Phytophthora infestans: populations,pathogenicity and phenylamides

Isolates of Phytophthora infestans (Mont) de Bary (the potato and tomato late blight pathogen) resistant to phenylamides appeared in Europe and North America in the late 1970s and early 1990s respectively. Concurrent, but coincidentally, with both these events there were radical structural shifts in the pathogen populations as immigrant genotypes from Mexico displaced the indigenous populations. Both A1 and A2 mating type isolates are now present in blighted crops, permitting alternative inoculum via germinating sexually produced oospores to influence dynamics of late blight populations. Studies of inheritance of ploidy, host-specific pathogenicity, mating type and resistance to antibiotics and phenylamide fungicides have provided insight into mechanisms of variation in this potent pathogen.     

Phytophthora infestans populations in central,eastern and southern African countries consist of two major clonal lineages

Limited knowledge is available on Phytophthora infestans populations in Sub‐Saharan Africa (SSA). Therefore, and in response to recent severe late blight epidemics, P. infestans isolates from potato, tomato and Petunia × hybrida from eight SSA countries were characterized. Isolates were characterized with ‘old’ markers, including mating type (176 isolates), mitochondrial DNA haplotype (mtDNA) (281 isolates), glucose‐6‐phosphate isomerase (Gpi) (70 isolates), restriction fragment length polymorphism analysis with probe RG‐57 (49 isolates), and by metalaxyl sensitivity (64 isolates). Most isolates belonged to the US‐1 genotype or its variants (US‐1.10 and US‐1.11). The exceptions were genotype KE‐1 isolates (A1 mating type, mtDNA haplotype Ia, Gpi 90/100 and unique RG‐57 genotype), identified in two fields in Kenya, which are related to genotypes previously identified in Rwanda (RW‐1 and RW‐2), Ecuador and Europe. Metalaxyl‐resistant P. infestans isolates from potato were present in all the countries except Malawi, whereas all the isolates from tomato were sensitive. Genotyping of 176 isolates with seven simple sequence repeat (SSR) markers, including locus D13 that was difficult to score, revealed 79 multilocus genotypes (MLGs) in SSA. When this locus was excluded, 35 MLGs were identified. Genetic differentiation estimates between regional populations from SAA were significant when locus D13 was either excluded (P = 0·05) or included (P = 0·007), but population differentiation was only low to moderate (F_ST = 0·044 and 0·053, respectively).     

Phenotypic variation in Nordic populations of Phytophthora infestans in 2003

A total of 743 single-lesion isolates of Phytophthora infestans were collected in summer 2003 from Denmark, Finland, Norway and Sweden. Most of the isolates were tested for mating type, and subsets were tested for sensitivity to fungicides and virulence (host specific pathogenicity). Approximately 60% of the isolates were A1 mating type in each country. Both mating types were present in 40% of the fields where more than one isolate was tested, indicating strong potential for sexual reproduction. The proportion of metalaxyl-resistant isolates dropped to under 15% from the 60% observed in the early 1990s in Norway and Finland, possibly due to lower selection pressure because of decreased use of metalaxyl. Propamocarb-HCl sensitivity remained unchanged in the Nordic countries compared to the situation in 1997–2000 in Finland. Four isolates collected from Finland and Sweden were able to sporulate in the presence of this fungicide at a concentration of 1000 mg L⁻¹. In Norway and Finland the frequencies of virulence factors and pathotypes remained nearly unchanged since the 1990s, but the mean number of virulence factors per isolate increased from 5·6 to 6·3. In Denmark and Sweden virulence factors 2 and especially 6 were more common than in Norway and Finland. In addition, in the Swedish population the frequencies of pathotypes were quite even while in other countries pathotype 1,3,4,7,10,11 was most prevalent.     

Characterization of some Asian isolates of Phytophthora infestans

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

Novel microsatellite markers for the analysis of Phytophthora infestans populations

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

宁夏南部山区马铃薯晚疫病菌寄生适合度测定

为了解宁夏南部山区马铃薯主栽品种对马铃薯晚疫病菌的抗病性情况,明确不同菌株的寄生适合度,采用离体叶片接种法,用4个马铃薯品种对采自宁夏南部山区的5个马铃薯晚疫病菌分离株进行了侵染率、病斑面积、产孢能力的测定和寄生适合度的计算。结果表明,‘克新18号’对供试的晚疫病菌分离株具有较高的抗病性,其次为‘中薯3号’和‘克新1号’,‘费乌瑞它’的抗病性最差;来源于固原原州区的分离株(GL2和GL7)寄生适合度较高,来源于泾源县的分离株(JZ)寄生适合度较弱。采自宁夏不同地区的晚疫病菌分离株对同一马铃薯品种的寄生适合度有较大差异,同一晚疫病菌株在不同马铃薯品种上的寄生适合度也有一定的差异,表明宁夏南部山区马铃薯晚疫病菌分离株存在一定的致病性分化。     

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

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

Genotypic and phenotypic characterization of Phytophthora infestans populations on Java,Indonesia

Phytophthora infestans is endemic to Indonesia and can infect potato crops at any stage in the growing season. Little is known about P. infestans populations in Indonesia. The objectives of this study were first to identify the genotypes causing late blight in the main potato-growing regions on Java in Indonesia, and secondly to examine genotypic diversity in the P. infestans populations in those regions. Samples were collected on FTA cards (n = 140) or in tubers (n = 6) from 15 locations in nine regencies over four years (2016–2019). Microsatellite analysis revealed that late blight outbreaks in these regencies were caused by EU_2_A1 and other genotypes that are unique to Indonesia. Eighty percent of the samples that amplified with CAPS markers were the A1 mating type. Cultures of six isolates were determined to be the A1 mating type based on the pairing test, and of these, two isolates were intermediate and four were sensitive to metalaxyl-M (mefenoxam). The mode of reproduction of the P. infestans population on Java, Indonesia, was found to be clonal. However, as the sample size in this study was small, more isolates need to be tested to confirm this. Microsatellite analysis revealed that 90% of Indonesian samples had trisomic loci. A high number of multilocus genotypes (MLGs) were found in all nine regencies (131 MLGs out of 146 samples). Results indicate that there is ongoing polyploidization in these populations due to a high mutation rate and no selection pressure from the susceptible potato hosts that are being grown in Indonesia.     

Large subclonal variation in Phytophthora infestans populations associated with Ecuadorian potato landraces

The population of Phytophthora infestans on potato landraces in three provinces (Carchi, Chimborazo and Loja) of Ecuador was analysed. All isolates (n = 66) were of the A1 mating type. Simple sequence repeats (SSR) were used to assess the genetic diversity of the isolates. The P. infestans isolates from the potato landraces grouped in a single clade together with reference isolates belonging to the clonal lineage EC‐1. In the 66 SSR profiles obtained, 31 multilocus genotypes were identified. The 66 isolates constituted 49 different races according to the Solanum demissum differential set ( R1 to R11). The P. infestans population was complex and virulent on 4 to 11 R genes. Analysis showed that the subclonal variation in the Ecuadorian EC‐1 clone is increasing over time and is much larger than clonal variation in lineages in the Netherlands and Nicaragua, suggesting high mutation rates and little or no selection in Ecuador.     

Phenotypic and genotypic variation in Michigan populations of Phytophthora infestans from 2008 to 2010

Severe potato and tomato late blight epidemics in Michigan since 2008 prompted characterization of Phytophthora infestans isolates from the region. From eight counties in Michigan, 124 isolates were collected from infected potato and tomato plants from 2008 to 2010 and characterized using ‘classical’ markers and microsatellites. The classical markers included mating type, Gpi allozyme, mitochondrial DNA haplotype, sensitivity to metalaxyl‐M and tuber pathogenicity. All isolates from 2008 to 2010 were A2 mating type and Ia mtDNA haplotype (124 isolates), 105 isolates had Gpi profile 100/122, 17 isolates had the profile 100/100/111 and the remaining two isolates had 100/111/122. Sensitivity to metalaxyl‐M, expressed as EC₅₀ for mycelial growth in vitro, ranged from <0·1 to 91 μg mL^?1, where 95 and 96% of isolates were classified as either sensitive or intermediate in 2008–2009 and 2010 respectively. The metalaxyl‐M sensitivity and dominant Gpi profile were typical of clonal lineage US‐22, first isolated in 2008 in North America from tomato plants. Tuber pathogenicity, characterized as severity of tuber late blight, was also variable among isolates; however, isolates were less aggressive than previous genotypes present in Michigan, such as US‐8. Microsatellites (simple sequence repeats; SSRs) revealed a shift in the population, characterized by two clusters differentiated over time. These results suggested displacement of the US‐8 genotype by US‐22 from 2008 to 2010 in Michigan. Continuous tracking of changes within P. infestans populations provides evidence of genetic shifts due to migration, prompting modification of management strategies based on the phenotypic characteristics of causal genotypes.     

Distribution of sensitivity responses to cymoxanil within global populations of Phytophthora infestans

In-vitro and in-vivo results show no decrease in Phytophthora infestans sensitivity to cymoxanil after more than 16 years of intensive use, and that a single, sensitive population exists within the countries sampled. © 1998 SCI     

Sensitivity profiles of Mycosphaerella graminicola and Phytophthora infestans populations to different classes of fungicides

Prior to the use of fungicides, the baseline sensitivity of individuals in a pathogen population may already differ by a factor of 10 to 100 between the least and the most sensitive isolates. In Mycosphaerella graminicola populations, this factor, measured in vitro, was 5 to 20 for both the strobilurin analogue azoxystrobin (baseline) and the triazole cyproconazole which has been in use for several years. In Phytophthora infestans populations, this factor, measured in a leaf disc assay, was about 100 for azoxystrobin (baseline), up to 1000 for the cyanoacetamide cymoxanil and >10000 for the phenylamide oxadixyl; both of the latter have been used for many years. In M. graminicola, cross-sensitivity was present between all azole fungicides for the majority of the isolates, whereas no correlation was found between triazoles and azoxystrobin. Despite the existence of cross-sensitivity between azoles, ‘box-and-whiskers’ plots revealed large variations in the sensitivity profiles of some triazoles; isolates resistant to triazoles have not been detected in M. graminicola populations. In P. infestans populations, the proportion of the phenylamide-resistant sub-population increased during the season more rapidly in treated than in untreated fields, but it was low at the beginning of the next season in all fields. During disease epidemics, the fitness of phenylamide-resistant P. infestans isolates, as characterised by lesion size, was higher than that of the sensitive isolates, but after the overwintering period, the recovery of resistant isolates was apparently lower. The presence of both A1 and A2 mating types of P. infestans in European populations, although at different frequencies, allows sexual recombination and increased genetic diversity, affecting sensitivity and fitness. Such mixed populations can still be adequately controlled by using sound anti-resistance strategies. ©1997 SCI     

Potential of sexual reproduction among host-adapted populations of Phytophthora infestans sensu lato in Ecuador

To determine the potential of sexual reproduction among host-adapted populations of Phytophthora infestans sensu lato in Ecuador, 13 A1 isolates belonging to clonal lineages US-1, EC-1 and EC-3, and 11 A2 isolates belonging to the clonal lineage EC-2, were paired on agar plates to induce crossing. In the first experiment, six A1 isolates (three US-1, two EC-1 and one EC-3) were each crossed with three A2 isolates (total = 18 crosses). Matings involving isolates of the EC-1 lineage produced more oospores of healthy appearance than did matings with isolates of US-1 or EC-3. In the second experiment, the oospores of 35 crosses (21 EC-1 × EC-2; 10 US-1 × EC-2; four EC-3 × EC-2) were dispersed on water agar to assess oospore germination. Overall, germination percentages were low. Only one cross produced enough progeny for evaluation. Twenty-three single-oospore offspring were isolated and evaluated for mating type; electrophoretic patterns of glucose-6-phosphate isomerase ( Gpi ) and peptidase ( Pep ) alloenzyme loci; mitochondrial DNA haplotype; and genomic DNA fingerprint. Multilocus genotype data indicated that all 23 isolates resulted from meiotic recombination. Four progeny with homothallic phenotype appeared to be unstable heterokaryons. Markers at several loci segregated according to simple Mendelian expectations for a diploid organism, but the ratios of three RFLP loci and the Pep locus were not consistent with Mendelian expectations. All progeny were nonpathogenic on hosts of the parental genotypes. Reduced mating success and reduced pathogenic fitness of progeny appear to be postmating mechanisms of reproductive isolation in populations of P. infestans sensu lato in Ecuador.     

Aggressiveness and other factors relating to displacement of populations of Phytophthora infestans in England and Wales

Mating type, in vitro sensitivity to the phenylamide fungicide metalaxyl, and mitochondrial (mtDNA) haplotype were determined in some or all of 618 isolates of Phytophthora infestans from the years between 1978 and 1995. A2 mating type occurred infrequently in most but not all years and insensitivity to metalaxyl increased over time. After 1982, the mtDNA Ib haplotype was largely replaced (except for one isolate in 1986 and one in 1995) by two new haplotypes, Ia and IIa. Type Ia was much more common than type IIa.Approximately one quarter of these isolates (165) were compared using two components of fitness associated with aggressiveness (infection frequency × number of sporangia per lesion) on detached leaves of cultivars Maris Piper, Cara and Stirling, which were chosen as exhibiting increasing levels of race non-specific resistance. Isolates were compared with three standard isolates of low, intermediate or high aggressiveness, and the data standardised for comparison between experiments. On cvs. Cara and Stirling, but not on Maris Piper, mtDNA Ia and IIa haplotypes were more aggressive than type Ib in several separate experiments. Similarly, metalaxyl sensitive phenotypes were more aggressive than insensitive phenotypes on Cara and Stirling but not on Maris Piper. The displacement of mtDNA type Ib by types Ia and IIa over this period may have been a result of the lower aggressiveness and lack of complete insensitivity to metalaxyl in type Ib isolates.     

Phenotypic and genotypic diversity of Phytophthora infestans populations in Scotland (1995–97)

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.     

Characterization of isolates of Phytophthora infestans collected in northwestern France from 1988 to 1992

Isolates of the potato late-blight pathogen Phytophthora infestans , collected in several French regions from 1988 to 1992, were characterized in order to determine the structure and pathogenicity of French populations and to compare them with other European samples. All 254 French isolates tested were of the Al mating type, as were two Sicilian, five Portuguese and one Spanish isolate. Metalaxyl sensitivity of 157 isolates was investigated. The frequency of resistance to this chemical decreased in western France from 1989 to 1992. Two glucose phosphate isomerase phenotypes (90/100 and 90/90) were detected among the 46 isolates tested; the former largerly predominated (75–80%) in the samples investigated. Both phenotypes corresponded to the population recently introduced into Europe. A third genotype (86/100), typical of the former European population, was found in one isolate from Picardy, originating from an artificially infected field trial. Five isolates from Portugal and Italy were of the 100/100 phenotype. Race patterns among 26 isolates from western France were very similar, and could not be explained by local selection. The unusual composition of the Brittany population, consisting of 'new' genotypes but all belonging to the Al mating type, is possibly related to the metapopulation type of structure of P. infestans in Europe.     

Survival of Phytophthora infestans in Surface Water

ABSTRACT Coverless petri dishes with water suspensions of sporangia and zoospores of Phytophthora infestans were embedded in sandy soil in eastern Washington in July and October 2001 and July 2002 to quantify longevity of spores in water under natural conditions. Effects of solar radiation intensity, presence of soil in petri dishes (15 g per dish), and a 2-h chill period on survival of isolates of clonal lineages US-8 and US-11 were investigated. Spores in water suspensions survived 0 to 16 days under nonshaded conditions and 2 to 20 days under shaded conditions. Mean spore survival significantly increased from 1.7 to 5.8 days when soil was added to the water. Maximum survival time of spores in water without soil exposed to direct sunlight was 2 to 3 days in July and 6 to 8 days in October. Mean duration of survival did not differ significantly between chilled and nonchilled sporangia, but significantly fewer chilled spores survived for extended periods than that of nonchilled spores. Spores of US-11 and US-8 isolates did not differ in mean duration of survival, but significantly greater numbers of sporangia of US-8 survived than did sporangia of US-11 in one of three trials.     

Diversity of Phytophthora infestans from Poland

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

Diversity of populations of Phytophthora infestans in relation to patterns of potato crop management in Latvia and Lithuania

Potato crop losses can be substantial when conditions for late blight (Phytophthora infestans) development and spread are favourable. In this study, drivers of differences between the P. infestans population structures in Latvia and Lithuania, two neighbouring countries with similar potato-growing traditions, were investigated. Genotypes of P. infestans and population genetic diversity were analysed using a 12-plex simple sequence repeat (SSR) marker assay. High genetic diversity was demonstrated in both populations, with population diversity being higher in Latvia. It would appear that local populations established from soilborne oospores early in the season are well adapted to the conditions in the region. However, somewhat greater spread and survival of local clones was detected in Lithuania, suggesting that potato cropping there is more vulnerable to clonal invasion than in Latvia. For effective disease management, current strategies should be adjusted according to the specific pathogen populations in the region, considering the reproduction and survival of the pathogen. Potato growers should implement late blight preventive measures such as longer field rotation to prevent oospore infections, especially in Latvia, and should use more disease resistant cultivars and high-quality seed potatoes.