of populations, because there is little variation and only a low level of polymorphism in mitochondrial DNA. Both SSR and AFLP methods provide high levels of polymorphism and can detect gene (allele) frequencies in populations rather easily. However, they could not be correlated to phenotypic traits such as mating type, sensitivity to phenylamide fungicides, geographic origin, virulence or aggressiveness. Therefore it is not surprising that there was a wide range of aggressiveness and different levels of sensitivity to phenylamide fungicides within the SSR genotypes A-03 and A-06 (66 and 33 isolates tested, respectively), although the former was predominantly resistant (46 of 66 isolates) and generally more aggressive than the latter, which was predominantly sensitive (25 of 33 isolates). The analysis of populations with SSR and AFLP markers resulted in different genotypic structures, because SSR analyses were based on 10 alleles at two loci, whereas AFLP analyses used 31 markers distributed over the entire genome. In addition, SSRs are codominant marker loci of tandem repeats of short nucleotide motif, whereas AFLPs are dominant markers of DNA fragments obtained from endonuclease restriction and selective amplification. SSR markers represent a more rapid and robust PCR-based technique compared to AFLP, which is much more time-consuming and technically difficult. SSR is therefore considered to be a better technique, although heterozygotic isolates may be misclassified when null alleles occur due to mutation in the primer annealing site, as was the case for the microsatellite Pi4G and the SSR genotypes A-01, B-01, D-01, F-05 and G-01 (Table 2). With the described SSR markers, it has been shown that the populations of P. infestans existing in 1996 and 1997 in Switzerland and France are characterized by broad diversity, demonstrating the large plasticity of this pathogen.

Acknowledgements

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

We express our thanks to many colleagues who have supported different areas of this work, including David S. Shaw (University of Wales, Bangor, UK) and Isabel Tenzer (formerly Federal Institute of Technology Zürich, CH) for support in generating molecular markers, Brigitte Mauch-Mani (University of Neuchatel, CH) and Hans Hohl (formerly University of Zürich, CH) for providing some of the reference isolates, Alexandra Schlenzig, Helge Sierotzki, Ruth Küng-Färber, Maya Waldner-Zulauf, Eliane Thiffault and Patrick Wille for many suggestions and experimental support, and finally the Novartis RAB Foundation for financial support of the Phytophthora Genomics Consortium and the Project ‘Host specificity and mating in P. infestans’ granted to U. Gisi at the University of Basel, Switzerland.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Andersson B, Sandström M, Strömberg A, 1998. Indications of soil borne inoculum of Phytophthora infestans. Potato Research 41, 30510.
  • Andrivon D, 1994. Race structure and dynamics in populations of Phytophthora infestans. Canadian Journal of Botany 72, 16817.
  • Carter DA, Archer SA, Buck KW, Shaw DS, Shattock RC, 1991. DNA polymorphism in Phytophthora infestans: the UK experience. In: LucasJA, ShattockRC, ShawDS, CookeLR, eds. Phytophthora. Cambridge, UK: Cambridge University Press, 27294.
  • Cohen Y, Farkash S, Reshit Z, Baider A, 1997. Oospore production of Phytophthora infestans in potato and tomato leaves. Phytopathology 87, 1916.
  • Dagget SS, Götz E, Therrien CD, 1993. Phenotypic changes in populations of Phytophthora infestans from eastern Germany. Phytopathology 83, 31923.
  • Day JP, Shattock RC, 1997. Aggressiveness and other factors relating to displacement of populations of Phytophthora infestans in England and Wales. European Journal of Plant Pathology 103, 37991.
  • Day JP, Hardman N, Shattock RC, Shaw DS, 2001. Diversity of Phytophthora infestans in the United Kingdom 1996–98: phenotypic and molecular variation in small samples from many sites reveal clonal lineages and related non-clonal variants. Biodiversity in Plant Pathology. Proceedings of the 5th Congress of the European Foundation for Plant Pathology, Taormina, Italy . Pisa, Italy: SIPV, 15.
  • Drenth A, Turkensteen LJ, Govers F, 1993a. The occurrence of the A2 mating type of Phytophthora infestans in the Netherlands: significance and consequences. Netherlands Journal of Plant Pathology 99, 5767.
  • Drenth A, Goodwin SB, Fry WE, Davidse LC, 1993b. Genotypic diversity of Phytophthora infestans in the Netherlands revealed by DNA polymorphisms. Phytopathology 83, 108792.
  • Drenth A, Tas ICQ, Govers F, 1994. DNA fingerprinting uncovers a new sexually reproducing population of Phytophthora infestans in the Netherlands. European Journal of Plant Pathology 100, 97107.
  • Duvauchelle S, L’Herbier V, Emery D, Sarniguet C, Lebreton L, Andrivon D, Gisi U, Knapova G, Edel D, 1997. Répartition des souches A2 de Phytophthora infestans en France en 1996. 5ème Conférence Internationale sur les Maladies des Plantes, Tours, France. Paris, France: Annales ANPP, 36974.
  • Flier WG, Turkensteen LJ, 1999. Foliar aggressiveness of Phytophthora infestans in three potato growing regions in the Netherlands. European Journal of Plant Pathology 105, 3818.
  • Frinking HD, Davidse LC, Limburg H, 1987. Oospore formation by Phytophthora infestans in host tissue after inoculation with isolates of opposite mating type found in the Netherlands. Netherlands Journal of Plant Pathology 93, 1479.
  • Fry WE, Goodwin SB, Dyer AT, Matuszak JM, Drenth A, Tooley PW, Sujkowski LS, Koh YJ, Cohen BA, Spielman LJ, Deahl KL, Inglis DA, Sandlan KP, 1993. Historical and recent migrations of Phytophthora infestans: chronology, pathways, and implications. Plant Disease 77, 65361.
  • Gavino PD, Smart CD, Sandrock RW, Miller JS, Hamm PB, Yun Lee T, Davis RM, Fry WE, 2000. Implications of sexual reproduction for Phytophthora infestans in the United States: generation of an aggressive lineage. Plant Disease 84, 7315.
  • Gisi U, Cohen Y, 1996. Resistance to phenylamide fungicides: a case study with Phytophthora infestans involving mating type and race structure. Annual Review of Phytopathology 34, 54972.
  • Gisi U, Hermann D, Ohl L, Steden C, 1997. Sensitivity profiles of Mycosphaerella graminicola and Phytophthora infestans populations to different classes of fungicides. Pesticide Science 51, 2908.
  • Goodwin SB, Drenth A, Fry WE, 1992. Cloning and genetic analyses of two highly polymorphic, moderately repetitive nuclear DNAs from Phytophthora infestans. Current Genetics 22, 10715.
  • Griffith GW, Shaw DS, 1998. Polymorphism in Phytophthora infestans: four mitochondrial haplotypes are detected after PCR amplification of DNA from pure cultures or from host lesions. Applied and Environmental Microbiology 64, 400714.
  • Hermansen A, Hannukkala A, Hafskjold Naerstad R, Brurberg MB, 2000. Variation in populations of Phytophthora infestans in Finland and Norway: mating type, metalaxyl resistance and virulence phenotype. Plant Pathology 49, 1122.
  • Hohl HR, Iselin K, 1984. Strains of Phytophthora infestans from Switzerland with A2 mating behaviour. Transactions of the British Mycological Society 83, 52930.
  • Knapova G, Schlenzig A, Gisi U, 2002. Crosses between isolates of Phytophthora infestans from potato and tomato and characterization of F1 and F2 progeny for phenotypic and molecular markers. Plant Pathology 51, in press.
  • Knapova G, Tenzer I, Gessler C, Gisi U, 2001. Characterisation of Phytophthora infestans from potato and tomato with molecular markers. Biodiversity in Plant Pathology. Proceedings of the 5th Congress of the European Foundation for Plant Pathology, Taormina, Italy . Pisa, Italy: SIPV, 69.
  • Lebreton L, Andrivon D, 1998. French isolates of Phytophthora infestans from potato and tomato differ in phenotype and genotype. European Journal of Plant Pathology 104, 58394.
  • Lebreton L, Duvauchelle S, Andrivon D, 1996. Occurrence in France and Belgium of A2 mating type isolates of Phytophthora infestans. In: 13th Triennal Conference of the European Association for Potato Research, 1995, Veldhoven, the Netherlands, 2623.
  • Lebreton L, Laurent C, Andrivon D, 1998. Evolution of Phytophthora infestans populations in the two most important potato production areas of France during 1992–96. Plant Pathology 47, 42739.
  • Legard DE, Lee TY, Fry WE, 1995. Pathogenic specialization in Phytophthora infestans: aggressiveness on tomato. Phytopathology 85, 135661.
  • Malcolmson JF, Black W, 1966. New R genes in Solanum demissum Lindl. and their complementary races of Phytophthora infestans (Mont.) de Bary. Euphytica 15, 199203.
  • Niederhauser JS, 1991. Phytophthora infestans: the Mexican connection. In LucasJA, ShattockRC, ShawDS, CookeLR, eds. Phytophthora. Cambridge, UK: Cambridge University Press, 2545.
  • Purvis AI, Pipe ND, Day JP, Shattock RC, Shaw DS, Assinder SJ, 2001. AFLP and RFLP (RG57) fingerprints can give conflicting evidence about relatedness of isolates of Phytophthora infestans. Mycological Research 105, 132130.
  • Ribeiro OK, 1978. A Source Book of the Genus Phytophthora. Vaduz, Liechtenstein: Cramer.
  • Sozzi D, Schwinn FJ, Gisi U, 1992. Determination of the sensitivity of Phytophthora infestans to phenylamides. A leaf disc method. EPPO Bulletin 22, 3069.
  • Spielman LJ, Drenth A, Davidse LC, Sujkowski LJ, Gu W, Tooley PW, Fry WE, 1991. A second world-wide migration and population displacement of Phytophthora infestans? Plant Pathology 40, 42230.
  • Tantius PM, Fyfe AM, Shaw DS, Shattock RC, 1986. Occurrence of the A2 mating type and self-fertile isolates of Phytophthora infestans. England and Wales. Plant Pathology 35, 57881.
  • Therrien CD, Ritch DL, Sujkowski LS, Spielman LJ, Fry WE, 1993. Phytophthora infestans in Poland from 1987 to 1989; nuclear DNA content, mating type distribution and response to metalaxyl. Journal of Phytopathology 139, 6880.
  • Turkensteen LJ, Flier WG, Wanningen R, Mulder A, 2000. Production, survival and infectivity of oospores of Phytophthora infestans. Plant Pathology 49, 68896.
  • Van der Lee T, De Witte I, Drenth A, Afonso C, Govers F, 1997. AFLP linkage map of the oomycete Phytophthora infestans. Fungal Genetics and Biology 21, 27891.
  • Vega-Sanchez ME, Erselius LJ, Rodriguez AM, Bastidas O, Hohl HR, Ojiambo PS, Mukalazi J, Vermeulen T, Fry WE, Forbes GA, 2000. Host adaptation to potato and tomato within the US-1 clonal lineage of Phytophthora infestans in Uganda and Kenya. Plant Pathology 49, 5319.
  • Zolan ME, Pukkila PJ, 1986. Inheritance of DNA methylation in Coprinus cinerus. Molecular and Cell Biology 6, 195200.