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AFLP and RFLP (RG57) fingerprints can give conflicting evidence about the relatedness of isolates of Phytophthora infestans

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Abstract

Selected isolates of Phytophthora infestans from around England and Wales were fingerprinted using both RG57, a multi-locus RFLP probe, and Amplified Fragment Length Polymorphisms (AFLPs). The larger number of polymorphisms detectable with the AFLP method allowed resolution of several similar AFLP genotypes among isolates with identical RG57 fingerprints. However, some isolates with the same RG57 genotype had remarkably dissimilar AFLP genotypes, suggesting that there has been convergent evolution of some RG57 fingerprints. Also, some isolates with dissimilar RG57 fingerprints had similar or identical AFLP fingerprints. Both techniques distinguished isolates of mitochondrial DNA haplotype la from those of haplotype IIa. However, with AFLPs only, most of the isolates of A2 mating type were very similar and were distinguished from those of A1 mating type, suggesting that gene flow between A1 and A2 genotypes is limited and that sexual recombination is rare.

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... This study describes the establishment of large numbers of single oospore progeny from three matings of one A2 of UK common RG57 fingerprint genotype, RF 040, (Purvis et al., 2001;Shattock, 2002) and three A1s, of which two were UK isolates (RF 039 and RF 032, common and not common RG57 fingerprint genotype, respectively) and one American isolate (P9463), RF genotype not identified. The genotypes of RG57 have the prefix 'RF' for 'RG57 Fingerprint'. ...
... A total of four parental isolates were selected to be used in the study. Three isolates 96.69, 96.89.43 (Purvis, 2000;Purvis et al., 2001) and P9463 were A1-mating type. All these were mated with a single A2 isolate 96.70 (Purvis, 2000;Purvis et al., 2001). ...
... Three isolates 96.69, 96.89.43 (Purvis, 2000;Purvis et al., 2001) and P9463 were A1-mating type. All these were mated with a single A2 isolate 96.70 (Purvis, 2000;Purvis et al., 2001). These are shown in Table 1. ...
Article
The aim of this study was to produce sexual progeny (oospores), of the causal agent of late blight, Phytophthora infestans, study the inheritance of mating type and morphological appearances of these progeny. This pseudofungus exists in two distinct mating types: A1 and A2 mainly in Mexico. However, reports indicate presence of both mating types in many countries all over the world which indicates possibilities of sexual reproduction. The experiment was conducted at the University of Wales, Bangor, in the School of Biological Sciences. Three A 1 parental isolates 96.89.43and 96.69 (both UK) and P9463 (California, USA) were mated with a common A 2 isolate 96.70 (UK). About 545 Single Oospore Progeny (SOP) were obtained. The A1:A2 ratio was 3:1 for cross #1; 1:1 for cross #2; and 3:1 for cross #3. All A1 SOPs and 7% of the A2 SOPs had non-lumpy (-) morphology, the rest (93%) of the A2 SOPs exhibited lumpiness of varying degrees. Lumpiness varied from slighty lumpy (+) (17%), lumpy (++) (56%), to being very lumpy (+++) (20%). No visible sporangiophores observed among the very lumpy progeny. The A2 SOP were lumpy as opposed to non-lumpy A2 parent, which was a fluffy isolate just like the A1 parents. Scanning electron microscope revealed that the lumpiness was an aggregate of short-branching hyphae. Lumpiness of A2 isolates has not been reported in nature but observed in laboratories. This could be due to the failure by such isolates to survive as in this study such isolates were observed to produce very few sporangia.
... Although more time consuming than other methods, it is reproducible and yields many dominant markers per reaction (Milbourne et al. 1997;Schneider et al. 1998) without the need for prior knowledge of the organism's genome. In Phytophthora, AFLPs have been utilized to examine speciation processes (Brasier et al. 1999;Bonants et al. 2000), for mapping (Van der Lee et al. 1997) and population analyses (Lamour and Hausbeck 2001;Purvis et al. 2001;Cooke et al. 2003). ...
... As in other AFLP-based studies of genetic diversity in plant pathogens (e.g. Schneider et al. 1998;Lamour and Hausbeck 2001;Purvis et al. 2001) many AFLP profiles were observed. Only two isolates shared the same fingerprint. ...
... AFLP analysis of more than 600 P. capsici isolates from Michigan showed 57-61% of AFLP bands were polymorphic and genetic differences between local populations were observed (Lamour and Hausbeck 2001). In the case of P. infestans, however, only 17-20% of AFLP bands were polymorphic within natural populations (Purvis et al. 2001;Cooke et al. 2003) and also within the progeny of a cross for genetic mapping (Van der Lee et al. 1997). Unlike P. capsici, P. infestans showed no regional substructuring of genetic diversity in its populations. ...
Article
The recently discovered oak-specific fine root plant pathogen Phytophthora quercina is a significant factor in the current phase of European oak decline but its origins and ecology are poorly understood. A genome-wide analysis of 260 amplified fragment length polymorphism (AFLP) markers was used to examine the genetic diversity of 72 isolates from five oak species at 28 sites in Germany (particularly Bavaria), Italy, France, Hungary and the UK. Within-site diversity was examined at 16 sites. The limited genetic diversity (within and between sites) and lack of genetic substructuring according to geographic origin or host species suggest the rapid spread of a relatively recently introduced species. Two subgroups were distinguished and these may reflect an initial introduction of isolates of two different genetic backgrounds. The relatively low genetic diversity is probably because of the predominantly inbreeding (homothallic) nature of P. quercina. However, evidence of limited intra-site diversity, temporal variation and the lack of clonality within the European population suggest that some diversity is being maintained by occasional outcrossing and turnover of a reservoir of long-lived soil-borne oospore (sexually derived) inoculum.
... A possible hypothesis is that a cluster of similar genotypes of higher frequency represents a clonal lineage. Previously, Purvis et al. (2001) presented dendrograms constructed for 98 isolates selected from a core of the 1995-97 isolates which included three of the four most common RG57 fingerprints described in this study. In some cases, isolates with the same RG57 fingerprint (e.g. ...
... In a contemporary study, four particularly common RG57 genotypes were found in southern Flevoland in the Netherlands in 1994 and 1995 (Zwankhuizen et al., 2000). In this Dutch study, the commonest clonal lineage, NL-41, was identical to RF039, and likewise NL-75 was identical to RF026, a fairly common genotype in Great Britain and analysed more comprehensively (as RF26) by Purvis et al. (2001). In the Netherlands, common genotypes were most frequently found within refuse piles and there was evidence of spread of these genotypes to adjacent, commercial fields. ...
... Characterization of these isolates with sensitive and reliable molecular markers such as AFLPs and microsatellites (e.g. Purvis et al., 2001;Abu-El Samen et al., 2003;Cooke et al., 2003a) should show whether nuclear genotypes of the rare variants were recombinant, and therefore a product of sexual mating, or were identical to the supposed parental genotypes. There is some evidence that common clonal lineages of opposite mating type can generate recombinants within a growing crop of potatoes. ...
Article
Full-text available
A total of 2691 single-lesion isolates of Phytophthora infestans was established from samples of late-blight disease from 354 commercial and garden/allotment sites in Scotland, England and Wales over four growing seasons, 1995–98. The A2 mating type was rare (3·0% of isolates) and was detected at only 34 sites. In vitro tests of sensitivity to the phenylamide fungicide metalaxyl showed that 316 sites yielded isolates with some insensitivity (resistant and/or intermediate); these were more often commercial sites than garden/allotment sites. Over the four seasons, the frequency of isolates with intermediate fungicide sensitivity increased, while the frequency of resistant phenotypes decreased. Resistant isolates were always of A1 mating type. A subset of 1459 isolates from 326 sites was analysed for molecular diversity. Mitochondrial DNA (mtDNA) haplotype Ia predominated (91·0% of isolates); haplotype IIa was present at 54 sites and both haplotypes at 33 sites. The multilocus RFLP probe RG57 detected 30 fingerprints. Four fingerprints were particularly common (RF002, RF006, RF039 and RF040) and 10 were unique to a single site in a single year. The three commonest fingerprints (RF039 > RF002 > RF006) were of A1 mating type and the fourth (RF040) was A2. RF002 isolates were resistant to the phenylamide metalaxyl and were more common in Scotland than in England and Wales. Small sample sizes limited the usefulness of estimates of diversity. Although approximately half of all sites appeared to be colonized by RF039 genotypes, some sites (both commercial and garden/allotment) showed a higher diversity, having both common and unique genotypes. The genotypic diversity within isolates collected from commercial sites and those from garden/allotment sites were similar. The contributions of sexual reproduction and alternatives to sex to the generation of variation are discussed.
... A range of methods has been used to study the genetic structure of P. infestans populations; isozymes (Tooley et al ., 1985), mitochondrial DNA (mtDNA) restriction digest profiles (Carter et al ., 1990;Griffith & Shaw, 1998) and restriction fragment length polymorphisms (RFLP) with the RG57 probe (Goodwin et al ., 1992a). Amplified fragment length polymorphisms (AFLP) (Vos et al ., 1995) have proved useful in mapping studies (Van der Lee et al ., 1997) and, more recently, for population studies of P. infestans (Perez et al ., 2001;Purvis et al ., 2001) and other Phytophthora species (Lamour & Hausbeck, 2001). ...
... Whilst the phenotypic data were valuable, it was the molecular data that provided detailed information on the population structure. In this and a previous study (Purvis et al., 2001), AFLPs have proved a sensitive and reliable means of estimating P. infestans population diversity. Different primer combinations (including E19/M16) have yielded broadly similar results (Purvis et al., 2001) and problems of convergent RG57 patterns (Zwankhuizen, 1998) have been avoided. ...
... In this and a previous study (Purvis et al., 2001), AFLPs have proved a sensitive and reliable means of estimating P. infestans population diversity. Different primer combinations (including E19/M16) have yielded broadly similar results (Purvis et al., 2001) and problems of convergent RG57 patterns (Zwankhuizen, 1998) have been avoided. ...
Article
In a survey of Scottish potato late blight (Phytophthora infestans) populations from 1995 to 1997, nearly 500 isolates were collected from over 80 disease outbreaks in commercial potato crops and gardens/allotments. The isolates were characterized by mating type, resistance to the fungicide metalaxyl and almost 300 were examined by DNA-based AFLP fingerprinting. These data were examined alongside cropping details to determine the population structure in the context of existing disease management strategies. A1 and A2 mating type isolates were present in both commercial potato crops and gardens or allotments although they coexisted more frequently in the latter sites. One-fifth of the isolates collected were of the A2 mating type and the frequency was similar over the 3 years and amongst sites. In 1995 the proportions of isolates that were sensitive and resistant to metalaxyl were equal (∼40%) but, over the following 2 years, the frequency of resistant isolates decreased and that of intermediate isolates increased. The mating type response to metalaxyl differed markedly, with 52% of A1 and only 5% of A2 isolates being resistant. Considerable molecular diversity was observed, with over half of the isolates having unique AFLP patterns. Analysis of the molecular and phenotypic data revealed a broad clustering of the population into three groups. Many factors point to an A2 population restricted by its sensitivity to phenylamides. The majority of the A2 isolates were found in a single AFLP group, but the presence of mixed mating type samples, an increasing frequency of isolates of intermediate metalaxyl resistance and the extent of the AFLP diversity suggest occasional sexual recombination, and thus gene flow, between groups.
... These data suggest that the P. infestans population in Northern China represents a clonal lineage, despite the fact that AFLP fingerprinting revealed some variation. Similar to our observations, AFLP fingerprinting of P. infestans isolates from Scotland, England and Wales revealed many unique AFLP patterns (Purvis et al. 2001; Cooke et al. 2003) and not all of these AFLP genotypes were consistent with genotypes found with other fingerprint markers (Purvis et al. 2001). The low genotypic diversity is in sharp contrast with the high level of virulence diversity. ...
... These data suggest that the P. infestans population in Northern China represents a clonal lineage, despite the fact that AFLP fingerprinting revealed some variation. Similar to our observations, AFLP fingerprinting of P. infestans isolates from Scotland, England and Wales revealed many unique AFLP patterns (Purvis et al. 2001; Cooke et al. 2003) and not all of these AFLP genotypes were consistent with genotypes found with other fingerprint markers (Purvis et al. 2001). The low genotypic diversity is in sharp contrast with the high level of virulence diversity. ...
Article
Phenotypic and genotypic characteristics of 48 Phytophthora infestans isolates, collected in five provinces in Northern China between 1997 and 2003, were determined and compared with reference isolates. Characterisation included mating type, virulence, mitochondrial DNA (mtDNA) haplotype and DNA fingerprinting patterns based on simple sequence repeats (SSR) and amplified fragment length polymorphisms (AFLP). All isolates had the A1 mating type, mtDNA haplotype IIa and an identical SSR genotype (designated as SG-01-01) that differed from SSR genotypes found in the reference isolates, including those representing the 'old' US-1 lineage that dominated the P. infestans population worldwide prior to 1980. In contrast, the virulence spectra were highly variable and virulence to all resistance genes present in the standard differential set (R1 to R11) was found. AFLP analysis revealed some diversity; eight different AFLP genotypes were found that could be grouped into two major clusters. This study shows that there is very little genotypic diversity in the P. infestans population in Northern China. The occurrence of many different races within this rather uniform population is discussed in the framework of recent insights into the molecular determinants of avirulence in potato-P. infestans'gene-for-gene' interactions.
... Isozymes are co-dominant (Tooley et al., 1985) but there are problems with throughput, band nomination and level of polymorphism (Elansky et al., 2001). Single-locus RFLPs are co-dominant and have been used in linkage studies (Carter et al., 1999), whilst the moderately repetitive multilocus RFLP probe RG57 (Goodwin et al., 1992a) has been useful in investigating genetic diversity of P. infestans populations with varying success (Goodwin et al., 1992b;Forbes et al., 1998;Purvis et al., 2001;Cooke et al., 2003;Day et al., 2004). AFLPs have been scored as co-dominant, but scoring relies on band intensities and is challenging and therefore unreliable. ...
... AFLPs have been scored as co-dominant, but scoring relies on band intensities and is challenging and therefore unreliable. However, they have been used in various studies such as mapping (van der Lee et al., 2001a) and diversity (Purvis et al., 2001;Cooke et al., 2003). These molecular techniques, however, require large quantities of DNA, and often the use of radioactivity (Rosendahl & Taylor, 1997), and can be laborious. ...
Article
Full-text available
Co-dominant microsatellite molecular markers for Phytophthora infestans were developed and their potential for monitoring the genetic variation in populations was demonstrated in the UK, across Europe and worldwide. Markers were developed according to two strategies. First, several thousand P. infestans expressed sequence tag (EST) and bacterial artificial chromosome (BAC) sequences were screened for the presence of simple sequence repeat (SSR) motifs, and, of these, 100 candidate loci were selected for further investigation. Primer pairs developed to these loci were tested against a panel of 10 P. infestans isolates and approximately 10% were shown to be polymorphic and therefore appropriate for further testing. Secondly, the construction and screening of a partial genomic library resulted in the development of one additional polymorphic marker. The resulting 12 SSR markers were converted to higher-throughput fluorescence-based assays and used in combination with two previously published markers to characterize a wider collection of 90 P. infestans isolates from the UK and six other countries. Several isolates from the closely related species P. mirabilis, P. ipomoea and P. phaseoli collected from around the world were also genotyped using these markers. Amongst the 90 isolates of P. infestans examined, considerable SSR diversity was observed, with 68 different genotypes and an average of 3·9 (range 2–9) alleles per locus. When other Phytophthora species were genotyped, all loci were successfully amplified and the majority were polymorphic, indicating their transferability for the potential study of other closely related taxa.
... Thus Griffin et al. (2002) found their type IE-1 to be the most common among Irish isolates (65%) from 1995 to 1999; this is identical to the most common Northern Ireland RG57 type, NI-1 (51% of isolates), in terms of RG57 fingerprint, mating type and mtDNA haplotype (IIa). The same RG57 fingerprint, named as RF06 and associated only with haplotype IIa, was found by Purvis et al. (2001) to be one of the most common among isolates from England and Wales. Cooke et al. (2003) reported this fingerprint (their type b) in mtDNA IIa isolates from Scotland. ...
... The most frequent RG57 fingerprint found among isolates from Poland collected in 1989–91 by Sujkowski et al. (1994) differs from NI-1 only in having band 4 present and, since band 4 is not reproducibly detected by RG57 (Koh et al., 1994), these fingerprints are probably the same. While convergent evolution can result in isolates having the same RG57 fingerprint on differing genetic backgrounds (Purvis et al., 2001 ), the widespread occurrence of this fingerprint in northern European P. infestans populations and its strong association with a single mtDNA haplotype and mating type suggest that it may represent a widely distributed clonal lineage. The two NI-1a isolates had an identical fingerprint to the IE-1b of Griffin et al. (2002), which represented 7% of isolates from the Republic of Ireland in their study. ...
Article
A total of 204 isolates of Phytophthora infestans from Northern Ireland, almost all from commercial potato crops, were collected over 5 years (1998-2002). Phenotypic diversity was assessed using mating type and metalaxyl resistance; genotypic diversity was assessed using two allozyme loci (glucose-6-phosphate isomerase, Gpi, and peptidase, Pep), mitochondrial DNA haplotype and the multilocus RFLP probe RG57. All isolates were A1 mating type and Gpi 100/100. The majority were Pep 100/100, but four Pep 83/100 and six Pep 96/100 isolates were identified. Three mtDNA haplotypes were detected; haplotype IIa was the most common, but each year up to 2001 its frequency declined, with a concomitant increase in Ia isolates. Three isolates had the rare haplotype IIb, but this was only detected in 1998. Metalaxyl resistance and mtDNA haplotype were markedly associated: most haplotype Ia isolates were metalaxyl-resistant, whereas haplotype IIa was more commonly associated with metalaxyl sensitivity. Analysis of a subsample of 91 isolates revealed nine RG57 genotypes, three associated exclusively with haplotype IIa and six exclusively with haplotype Ia. The most common RG57 genotype (51% of isolates) comprised both metalaxyl-resistant and -sensitive haplotype IIa isolates. However, of haplotype Ia isolates, all metalaxyl-resistant phenotypes belonged to one of four RG57 types, one of which was the second most frequent overall (29% of isolates), while all metalaxyl-sensitive isolates belonged to one of two other types. The P. infestans population in Northern Ireland appears to consist of a limited number of clones related to, but differentiated from, the populations in mainland Britain and elsewhere in Europe.
... Such apparent 'identity' occurs by chance alone, rather than common descent, and will confound genetic analysis. AFLP fingerprinting, for example, discriminates isolates considered identical based on RG57 fingerprint (Purvis et al ., 2001) and two SSR markers (Knapova & Gisi, 2002). The converse, where a high proportion of isolates within a population have unique genetic fingerprints (e.g. ...
... Although it is a powerful tool for the phylogeographic analysis of many organisms, P. infestans mtDNA diversity is relatively limited, with the vast majority of tested isolates falling into two [ Ia(A) and IIa(B)] of the six defined haplotypes (Griffith & Shaw, 1998;Gavino & Fry, 2002). Marked regional variation in mtDNA haplotype frequency (Forbes et al., 1998;Griffith & Shaw, 1998) and associations between haplotype and nuclear DNA fingerprint have been observed (Purvis et al., 2001), but neither the cause nor the functional significance (if any) is known. There is no known mechanism of selection acting on the mtDNA (Gavino & Fry, 2002), but the emergence of mtDNA-based resistance to QoI fungicides in other oomycetes indicates a potential selection pressure to consider in future monitoring. ...
Article
Late blight, caused by Phytophthora infestans, is an ongoing threat to potato and tomato crop production worldwide and considerable fundamental and applied research is conducted with the long-term aim of improved disease control. Understanding the mechanisms, processes and rates of P. infestans evolution is an important factor in predicting the effectiveness and durability of new management practices. A range of phenotypic and genotypic tests has been applied to achieve this goal, but each has limitations and new methods are sought. Recent progress in P. infestans genomics is providing the raw data for such methods and new high-throughput codominant biomolecular markers are currently being developed that have tremendous potential in the study of P. infestans population biology, epidemiology, ecology, genetics and evolution. This paper reviews some key applications, recommends some changes in approach and reports on the status and potential of new and existing methods for probing P. infestans genetic diversity.
... Amplification of only 1/16th of EcoRI-MseI fragments occurs. AFLP fingerprinting, for example, discriminates isolates considered identical based on RG57 fingerprint (Purvis et al., 2001) and two SSR markers (Knapova and Gisi, 2002). The converse, where a high proportion of isolates within a population have unique genetic fingerprints (e.g. ...
... Although it is a powerful tool for the phylogeographic analysis of many organisms, P. infestans mtDNA diversity is relatively limited, with the vast majority of tested isolates falling into two [Ia(A) and IIa(B)] of the six defined haplotypes (Griffith and Shaw, 1998;Gavino and Fry, 2002). Marked regional variation in mtDNA haplotype frequency (Forbes et al., 1998;Griffith and Shaw, 1998) and associations between haplotype and nuclear DNA fingerprint have been observed (Purvis et al., 2001), but neither the cause nor the functional significance (if any) is known. There is no known mechanism of selection acting on the mtDNA (Gavino and Fry, 2002), but the emergence of mtDNA-based resistance to QoI fungicides in other oomycetes (Gisi et al., 2002) indicates a potential selection pressure to consider in future monitoring. ...
Article
Molecular epidemiology is a science that focuses on the contribution of potential genetic and environmental risk factors, identified at the molecular level, to the etiology, distribution and prevention of disease Molecular epidemiology provides the ‗tools‘ (both laboratory and analytical) that have predictive significance and that epidemiologists can use to better define the etiology of specific diseases, and work towards their control. Application of these molecular techniques has increased the understanding of the epidemiology of the most important infectious agents, Phytophthora infestans. Recent progress in P. infestans genomics is providing the raw data for such methods and new bio molecular markers are currently being developed which have tremendous potential in the study of P. infestans. Closer collaborations between specialists in the fields of plant pathology, epidemiology, population genetics / molecular ecology, P. infestans molecular biology and plant breeding are advocated to enable such progress. Molecular techniques help to stratify and to refine data by providing more sensitive and specific measurements which facilitate epidemiologic activities including disease surveillance, outbreak investigations, identifying transmission patterns and risk factors among apparently disparate cases characterizing host pathogen interactions and providing better understanding of disease pathogenesis at the molecular level.
... high-resolution markers for characterizing Phytophthora isolates and for tracking clonal lineages within populations (36,38,43,66,93). These studies provided invaluable new insights into both population structure within species and variation between species. ...
Article
Full-text available
Plant diseases caused by Phytophthora species will remain an ever increasing threat to agriculture and natural ecosystems. Phytophthora literally means plant destroyer, a name coined in the 19th century by Anton de Bary when he investigated the potato disease that set the stage for the Great Irish Famine. Phytophthora infestans, the causal agent of potato late blight, was the first species in a genus that at present has over 100 recognized members. In the last decade, the number of recognized Phytophthora species has nearly doubled and new species are added almost on a monthly basis. Here we present an overview of the 10 clades that are currently distinguished within the genus Phytophthora with special emphasis on new species that have been described since 1996 when Erwin and Ribeiro published the valuable monograph 'Phytophthora diseases worldwide' (35).
... Molecular analysis of UK and Irish populations in the 1990s identified several common genotypes which were detected at many sites (Carlisle et al. 2001;Cooke et al. 2003Day et al. 2004;Griffin et al. 2002;Purvis et al. 2001;Shaw and Wattier 2002). These were thought to be separate clonal lineages, widely but not evenly distributed over the region. ...
Article
Full-text available
Phytophthora infestans, the causal agent of late blight, is a major threat to potato production in northwestern Europe. Before 1980, the worldwide population of P. infestans outside Mexico appeared to be asexual and to consist of a single clonal lineage of A1 mating type characterized by a single genotype. It is widely believed that new strains migrated into Europe in 1976 and that this led to subsequent population changes including the introduction of the A2 mating type. The population characteristics of recently collected isolates in NW Europe show a diverse population including both mating types, sexual reproduction and oospores, although differences are observed between regions. Although it is difficult to find direct evidence that new strains are more aggressive, there are several indications from experiments and field epidemics that the aggressiveness of P. infestans has increased in the past 20years. The relative importance of the different primary inoculum sources and specific measures for reducing their role, such as covering dumps with plastic and preventing seed tubers from becoming infected, is described for the different regions. In NW Europe, varieties with greater resistance tend not to be grown on a large scale. From the grower’s perspective, the savings in fungicide input that can be achieved with these varieties are not compensated by the higher (perceived) risk of blight. Fungicides play a crucial role in the integrated control of late blight. The spray strategies in NW Europe and a table of the specific attributes of the most important fungicides in Europe are presented. The development and use of decision support systems (DSSs) in NW Europe are described. In The Netherlands, it is estimated that almost 40% of potato growers use recommendations based on commercially available DSS. In the Nordic countries, a new DSS concept with a fixed 7-day spray interval and a variable dose rate is being tested. In the UK, commercially available DSSs are used for c. 8% of the area. The validity of Smith Periods for the new population of P. infestans in the UK is currently being evaluated. KeywordsDecision support systems–Epidemiology–Fungicide–Mating type–Pest control– Phytophthora infestans
... The 10 bp primers can bind to any region in the genome that contain a matching sequence and generate polymorphism between two individuals, when the binding site of a primer is lost or modified in one individual but not in the other. This lack of association between different DNA markers was also observed by Purvis et al. (2001), who fingerprinted a collection of P. infestans isolates with AFLP markers and the RG57-RFLP marker. They found that some isolates with the same RG-57 genotype had remarkably dissimilar AFLP genotypes, and some isolates with dissimilar RG-57 fingerprints had similar or identical AFLP fingerprints. ...
Article
Genotypic variation among 32 single-zoospore isolates (SZI) of Phytophthora infestans, derived asexually from two hyphal-tip parental isolates (PI-105 and PI-1) of the US-8 genotype, was assessed with 80 random amplified polymorphic DNA (RAPD) primers and 18 amplified fragment length polymorphic DNA (AFLP) primer pairs. In previous investigations, the SZIs from parental isolate PI-105 showed high levels of virulence variability and were differentiated into 14 races, whereas the SZIs from PI-1 showed identical virulence to the parent. The purpose of this investigation was to determine if phenotypic variation observed among SZIs of P. infestans could be detected at the DNA level in these isolates. Polymorphism was detected with 51 RAPD primers and with all 18 AFLP primer pairs in PI-105 SZIs. In SZIs from PI-1, polymorphism was also detected with 25 RAPD primers and 17 AFLP primer pairs. Cluster analysis using the unweighted pair-group method with arithmetic averages (UPGMA) separated the SZIs from parent PI-105 into six virulence groups, 11 RAPD groups and three AFLP groups. Cluster analysis of PI-1 SZIs, which all belong to the same virulence group, differentiated them into four RAPD groups and six AFLP groups. No close correlation among RAPD, AFLP and virulence groups could be established within the two progenies of SZIs. Results of this study suggest that there is a considerable level of inherent genetic variability among SZIs derived asexually from the same parental isolate. The possible mechanisms and implications of this genetic variation are discussed.
... 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). ...
Article
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.
... Although homothallic taxa such as P. sojae have been proven to out-cross (Whisson et al., 1994), the likelihood of out-crossing in invasive pathogens such as Pff with limited diversity as a result of passing through a narrow genetic bottleneck, is severely reduced. Fewer than 5% of the P. fragariae AFLP bands were polymorphic in this study compared to 20-62% noted amongst surveys of P. infestans (Purvis et al., 2001;Cooke et al., 2003), P. capsici (Lamour & Hausbeck, 2001) or P. quercina (Cooke et al., 2005). It should be noted however, that this study examined only a small sample of isolates derived from a limited population of oospores within a single field. ...
Article
An experimental field infested with Phytophthora fragariae var. fragariae (Pff) and used for strawberry red core fungicide and cultivar resistance trials until 1981 was surveyed for the presence of inoculum of the pathogen 11 and 12 years later. Alpine strawberries, highly susceptible to all races of Pff, were grown from true seed and planted as a bait crop on a 0·5 m-spaced grid. Rapid and widespread red core infection was observed, which provided good evidence that oospores had survived in soil for this extended period. Site elevation and the distribution of red core infected plants showed a strong correlation, with a higher frequency of infected and dead plants in the lowest areas of the field. The race designation of 18 recovered isolates were determined and AFLP fingerprint patterns of some of these and their single-spore derivatives were analysed. The isolates differed little in race type, and the majority were genetically identical at 433 AFLP loci. Races used to inoculate the site in the 1970s were recovered. The fingerprints of the single variant isolate matched that of an isolation made by Hickman in the 1950s, originally used to inoculate the site. Clearly Pff is a very stable and long-lived pathogen able to retain its genetic integrity and lie dormant in soil for many years, ensuring its survival between epidemiologically favourable conditions which occur erratically.
... The Chinese multilocus genotype CN-3 had the same RFLP as MO20 (from Moscow, 1995), as reported by Elansky et al. (2001), but the mtDNA haplotype was different: CN-3 is Ia, whereas MO20 is IIa. This may be an example of the possibility of convergent evolution in the same RG57 fingerprints (Purvis et al., 2001). Koh et al. (1994) identified almost all isolates from Korean populations of P. infestans as JP-1 in 1991. ...
Article
A total of 401 isolates of Phytophthora infestans were collected from eight Asian regions (Korea, India, Taiwan, Indonesia, Thailand, Nepal, China and Japan) between 1992 and 2000 – 318 from potato and 83 from tomato. The isolates were analysed for mating type, metalaxyl resistance, RG57 fingerprinting, mitochondrial DNA (mtDNA) haplotype and the polymorphism of three allozyme loci, i.e. glucose-6-phosphate isomerase (Gpi), peptidase (Pep) and malic enzyme (Me). The isolates were multilocus-genotyped based on RFLP (RG57) fingerprint, dilocus allozyme genotype, mtDNA haplotype and mating type. Twenty multilocus genotypes were identified among 125 isolates. Of these genotypes, 14 had not been previously reported. Some of the multilocus genotypes were common to isolates from several geographical regions, suggesting migration. The metalaxyl-resistant isolates belonged to the multilocus genotypes JP-1, JP-2, and JP-3. Multilocus genotypes coexisting in a single field were found in following regions: Thailand (1994), central China (1996), Nepal (1997) and Japan (1998 and 2000). The possible origins of certain genotypes are discussed, including the possibility of sexual recombination within the P. infestans populations in Nepal and perhaps Thailand.
... Conversely, analyses of other populations indicate a dominance of asexual clones and numerous rare genotypes Day et al., 2004;Knapova and Gisi, 2002). Careful scrutiny of AFLP diversity revealed a clear substructure of the U.K. population that corresponded to lineages defined by mating type and mtDNA haplotype (Purvis et al., 2001). This and other observations, such as the historical absence of metalaxyl resistance in European A2 lineages , is consistent with an absence of sexual processes that would reassort such traits. ...
... Partition of the two clusters could reflect the two different mating types of P. leucotricha demonstrated by Coyier (1974) to be heterothallic. As shown by Purvis et al. (2001) for Phytophthora infestans, two major clusters based on AFLP marker data clearly separated the two mating types A1 and A2, suggesting that gene flow between A1 and A2 genotypes is limited and that sexual reproduction may be rare in this particular pathogen. ...
Article
The development of apple varieties displaying durable resistance against powdery mildew is one of the major aims in apple breeding programmes worldwide. For a reliable judgment of the resistance of different Malus genotypes, an extended knowledge about the virulence of the pathogen is necessary. To prove the existence of physiological races of Podosphaera leucotricha, 31 monoconidial isolates of the obligate biotrophic fungus representing five locations within Europe have been established and maintained over a period of 3–4 years. The isolates were maintained on in vitro shoots of the highly susceptible apple cv. Gibb's Golden Gage. An AFLP-based DNA fingerprinting protocol was developed and, using 54 stably reproducible AFLP markers, a dendrogram revealed genetic variability among different isolates of P. leucotricha. Although the molecular characterization of the isolates showed an overall low level of genetic variability, the high phenotypic diversity among European isolates suggest that sexual reproduction may also be involved in the disease cycle of the pathogen in Europe. Phytopathological tests using detached leaves of a collection of 36 Malus genotypes allowed the differentiation of five selected isolates by their virulence patterns. A high level of diversity in terms of virulence was obtained in P. leucotricha. From the present study, based on apple breeding germplasm, cultivars and Malus species, it can be concluded that physiological races of P. leucotricha do indeed exist in Europe.
... The random decamer primers can bind to any region in the genome that contain complementary sequence and generate polymorphism between two individuals. The lack of association between different DNA markers was also observed by Purvis et al. (2001), who fingerprinted P. infestans isolates with AFLP and RFLP markers. An additional factor affecting genetic diversity assayed by different marker techniques is the number of markers or probes used in an analysis (Messmer et al. 1991). ...
Article
The oomycetous fungus Phytophthora colocasiae that causes taro leaf blight is one of the most devastating diseases of taro and is widely distributed in India. Molecular and cultural techniques were employed for assessing and exploiting the genetic variability among isolates of P. colocasiae obtained from different geographical regions of India. Analysis of the 5.8-ITS region revealed detectable intraspecific variation among isolates. Ten random amplified polymorphic DNA (RAPD) and eight amplified fragment length polymorphism (AFLP) primers produced 198 and 510 reproducible fragments, respectively. AFLP produced 100 % polymorphism, whereas RAPD showed 93.5 % polymorphism. The average value of the number of observed alleles, the number of effective alleles, mean Nei’s genetic diversity, and Shannon’s information index were 2.00–1.94, 1.53–1.36, 0.31–0.24, and 0.47–0.40, respectively, for two DNA markers used. Analysis of molecular variance (AMOVA) for both markers produced similar results with the majority (85 %, AFLP; 89 %, RAPD) of the diversity present within population of P. colocasiae. Dendrograms based on twomolecular data using the unweighted pair group method with arithmetic mean (UPGMA) was incongruent and classified the P. colocasiae isolates into one and two major clusters. Cophenetic correlation coefficient between dendrogram and original similarity matrix were significant for RAPD (r=0.904) and AFLP (r=0.825). The results of this study displayed a high level of genetic variation among the isolates irrespective of the geographical origin. The possible mechanisms and implications of this genetic variation are discussed.
... In general, PCR-based techniques have the major disadvantage of being sensitive to the 2 C.-H. Duan et al. 296 reaction conditions, DNA quality and PCR temperatures (Vos et al. 1995). Amplified fragment length polymorphism (AFLP) is a recent DNA fingerprinting technique using PCR that evolved in the 1990s and has been used to assess genetic variation among species of Phytophthora (Lamour and Hausbeck 2001;Purvis et al. 2001;Werres et al. 2001). In contrast to the random amplification of polymorphic DNA (RAPD) technique, AFLP uses sequence-specific primers, which eliminates the variability associated with non-specific primers (Vos et al. 1995). ...
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Fatty acid methyl ester (FAME) profiles and amplified fragment length polymorphisms (AFLPs) were evaluated as tools for identifying species of Phytophthora. Five isolates of each of Phytophthora cactorum, Phytophthora citrophthora, Phytophthora cinnamomi, Phytophthora nicotianae and Phytophthora cryptogea were subjected to both analyses to examine variation among and within species. In FAME analysis, isolates of P. cactorum, P. cinnamomi and P.nicotianae were clustered by species, but isolates of P. citrophthora and P.cryptogea were divided into multiple clusters based on greater variations within these two species. The AFLP analysis differentiated all five species of Phytophthora. The five isolates of each species were grouped in a separate terminal cluster, but diversity within a species cluster varied considerably with variation greater in P. cryptogea and P. citrophthora. Comparing the dendrograms based on FAME and AFLP analyses, the overall patterns of both were similar. The P. cactorum cluster was distinct from clusters of the other four species, which formed one large cluster. The higher values of percentages of polymorphic loci and gene diversity in AFLP analysis substantiated diversity observed among isolates of P. citrophthora and P. cryptogea in FAME and AFLP dendrograms. Both FAME and AFLP appear to be useful tools for identifying species of Phytophthora, but only AFLP analysis has potential to study genetic and phylogenetic relationships within and among species in this genus.
Article
Members of the genus Phytophthora are destructive plant pathogens, causing significant economic losses worldwide. Extensive background knowledge exists on the transmission biology of the genus, but it is only in the last decade that the mechanisms underlying the success of the Phytophthoras as plant pathogens have begun to be elucidated at the cellular and molecular level. Using molecular genetic tools, substantial progress has been made in identifying key molecules involved in the pathogen infection cycle. Genomic approaches are now opening up new routes to gene discovery and promise to have a significant impact upon our understanding of the biology and pathology of this important group of organisms. This article reviews the tools and resources available for molecular genetic and genomic studies in Phytophthora and discusses the insights that these studies are giving into the molecular basis of the plant-pathogen relationship.
Article
Forty-seven isolates of Phytoplithora cactorum from North America and Germany were subjected to amplified fragment length polymorphism (AFLP) analysis to investigate genetic diversity among isolates and geographical populations; 42 isolates were recovered from cultivated strawberry plants (Fragaria x ananassa), and five isolates had been recovered from plants in four other genera (Syringa, Abies, Malus, and Panax). From all isolates evaluated, 226 out of 264 markers (85.6%) were polymorphic and provided 42 unique AFLP profiles. The genetic diversity among isolates of P. cactorum from strawberry was greater than that among isolates from the other hosts. Isolates collected during recent crown rot epidemics in strawberry fields in South Carolina were genetically diverse and scattered among isolates from other geographical areas in an unweighted pair-group mean analysis (UPGMA) dendrogram. Isolates collected during recent crown rot epidemics in North Carolina also were genetically diverse, but most isolates clustered with isolates collected in 1997 from Florida strawberry fields. These data suggest that recent outbreaks of Phytophthora crown rot in the southeastern United States resulted from use of transplants already infected or infested with P. cactorum rather than from endemic populations of this pathogen, which would affect recommendations for disease management.
Article
Late blight, caused by Phytophthora infestans, is a major disease of tomato in cool and wet environments. In this study, we report on the host specificity, race composition, and variation among races revealed by amplified fragment length polymorphism (AFLP) of P. infestans isolated from tomato production areas in Taiwan. In all, 177 R infestans isolates were collected in Taiwan during 2004 and 2005. All were aggressive on both potato and tomato. Nine physiological races were identified based on disease response on a set of tomato differentials developed by the Asian Vegetable Research and Development Center-The World Vegetable Center. Eighty-seven polymorphic bands from 32 isolates of four races were detected by AFLP. No significant correlation between the polymorphism and the races was found using cluster analysis. This study revealed that a high variability of race composition among the asexual population of P. infestans isolates existed in Taiwan during 2004 and 2005. Breeding new tomato cultivars for resistance to P. infestans is an urgent and ongoing need because new races of the pathogen appeared continuously in Taiwan in past years. Further analysis of the genomic diversity is necessary to determine whether the high genetic variation of P. infestans is related to the complex race composition.
Article
The mating type, glucose-6-phosphate isomerase (Gpi) and peptidase (Pep) genotypes, RG57 fingerprint, and mitochondrial DNA (mtDNA) haplotype of Chinese isolates of Phytophthora infestans collected in Hebei and Gansu in 1996 were compared with those of Japanese isolates collected during 1997–2000. The Chinese isolates were divided into four genotypes, one of which was identical to the dominant Japanese genotype, A1-A (mating type A1; Gpi 100/100; Pep 100/100; RG57 100010001100110100011001110: 1–25, 14a, and 24a; and mtDNA haplotype IIa). Comparison of the genotypes with reported data revealed that some completely and partially identical genotypes occur in Russia and parts of Europe. The other two A1 genotypes and one A2 genotype were also detected in Gansu (Gpi 100/100, Pep 100/100, and mtDNA haplotype Ia), which were regarded as unique to this region.
Article
Phytophthora infestans, the causal agent of potato- and tomato late blight, remains a serious threat for (commercial) potato and tomato production. In North Western Europe, frequent fungicide applications, mostly aimed to prevent infection, form the back bone of potato late blight control. Modern protectants such as Shirlan (a.i. fluazinam) are highly effective against (germinating) P. infestans sporangia and zoospores. Zoospores in particular are so sensitive to low concentrations that the many applications over the past two decades may well have exerted sufficient selection to pressure against the formation of zoospores. Thus, over the years the balance between direct and indirect germination may have shifted towards direct germination. This hypothesis was investigated at Bayer Crop Science and Plant Research International
Article
Phytophthora auercina, a new species associated with oak decline in Europe, has been assigned to Waterhouse's Group I of Phytophthora. The level of intraspecific variation and evidence of affinities to other Group I species and another, as yet unidentified, species from oak, were examined at the molecular level using four random ten-mer primers to amplify total DNA (RAPDs). Sequences and restriction fragment length polymorphisms of a 900 bp PCR product consisting of the ITS1 and ITS2 regions, and the 5.8S subunit of ribosomal DNA were also examined to estimate relatedness to a broader range of Phytophthora species. The RAPD banding patterns of ten isolates of P. quercina from eight sites in Germany, Hungary and Italy were almost identical and distinct from all the other species tested. Their ITS restriction fragment patterns were also identical, as were the ITS sequences of four selected isolates. Phylogenetic analysis of the ITS sequence data confirmed its unique position in this section of the genus which comprises P. quercina, another five Group I species, P. infestans (Group IV) and P. nicotianae (Group II). Isolates of P. quercina formed a distinct branch at the base of this clade showing no close affinities with any other species. Such data support morphological, physiological and pathological evidence that P. quercina is distinct, although it has some affinity with the other Group I species. The results support earlier reports that Waterhouse's groupings of morphospecies do not fully correspond to phylogenetic relationships indicated by molecular studies. The unidentified Phytophthora sp. 2 from oak was closely related to P. ilicis (Group IV); both were distinct on molecular criteria from all other species in the study.
Article
The biology of late blight of potato and tomato, caused by Phytophthora infestans, changed when sexual reproduction by the pathogen became possible in many parts of the world, including Europe. In northern Europe, especially Scandinavia, there is increasing evidence that the pathogen is reproducing sexually on a regular basis, although in other regions further south or to the west it appears to reproduce primarily in a clonal manner. The presence of both mating types, the production of viable oospores, and observations of fields with soilborne sources of inoculum are consistent with sexual reproduction. Studies with different marker systems have revealed a population structure without any dominating clonal lineages in Scandinavia, and that is most easily explained by sexual reproduction. Phytophthora infestans recovered from the soil can also be linked to parental genotypes using likelihood-based methods when codominant markers are used. A synthesis of all the available data points to a second centre of sexual reproduction in northern Europe.
Chapter
The aim of this chapter is to introduce recent information about the late blight disease in potatoes (Solanum tuberosum L.) which is considered one of the most famous diseases in agriculture. Late blight disease is caused by the oomycete pathogen Phytophthora infestans. This chapter focus also, assessment the economic importance of the disease. Also identification of the fungus using the most vital methods distinguishes between the new isolates of the fungus by numerous techniques, (a) the traditional method (b) using DNA markers and (c) bioinformatics and then control fungus by the best practices of chemical pesticides. Meanwhile, some chemicals are toxic and dangerous for both the environment and human health. Therefore, we will go to resistant cultivars and alternative methods such as plant oils and extracts as well as the use of nanotechnology and biocontrol compared to chemical fungicides to reduce the environmental problems on the plants, animals and then humans.
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A number of PCR-based techniques can be used to detect polymorphisms in plants. For their wide-scale usage in germplasm characterisation and breeding it is important that these marker technologies can be exchanged between laboratories, which in turn requires that they can be standardised to yield reproducible results, so that direct collation and comparison of the data are possible. This article describes a network experiment involving several European laboratories, in which the reproducibility of three popular molecular marker techniques was examined: random-amplified fragment length polymorphism (RAPD), amplified fragment length polymorphism (AFLP) and sequence-tagged microsatellites (SSR). For each technique, an optimal system was chosen, which had been standardised and routinely used by one laboratory. This system (genetic screening package) was distributed to different participating laboratories in the network and the results obtained compared with those of the original sender. Different experiences were gained in this exchange experiment with the different techniques. RAPDs proved difficult to reproduce. For AFLPs, a single-band difference was observed in one track, whilst SSR alleles were amplified by all laboratories, but small differences in their sizing were obtained.
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Prior to 1996, the A2 mating type of Phytophthora infestans was not detected on potato in France, but was found at one site on tomato in 1995. This finding lead to the question of the extent of differences and relationships existing between the populations of P. infestans present on each host. A collection of 76 isolates collected in France, mainly in 1996, from potato and tomato was characterised for mating type, allozyme genotype at the Gpi and Pep loci, and mitochondrial DNA haplotype; 74 of these isolates were also characterised for multilocus RFLP fingerprint, and 62 for virulence. All isolates except four showed allozyme genotypes (Gpi 90/100 or 100/100, Pep 83/100 or 100/100) and mtDNA haplotypes (Ia or IIa) characteristic of the populations introduced into Europe in the late 1970s. The four exceptions were isolates collected from tomato in Southern France in 1988-1991, which showed some characteristics of the former European populations (Gpi 86/100, Pep 92/100, mtDNA Ib). Both mating types were present among the collections from both hosts, but isolates with the A2 mating type were found on potato only in one garden crop, adjacent to tomato. Nine different RG57 fingerprints were observed, with a greater diversity among tomato isolates. Furthermore, tomato and potato collections differed markedly in the frequencies of genotypes present. Finally, tomato isolates generally had a lower virulence complexity than potato isolates. These data suggest that P. infestans populations on tomato and potato are largely separated, despite the occurrence of limited gene flow.
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The appearance of the A2 mating type (previously restricted to central Mexico) in Europe during the 1980s prompted an investigation of the genetic make-up of European populations using allozyme loci as genetic markers. The investigation shows that major genetic changes have occurred in populations of Phytophthora infestans in the Netherlands, Poland, and the British Isles. It now appears that a new type of population has been introduced into several locations, and has displaced or is displacing the original populations in these locations. The new and old population types are characterized by unique allozyme alleles and genotypes. The mechanism for displacement of the'old'by the'new'population is not yet known.
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A marker database was compiled for isolates of the potato and tomato late blight pathogen, Phytophthora infestans, originating from 41 locations which include 31 countries plus 10 regions within Mexico. Presently, the database contains information on 1,776 isolates for one or more of the following markers: restriction fragment length polymorphism (RFLP) fingerprint consisting of 23 bands; mating type; dilocus allozyme genotype; mitochondrial DNA haplotype; sensitivity to the fungicide metalaxyl; and virulence. In the database, 305 entries have unique RFLP fingerprints and 258 entries have unique multilocus genotypes based on RFLP fingerprint, dilocus allozyme genotype, and mating type. A nomenclature is described for naming multilocus genotypes based on the International Organization for Standardization (ISO) two-letter country code and a unique number, Forty-two previously published multilocus genotypes are represented in the database with references to publications. As a result of compilation of the database, seven new genotypes were identified and named. Cluster analysis of genotypes from clonally propagated populations worldwide generally confirmed a previously published classification of old and new genotypes. Genotypes from geographically distant countries were frequently clustered, and several old and new genotypes were found in two or more distant countries. The cluster analysis also demonstrated that A2 genotypes from Argentina differed from all others. The database is available via the Internet, and thus can serve as a resource for Phytophthora workers worldwide.
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ABSTRACT A wide range of commercially formulated fungicides cause in vitro effects on mating behavior in specific isolates of Phytophthora infestans, the causal agent of late blight of potato and tomato. Four isolates of P. infestans representing each of the four common US genotypes, US-1, US-6, US-7, and US-8 and varying in their sensitivity to metalaxyl, were exposed to a variety of fungicides used to control late blight in petri dish assays at concentrations ranging from 1 to 100 mug a.i./ml. Exposure of each of these normally heterothallic single mating type isolates of P. infestans to 9 of the 11 commercial fungicide formulations tested resulted in the formation of oospores after 2 to 4 weeks. The highest numbers of oospores were formed on media amended with Ridomil 2E (metalaxyl) and Ridomil Gold EC (mefenoxam) at 0.1 to 10 mug a.i./ml, averaging as many as 471 and 450 oospores per petri dish, respectively. Several other fungicides including Maneb, Manzate (Mancozeb), Curzate (cymoxanil + mancozeb), and Acrobat MZ (dimethomorph + mancozeb) also induced oospore formation, producing from 0 to 200 oospores per plate at fungicide concentrations from 0.1 to 10 mug a.i./ml. The metalaxyl resistant isolates formed oospores in response to the fungicides more often than the metalaxyl sensitive isolates. No oospores were formed on media amended with Bravo (chlorothalonil) or Tattoo C (chlorothalonil + propamocarb HCl) and these compounds completely suppressed growth of the isolates at 0.1 and 1 mug a.i./ml. Three metalaxyl resistant A2 isolates mated with both A1 and A2 isolates after exposure to the fungicides Ridomil 2E and Ridomil Gold EC. Alterations in mating type expression were also observed in a metalaxyl sensitive A1 isolate after exposure to Benlate (benomyl). Copious amounts of chemicals are applied annually to potato and tomato production areas to control late blight. Our results indicate that a wide range of chemically diverse fungicides can induce normally heterothallic metalaxyl resistant isolates of P. infestans to form oospores in vitro after short exposures to the fungicides.
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A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50—100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.
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Four pairs of primers were designed for PCR amplification of known polymorphic regions of the mitochondrial genome of Phytophthora infestans. Digestion of the amplified products with restriction enzymes allows identification of previously identified haplotypes. Product P2 cut with MspI uniquely identifies haplotypes Ib and IIa, while types Ia and IIb are differentiated by digestion of product P4 with EcoRI. Digestion of products P1 and P3 gave results similar to that with digestion of P4, but amplification of these products was less robust. Thus, all four common haplotypes are identified by amplifying and digesting products P2 and P4. Identification of haplotypes was also possible from DNA extracted directly from small, late-blight lesions on both tomato and potato leaves, making isolation of the fungus unnecessary. A rapid and efficient method of monitoring changes in the pathogen population is facilitated. These PCR primers were also useful for differentiating other Phytophthora species.
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Recently, DNA pairing analyses showed that Pseudomonas syringae pv. tomato and related pathovars, including P. syringae pv. maculicola, form a genomic species (Pseudomonas tomato) (L. Gardan, H. L. Shafik, and P. A. D. Grimont, p. 445-448, in K. Rudolph, T. J. Burr, J. W. Mansfield, D. Stead, A. Vivian, and J. von Kietzell, ed., Pseudomonas syringae Pathovars and Related Pathogens, 1997). The genetic diversity of 23 strains belonging to this genomic species and 4 outgroup strains was analyzed with randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphic (AFLP) techniques. Simple boiling of P. syringae cells was suitable for subsequent DNA amplification to obtain reliable patterns in RAPD and AFLP analyses. In general, the grouping of P. syringae strains by both analysis techniques corresponded well with the classification obtained from an RFLP analysis of ribosomal DNA operons, DNA pairing studies, and an analysis of pathogenicity data. However, two strains of P. syringae pv. maculicola produced distinct DNA patterns compared to the DNA patterns of other P. syringae pv. maculicola strains; these patterns led us to assume that horizontal transfer of DNA could occur between bacterial populations. Both techniques used in this study have high discriminating power because strains of P. syringae pv. tomato and P. syringae pv. maculicola which were indistinguishable by other techniques, including pathogenicity tests on tomato, were separated into two groups by both RAPD and AFLP analyses. In addition, data analysis showed that the AFLP method was more efficient for assessing intrapathovar diversity than RAPD analysis and allowed clear delineation between intraspecific and interspecific genetic distances, suggesting that it could be an alternative to DNA pairing studies. However, it was not possible to distinguish the two races of P. syringae pv. tomato on the basis of an analysis of the data provided by either the AFLP or RAPD technique.
Article
The amplified fragment length polymorphism (AFLP) technique was assessed for its effectiveness as a marker system in wheat, a hexaploid species with a large genome (1.6 x 1010 bp per haploid). The degree of polymorphism detected by AFLP among 11 pure lines of wheat was determined and compared with the polymorphism detected by RFLP. Cost comparison between AFLP and RFLP techniques was also made. The appropriate amount of wheat genomic DNA as template in pre-amplification was first determined. All 20 AFLP primer pairs tested resulted in amplification of bands which were polymorphic between all pairwise genotype comparisons. On average, 106 products per genotype were amplified, and for each primer pair used, more than 25 polymorphic fragments detected polymorphism among the 11 genotypes. In comparison, only 61% of 18 RFLP probes tested detected polymorphism among 10 of the 11 lines and each polymorphic probe detected an average of only three bands showing polymorphism between genotype pairs. The AFLP technique is more efficient but less expensive and requires less labor than the RFLP technique in wheat.
Article
Cellulose-acetate electrophoresis (CAE) provided excellent resolution of allozyme genotypes of Phytophthora infestans at the two loci Glucose-6-phosphate isomerase (Cpi) and Peplidase (Pep). The cellulose-acetate system has many advantages over traditional starch gel analyses. It is much faster, running in only 15 to 20 min compared to 16 to 18 h for starch gels, and because of the short run times the gels do not need to be cooled during electrophoresis. Cellulose acetate is purchased as precast plates, which eliminates the time required lo pour starch gels. Both Gpi and Pep can be analyzed using a single buffer in the CAE system, whereas four buffers are required to resolve these enzymes using starch gels. Finally, only very small amounts of tissue (e.g., 3,000 sporangia washed from lesions or infected tuber slices) are required for CAE, so it can be useful even when the fungus has not been isolated into axenic culture. These advantages may allow CAE to be useful as a diagnostic tool in field situations, where rapid determination of genotypes could aid disease management strategies. Because populations of P. infestans in the United States and Canada currently are highly clonal, mating type and response to metalaxyl are highly correlated with allozyme genotype. Therefore, CAE of allozyme genotypes could provide a rapid, accurate method for predicting mating types and metalaxyl sensitivities of P. infestans within fields.
Article
The variability in culture of mycelial isolates of Phytophthora infestans was studied by examining the variation among single zoospore, single sporangium, and single hyphal tip subcultures. Extensive variation in rate of growth and sporangium production on artificial medium was detected among the single zoospore progenies of three mass cultures. Differences in colony morphology and viability of zoospores were also apparent but were not studied in detail. Subcultures established by single sporangia or single hyphal tips were much more uniform than zoospore cultures, although significant differences in growth rate could still be detected. While, with continued culture, some of the single zoospore variants tended to revert to their parental type, others showed a remarkable degree of stability.Isolates established from single zoospores gave rise to as much variation in their asexual progenies as the original mass cultures. This persistence of variation was also observed with isolates whose derivation included two successive single zoospore propagations. Selection for high and low growth rate among the zoospore progeny of a single mass culture rapidly led to the production of populations of zoospore cultures with different mean growth rates. Such response to selection implies the existence of a genetic mechanism which allows the transmission of phenotypic characters from one asexual generation to the next.The origin of variation among single zoospore cultures is discussed with reference to five different asexual mechanisms of variation. It is suggested that variation is the result of cytoplasmic differences present in the original mass isolates, although the possibility of other mechanisms can not be completely dismissed. A review of the literature suggests that asexual variation associated with zoospore propagation is widespread in the genus Phytophthora.
Article
Progeny derived from selfed oospores of an Al (isolate 533) or A2 (isolate 550) field isolate of Phytophthora infestans induced by mating-type-specific hormones consisted of both the A1 and A2 mating types. Oospore progeny produced by selfing of the hybrid of these field isolates also contained the self-fertile A1A2 type in addition to the A1 and A2 types. Selfed-oospore progeny produced by six A1 cultures in S-1 of isolates 533 and 550 consisted of the A1, A2, and A1A2 types; of the Al and A2 types; or of the Al type only. One of the selfed progeny produced by A2 cultures in S-1 of isolate 533 consisted of the A1 and A2 types, while the other consisted of the A2 type only. Results from this study suggest that some individuals of the A2 mating type of P. infestans existing outside Mexico may have originated from selfed oospores produced by the A1 mating type after arriving at the present host countries.
Article
Genetic changes in populations of Phytophthora infestans in Poland were determined by analyzing 247 isolates collected from 1985 through 1991. All isolates collected from 1985 through 1987 were Al mating type and consisted of a single clonal lineage based on allozyme and DNA fingerprint analyses. The appearance of new genotypes in Poland, presumably due to migration, was first detected in 1988 with the identification of the A2 mating type, three allozyme, and a number of DNA fingerprint alleles that had not been detected previously. This migration resulted in dramatic changes. Gene and genotype diversity and the frequency of uniquely detected genotypes all increased beginning in 1988, and reached a peak in 1990 [...]
Article
Eighty single-oospore offspring of Phytophthora infestans from a mating of isolates, which had previously been analyzed for segregation of avirulence/virulence, were assessed for the inheritance of 20 RFLP markers. Three offspring were triploid; they inherited three alleles at all loci where this could be detected and when heterozygous, showed unequal intensities of hybridization with most probes. Twenty-four offspring were trisomic, as each had three doses of one or a few markers, evident from their inheritance of three alleles or from unequal hybridization to one probe. Coinheritance of the extra allele(s) and mitochondrial haplotype in the majority of trisomic offspring suggested that meiosis in oogonia was more aberrant than in antheridia. Linkage analysis was performed on 50 offspring, which were assumed to be euploid; six small linkage groups were detected and several avirulence loci were found to be linked. The origins of aberrant offspring are discussed.
Article
Isolates from potato in an isolated garden in North Wales in 1995 consisted of essentially two distinct phenotypes. Phenotypes P1 and P2, of A1 and A2 mating type respectively, had distinctive multi-locus (RG57) and telomeric RFLP fingerprints. In addition, about 6% of isolates forming abundant oospores were self-fertile and showed a combination of both fingerprints. Among a total of 40 single-hyphal-tip and single-sporangial propagations from two self-fertile isolates, 16 were P2 whereas only one propagation was P1; four propagations were still self-fertile and still carried the molecular markers typical of both P1 and P2. Most of the remainder were A1 but otherwise carried both P1 and P2 molecular markers. Uninucleate zoospore lines from two of these were also of A1 mating type but had segregated molecular markers typical of P2; a zoospore line from a third segregated both A2 mating type and markers typical of P2. The original self-fertile isolates appeared to have heterokaryotic hyphae and sporangia with P1 and P2 nuclei and also nuclei of A1 mating type but P2 markers. Tetraploid nuclei within a heterokaryon, resulting from fusion of P1 with P2 nuclei, could have segregated to yield the latter.
Article
A new PCR-based technique for the detection of inter- and intraspecific genetic variation has been tested on isolates of the fungal phytopathogens Cladosporium fulvum and Pyrenopeziza brassicae. The method is based on the selective PCR amplification of restriction fragments from digests of genomic DNA. We show that the technique is very efficient at detecting polymorphisms, even in species where very little variation could previously be found by RFLP analysis. 21 primer combinations were used on four isolates of P. brassicae, detecting a total of 162 polymorphisms (mean = 4·1 polymorphisms per primer combination per pair of isolates). Four primer combinations were used on eight isolates of C. fulvum, detecting a total of 32 polymorphisms (mean = 3·3 polymorphisms per primer combination per pair of isolates). Primer combinations varied in their ability to detect variation, ranging from 0 to 24 polymorphisms between P. brassicae isolates and 0 to 10 polymorphisms between C. fulvum isolates. AFLP fingerprints were highly reproducible and have great potential as a tool for evaluating genetic diversity of fungal pathogens.
Article
Here we present the first comprehensive genetic linkage map of the heterothallic oomycetous plant pathogenPhytophthora infestans.The map is based on polymorphic DNA markers generated by the DNA fingerprinting technique AFLP (Voset al.,1995,Nucleic Acids Res.23:4407–4414). AFLP fingerprints were made from single zoospore progeny and 73 F1 progeny from two field isolates ofP. infestans.The parental isolates appeared to be homokaryotic and diploid, their AFLP patterns were mitotically stable, and segregation ratios in the F1 progeny were largely Mendelian. In addition to 183 AFLP markers, 7 RFLP markers and the mating type locus were mapped. The linkage map comprises 10 major and 7 minor linkage groups covering a total of 827 cM. The major linkage groups are composed of markers derived from both parents, whereas the minor linkage groups contain markers from either the A1 or the A2 mating type parent. Non-Mendelian segregation ratios were found for the mating type locus and for 13 AFLP markers, all of which are located on the same linkage group as the mating type locus.
Article
Mitochondrial (mt) DNA has been prepared from 24 isolates of Phytophthora infestans from 11 countries and analysed for the occurrence of restriction fragment length polymorphisms using six restriction endonucleases. An insertion of about 2 kilobase pairs has been detected in mtDNA of five of the isolates and the approximate site of insertion located on the restriction map of P. infestans mtDNA. The insertion occurred in four out of eight isolates of A2 mating type, but in only one out of 15 isolates of A1 mating type. The insertion occurred in mtDNA from isolates from Brazil, Egypt, U.K. and U.S.A., suggesting a global distribution. Two further polymorphisms were detected which could have been due to single base changes or very small deletions or insertions. One of these, in mtDNA lacking the insertion, occurred in four isolates which, although obtained from four different countries (Brazil, Ireland, U.K. and U.S.A.), appeared fairly uniform with respect to nuclear markers and may be closely related.
Article
We tested the hypothesis that the population of Phytophthora infestans in the Toluca valley region is genetically differentiated according to habitat. Isolates were sampled in three habitats from (i) wild Solanum spp. (WILD), (ii) land-race varieties in low-input production systems (RURAL), and (iii) modern cultivars in high-input agriculture (VALLEY). Isolates were sampled in 1988-89 (n = 179) and in 1997-98 (n = 389). In both sampling periods, the greatest genetic diversity was observed in RURAL and VALLEY habitats. Based on the Glucose-6-phosphate isomerase and Peptidase allozymes, the subpopulations from the three habitats were significantly differentiated in both sampling periods. In contrast to allozyme data for 1997-98, no differences were found among the three subpopulations for sensitivity to metalaxyl. Two groups of isolates identical for allozyme and mating type were further investigated by restriction fragment length polymorphism fingerprinting; 65% of one group and 85% of another group were demonstrated to be unique. The genetic diversity data and the chronology of disease occurrence during the season are consistent with the hypothesis that populations of P infestans on wild Solanum populations are derived from populations on cultivated potatoes in the central highlands of Mexico near Toluca
Article
Genotypic changes in populations of Phytophthora infestans in Southern Flevoland (150 km2) were analysed by characterising isolates from potato refuse piles, conventional and organic potato fields, and potatoes and tomatoes in allotment gardens for mating type (1712 isolates) and DNA fingerprint pattern using probe RG57 (1048 isolates). The overall percentages of genotypes (and of isolates) that were A2 varied from 32 (4) in 1994 to 45 (56) in 1996. Among the 1048 isolates 170 different genotypes were identified, of which 138 (81%) were 'rare' (i.e., detected in only one sampling site in the research area during 1993-1996). Many rare genotypes were encountered in organic potato fields and in allotment gardens. In 1994 and 1995, four genotypes were abundant. The highest percentages of isolates with these 'common' genotypes were encountered in refuse piles and conventional potato fields. The common genotypes were nearly absent in 1996, suggesting that the population may have passed through a bottleneck at the transition from 1995 to 1996. The Shannon index of genotypic diversity was high in allotment gardens and in organic potato fields. For the total populations the normalised Shannon index of genotypic diversity increased from 0.34 in 1994, with weather favourable to late blight, to 0.61 in 1996, with unfavourable weather. The high numbers of rare genotypes detected every year indicate that oospores may act as an infection source in commercial potato fields. However, refuse piles were identified as the most important infection sources for commercial fields in 1994 and 1995. In 1996 disease in commercial organic fields was probably initiated by a few genotypes originating from seed tubers. In allotment gardens oospores were probably the most important infection source. Abbreviation: Rc - refuse pile, Fc - conventional potato field, Fo - organic potato field, A p - potato plot in allotment garden, At - tomato plot in allotment garden.
Article
ABSTRACT Genetic diversity among isolates of Claviceps africana, the sorghum ergot pathogen, and isolates of other Claviceps spp. causing ergot on sorghum or other hosts, was analyzed by random amplified microsatellite (RAM) and amplified fragment length polymorphism (AFLP) analyses. Of the RAM primer sets tested, one revealed polymorphism in C. africana isolates, with Australian and Indian isolates possessing a unique fragment. AFLP analysis, in addition to clearly distinguishing Claviceps spp., revealed polymorphisms in C. africana. A group of isolates from the United States, Puerto Rico, and South Africa exhibited 95 to 100% similarity with one another. Several isolates from Isabela, Puerto Rico were 100% similar to an isolate from Texas, and another isolate from Puerto Rico was identical with one from Nebraska. Australian and Indian isolates showed greater than 90% similarity with isolates from the United States., Puerto Rico, and South Africa. A number of polymorphisms existed in the United States group, indicating that the recently introduced population contains multiple genotypes. Isolates of C. sorghicola, a newly described sorghum pathogen from Japan, were very distinct from other species via RAM and AFLP analyses, as were isolates from outgroups C. purpurea and C. fusiformis. Both RAM and AFLP analysis will be useful in determining future patterns of intercontinental migration of the sorghum ergot pathogen, with the AFLP method showing greater ability to characterize levels of intraspecific variation.
Article
Randomly selected clones from a Phytophthora infestans partial genomic library were characterized by hybridizing individual clones to Southern blots of total genomic DNA digested with the restriction enzyme EcoRI. Among 59 clones that were screened on seven different central-Mexican isolates, five revealed a unique banding pattern for each isolate tested. Two of these clones were tested further; the banding patterns produced by both were somatically stable when probed to DNA from 63 single-zoospore (asexual) progeny from five different "parent" isolates. For one probe, RG57, each band appeared to represent a unique genetic locus in three different crosses, and each locus segregated for the presence or absence of a band. No bands were found to be allelic, but two pairs of cosegregating loci were identified. Genetic analyses of the other probe (RG7) revealed many more pairs of cosegregating bands and some bands which were allelic. When these probes were hybridized to DNA from the other five species in Phytophthora group IV, probe RG57 hybridized strongly to DNA from P. colocasiae, P. phaseoli and P. mirabilis, but weakly or not at all to that of P. hibernalis and P. ilicis. Probe RG7 hybridized fairly strongly to DNA from all six species. Because the sequence recognized by probe RG57 appears to be evolutionarily conserved, and is dispersed, moderately repetitive and highly polymorphic, it could be very useful in additional studies on the genetics and population biology of P. infestans.
Article
The oomyceteous fungus Phytophthora infestans, which causes the late blight diseases of potato and tomato, has a history that is closely associated with that of mycology and plant pathology. Nevertheless, P. infestans and other oomycetes remain poorly understood relative to fungi in other groups. A resurgence in the worldwide impact of late blight has recently increased interest in the species. Fortunately, over the past decade improved tools for laboratory analysis have been developed which provide an opportunity to advance our understanding of this important pathogen. Since oomycetes do not have a close taxonomic affinity with well-characterized organisms such as ascomycetes and basidiomycetes, it is likely that studies of P. infestans will yield novel biological findings. This review provides an update on the status of research into the fundamental aspects of the biology, genetics, and pathology of P. infestans and describes prospects for future advances.
Article
Multiple loci identified in DNA fingerprints were used to test for random association in two agricultural populations of S. sclerotiorum. In linkage disequilibrium tests among pairs of loci with frequencies between 0.1 and 0.9, 44.5 and 80.5% of pairs of loci were consistent with random association in the clone-corrected samples of the Canadian canola and the North Carolina cabbage populations, respectively. In estimates of corrected (Bonferroni) P value, 70.66 and 98.89% of pairs of loci were in random association. All four possible genotypes for each pair of loci were observed in the Canadian canola sample, consistent with random association among loci. In multilocus association tests across all loci, however, significant association was observed in both populations. In the Canadian canola population, 40 possible heterokaryons were identified. Our data suggest that populations of S. sclerotiorum are predominantly clonal and that occasional genetic exchange and recombination, and not mutation alone, may be a source of new genotypes.
Molecular Variability of Fungal Pathogens Spontaneous variability of single isolates of Phytophthora infestans. I. Cultural variation
  • P D Bridge
  • Y Couteaudier
  • J M Clarkson
Bridge, P. D., Couteaudier, Y. & Clarkson, J. M. (1998) Molecular Variability of Fungal Pathogens. CAB International, Wallingford. Caten, C. E. & Jinks, J. L. (1968) Spontaneous variability of single isolates of Phytophthora infestans. I. Cultural variation. Canadian Journal of Botany 46 : 329–348.
European patent application
  • M Zabeau
  • P Vos
Zabeau, M. & Vos, P. (1993) European patent application. Publication no. EP 0534858.
Telomere-associated restriction fragment length polymorphisms in Phytophthora infestans
  • N D Pipe
  • D S Shaw
Pipe, N. D. & Shaw, D. S. (1997) Telomere-associated restriction fragment length polymorphisms in Phytophthora infestans. Molecular Plant Pathology On-Line http :\\www.bspp.org.uk\mppol\1997\1124pipe Pipe, N. D., Azcoitia, V. & Shaw, D. S. (2000) Self-fertility in Phytophthora infestans : heterokaryons segregate several phenotypes. Mycological Research 104 : 676–680.
Rapid preparation of DNA from filamentous fungi
  • Raeder