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.     

Assessing host specialization of Botrytis cinerea on lettuce and tomato by genotypic and phenotypic characterization

This study tested the hypothesis that Botyrtis cinerea shows host specialization on tomato and lettuce, using phenotypic and genotypic tools. Strains were isolated from tomato and lettuce grown together in the same greenhouse. Forty‐four lettuce strains and 42 tomato strains were investigated for their genetic diversity and their aggressiveness. Both gene diversity and allelic richness were significantly higher in lettuce strains than in tomato strains (P = 0·01). Cluster analysis revealed a clear division of the strains under study into two clusters. However, this structure did not separate the strains according to their host of origin. Tomato strains were significantly more aggressive than lettuce strains when inoculated on tomatoes (P = 0·001), but no significant differences in aggressiveness were observed when the strains were inoculated on lettuce (P = 0·17) or on apple (P = 0·87). The results suggest an absence of clear host specialization of B. cinerea on tomato and lettuce.     

Evidence for selection pressure from resistant potato genotypes but not from fungicide application within a clonal Phytophthora infestans population

Insight into pathogen population dynamics provides a key input for effective disease management of the potato late blight pathogen Phytophthora infestans. Phytophthora infestans populations vary from genetically complex to more simple with a few clonal lineages. The presence or absence of certain strains of P. infestans may impact the efficacy of fungicides or host resistance. Current evidence indicates that genetically, the Irish populations of P. infestans are relatively simple with a few clonal lineages. In this study, P. infestans populations were genetically characterized based on samples collected at the national centre for potato breeding during the period 2012–16. The dominance of clonal lineages within this P. infestans population was confirmed and the potential selection pressure of fungicide treatment (2013–15) and host resistance (2016) on this clonal P. infestans population was then investigated. It was found that fungicide products did not notably affect the genetic structure of sampled populations relative to samples from untreated control plants. In contrast, samples taken from several resistant potato genotypes were found to be more often of the EU_13_A2 lineage than those taken from control King Edward plants or potato genotypes with low resistance ratings. Resistant potato varieties Sarpo Mira and Bionica, containing characterized R genes, were found to strongly select for EU_13_A2 strains.     

Diversity of Avr‐vnt1 and AvrSmira1 effector genes in Polish and Norwegian populations of Phytophthora infestans

The oomycete Phytophthora infestans, the cause of late blight, is one of the most important potato pathogens. During infection, it secretes effector proteins that manipulate host cell function, thus contributing to pathogenicity. This study examines sequence differentiation of two P. infestans effectors from 91 isolates collected in Poland and Norway and five reference isolates. A gene encoding the Avr‐vnt1 effector, recognized by the potato Rpi‐phu1 resistance gene product, is conserved. In contrast, the second effector, AvrSmira1 recognized by Rpi‐Smira1, is highly diverse. Both effectors contain positively selected amino acids. A majority of the polymorphisms and all selected sites are located in the effector C‐terminal region, which is responsible for their function inside host cells. Hence it is concluded that they are associated with a response to diversified target protein or recognition avoidance. Diversification of the AvrSmira1 effector sequences, which existed prior to the large‐scale cultivation of plants containing the Rpi‐Smira1 gene, may reduce the predicted durability of resistance provided by this gene. Although no isolates virulent to plants with the Rpi‐phu1 gene were found, the corresponding Avr‐vnt1 effector has undergone selection, providing evidence for an ongoing ‘arms race’ between the host and pathogen. Both genes remain valuable components for resistance gene pyramiding.     

Genetic diversity and host differentiation among isolates of Phytophthora infestans from cultivated potato and wild solanaceous hosts in Peru

To test the hypothesis that isolates of Phytophthora infestans attacking wild Solanaceae exhibit specialization for particular host species, 115 isolates of P. infestans were collected from cultivated potatoes, nontuber-bearing Solanum spp. of the Basarthrum section and wild tomatoes from five departments in the northern and central highlands of Peru, and characterized using several neutral markers. All isolates belonged to one of four clonal lineages described previously in Peru: EC-1, US-1, PE-3 and PE-7. There was a strong association of three lineages with host species: PE-3 was only isolated from cultivated potato, while PE-7 and US-1 were only isolated from nontuber-bearing Solanum spp. ( Basarthrum section and wild tomatoes). EC-1 was isolated from all host groups sampled. A subset ( n  = 74) of the isolates was evaluated for metalaxyl resistance. High levels of resistance were found almost exclusively in EC-1 and PE-3, while US-1 and PE-7 isolates were generally sensitive. In a detached-leaf assay for lesion diameter using five EC-1 isolates from S. caripense and seven EC-1 isolates from cultivated potato, there was a significant interaction between isolate origin and inoculated host, caused by higher aggressiveness of EC-1 from cultivated potato on its host of origin. In a comparison of EC-1 (seven isolates from cultivated potato) and US-1 (three isolates from S. caripense ), each pathogen lineage was more aggressive on its original host species, causing a highly significant interaction between isolate origin and inoculated host. Wild tomatoes and nontuber-bearing Solanum spp. harbour several pathogen lineages in Peru and could serve as reservoirs of inoculum that might contribute to epidemics on potato or tomato. Potential risks associated with the use of wild Solanum hosts as sources of resistance to P. infestans are discussed .     

Severe outbreaks of late blight on potato and tomato in South India caused by recent changes in the Phytophthora infestans population

Late blight, caused by Phytophthora infestans, has emerged as the most destructive disease of potato and tomato in South India since 2008. One hundred and fifty‐seven isolates of Phytophthora infestans, 63 from potato and 94 from tomato, were collected from major potato and tomato production areas of South India between 2010 and 2012. Their phenotypic and genotypic characteristics were determined and compared with reference isolates. Isolates were characterized based on mating type, in vitro metalaxyl sensitivity, mitochondrial DNA haplotype, RG57 DNA fingerprinting patterns, SSR markers and aggressiveness on potato and tomato, in order to monitor population changes in P. infestans. All isolates were A2 mating type, metalaxyl resistant, mtDNA haplotype Ia and had RG57 and SSR fingerprints almost identical to the 13_A2 clonal lineage reported in Europe. Variation at the D13 and SSR4 loci allowed discrimination of minor variants, designated as 13_A2_3, 13_A2_3b, 13_A2_3c and 13_A2_1. A comparison of the lesion diameters caused by 157 isolates on detached leaflets of three potato and tomato cultivars showed all isolates to be equally aggressive, confirming that the same clonal population is infecting both hosts. This study demonstrates that the 13_A2 lineage was responsible for severe late blight outbreaks on potato and tomato in South India and has replaced the prior population represented by the US‐1 and other genotypes. Revised management strategies will be required to combat this destructive 13_A2 clonal lineage and monitoring of the population across other potato‐ and tomato‐growing regions of India is warranted.     

Markers, old and new, for examining Phytophthora infestans diversity

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.     

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.     

French isolates of Phytophthora infestans from potato and tomato differ in phenotype and genotype

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.     

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.     

Activation of defence responses to Phytophthora infestans in potato by BABA

Late blight caused by Phytophthora infestans is one of the most devastating diseases of the potato crop. Resistance breeding and current fungicides are unable to control the rapidly evolving P. infestans and new control strategies are urgently needed. This study examined mechanisms of dl ‐β‐aminobutyric acid (BABA)‐induced resistance (IR) in the potato–P. infestans system. Leaves from two cultivars that differ in their degree of resistance, Bintje and Ovatio, were analysed after foliar treatment with BABA. Rapid activation of various defence responses and a significant reduction in P. infestans growth were observed in leaves treated with BABA. In the more resistant cultivar, Ovatio, the activation was both faster and stronger than in Bintje. Microscopic analysis of leaves treated with BABA revealed induction of small hypersensitive response (HR)‐like lesions surrounded by callose, as well as production of hydrogen peroxide (H₂O₂). Molecular and chemical analyses revealed soluble phenols such as arbutin and chlorogenic acid and activation of PR‐1. These results show a direct activation of defence responses in potato, rather than priming as reported for other plant species. They also show that the efficiency of BABA‐IR differs between cultivars, which highlights the importance of taking all aspects into consideration when establishing new methods for disease management.     

Evidence for presence of the founder Ia mtDNA haplotype of Phytophthora infestans in 19th century potato tubers from the Rothamsted archives

Late blight remained a significant disease for potato growers in Europe long after the famine of the 1840s. Of the four mitochondrial haplotypes of Phytophthora infestans, only the Ia mitochondrial DNA (mtDNA) haplotype has been identified previously in infected potato leaves from famine‐era herbarium specimens collected in England, Ireland and Europe in the 19th century. Long‐term soil fertility experiments were conducted on potato between 1876 and 1901 in Rothamsted to investigate effects of combinations of organic manures and mineral fertilizers on disease and yield. This report identifies for the first time the same Ia mtDNA haplotype of P. infestans in three diseased tubers from 1877 from the long‐term Rothamsted trials, thus providing the earliest evidence of the presence of the founder Ia mtDNA haplotype of P. infestans in potato tubers in England. Soil amendments had a significant impact on disease and yield. A real‐time PCR assay was used to detect and quantify P. infestans in tubers. The level of pathogen DNA was greatest in tubers from highest yielding plots that received combinations of inorganic nitrogenous and mineral fertilizers and least in tubers from plots with organic farmyard manures or non‐nitrogenous mineral fertilizers. The Ia mtDNA haplotype was also confirmed from diseased potato leaves during the same time period. Thus, the founder Ia mtDNA haplotype survived in potato tubers after 1846 and was present over 30 years later in the UK.     

Tracking Phytophthora infestans with SSR markers within and between seasons – a field study in Sweden

The dynamics of a late blight epidemic and sexual reproduction in Phytophthora infestans were studied in an experimental field in mid‐Sweden. The field was inoculated with six isolates of P. infestans taken from another potato field where sexual reproduction of the pathogen was suspected. Three weeks after inoculation single‐lesion leaflets were sampled and the resulting isolates characterized using microsatellites (SSRs) and mating type as markers. Among the 151 isolates analysed, the inoculum genotypes constituted more than 80% of the genotypes found, with three other genotypes making up the remainder. The following year, P. infestans obtained from soil samples taken from this field were analysed, and six novel genotypes were identified. Genotypes from the previous summer’s population were not detected. Analysis of the genotypes recovered was consistent with them being recombinants, with the previous summer’s population acting as parents. These findings are consistent with the hypothesis that oospores produced during a summer epidemic in Sweden can overwinter and cause infection the next year.     

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.     

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.     

Effects of host and pathogen genotypes on inducibility of resistance in tomato (Solanum lycopersicum) to Phytophthora infestans

Thirteen tomato (Solanum lycopersicum) accessions were tested for inducibility of resistance against two isolates of Phytophthora infestans using BABA (dl ‐3‐amino butyric acid) as the inducing agent. In a more detailed trial, six of the accessions were assessed for inducibility of resistance to six P. infestans isolates on three leaves of different age per plant. Plants were inoculated 1 week after treatment with BABA. Area under the disease progress curve (AUDPC), sporulation capacity (SC) and infection efficiency (IE) were all affected by treatment with BABA. On leaves of all ages AUDPC was most affected by induction (43–100% reduction on the youngest leaves) followed by SC (14–100%) and IE (0–100% reduction). Tomato genotypes varied significantly in inducibility of resistance against P. infestans and the degree of induction generally decreased with increasing leaf age, whilst the absolute susceptibility with respect to AUDPC and SC rarely changed. The level of induction was not always related to the resistance level of the tomato accession and it was significantly influenced by the pathogen isolate used for challenge inoculation. The results show that inducibility of resistance is a selectable trait that is, however, isolate‐specific.     

Population genetics of Phytophthora infestans in Denmark reveals dominantly clonal populations and specific alleles linked to metalaxyl‐M resistance

Control of the potato late blight pathogen Phytophthora infestans relies heavily on chemicals. The fungicide metalaxyl‐M (Mefenoxam) has played an important role in controlling the disease, but insensitivity to the fungicide in certain isolates is now of major concern. A genetic basis for resistance to metalaxyl suggests the possibility for linking resistance phenotypes to specific population genetic markers, but in order to do this, the population genetic structure and mode of reproduction in a population must first be well described. The dynamics of metalaxyl‐M resistance in the Danish population of P. infestans was characterized over the course of the 2013 growing season, as was the population genetic structure, using simple sequence repeat (SSR) genotypes and single nucleotide polymorphism (SNP)‐based mitochondrial haplotyping of over 80 isolates. Both mating types A1 and A2 were present in most fields, but tests for recombination showed that clonal reproduction dominates in Danish populations. Genotype was not linked to haplotype and no differentiation was observed at the haplotype level, but rather between fields. Resistance phenotypes were linked to specific SSR alleles, demonstrating the potential for a more precise SNP‐based marker system for predicting resistance to metalaxyl‐M.