Understanding the Temporal Dynamics of Invasive Late Blight Populations in India for Improved Management Practices.

The microbial oomycete pathogen Phytophthora infestans causes severe epidemics of potato late blight in crops globally. Disease management benefits from an understanding of the diversity of pathogen populations. In this study, we explore the dynamics of P. infestans populations in the late blight-potato agro-ecosystem across the Indian subcontinent. Investigations of the macroecological observations at the field level and microbial ecological principles provided insights into future pathogen behavior. We use a comprehensive simple sequence repeat allele dataset to demonstrate that an invasive clonal lineage called EU_13_A2 has dominated populations over 14 years across India, Bangladesh, and Pakistan. Increasing levels of subclonal variation were tracked over time and space, and, for the first time, populations in Asia were also compared with the source populations from Europe. Within India, a regional pathogen population structure was observed with evidence for local migration, cross-border movement between surrounding countries, and introductions via imports. There was also evidence of genetic drift and between-season transmission of more strongly pathogenic subclones with a complete displacement of some subclonal types. The limited introduction of novel genotypes and the use of resistant potato cultivars could contribute to the dominance of the 13_A2 lineage. The insights will contribute to the management of the pathogen in these key global potato production regions.

The Variability of Phytophthora infestans Isolates Collected from Estonian Islands in the Baltic Sea.

Knowledge of a pathogen's genetic variability and population structure is of great importance to effective disease management. In this study, 193 isolates of Phytophthora infestans collected from three Estonian islands were characterized over 3 years using simple sequence repeat (SSR) marker data complemented by information on their mating type and resistance to metalaxyl. In combination with SSR marker data from samples in the neighboring Pskov region of Northwest Russia, the impact of regional and landscape structure on the level of genetic exchange was also examined. Among the 111 P. infestans isolates from Estonian islands, 49 alleles were detected among 12 SSR loci, and 59 SSR multilocus genotypes were found, of which 64% were unique. The genetic variation was higher among years than that among islands, as revealed by the analysis of molecular variance. The frequency of metalaxyl-resistant isolates increased from 9% in 2012 to 30% in 2014, and metalaxyl resistance was most frequent among A1 isolates. The test for isolation by distance among the studied regions was not significant, and coupled with the absence of genetic differentiation, the result revealed gene flow and the absence of local adaptation. The data are consistent with a sexual population in which diversity is driven by an annual germination of soilborne oospores. The absence of shared genotypes over the years has important implications when it comes to the management of diseases. Such population diversity can make it difficult to predict the nature of the outbreak in the coming year as the genetic makeup is different for each year.

First report of Phytophthora infestans lineage EU23 causing potato and tomato late blight in South Africa.

In South Africa, potato (Solanum tuberosum) late blight epidemics from 1996 to 2007 were caused by Phytophthora infestans clonal lineage US-1 (McLeod et al. 2001; Pule et al. 2013). Similarly, surveys on tomatoes in the mid-1990s only identified the US-1 clonal lineage in South Africa (McLeod et al., 2001). On potatoes, populations from the Southern Cape and Western Cape regions consisted of persistent mefenoxam-resistant populations (McLeod et al. 2001; Pule et al. 2013). Limited mefenoxam (R-enantiomer of metalaxyl) screening in 2021 in the Western Cape showed that potato isolates were sensitive, which prompted our study. Potato late blight samples were collected in 13 potato fields in the 2021 to 2023 seasons in the Western Cape (n = 4), Free State (n = 7), Limpopo (n = 1) and Kwazulu-Natal (n = 1) Provinces, and one tomato sample in 2022 in the Limpopo Province. Fourteen samples, one per field, were simple sequence repeat (SSR) genotyped for 12 loci (Li et al. 2013) using as DNA template, FTA cards, or genomic DNA extracted from cultures. P. infestans isolations from lesions and DNA culture extractions were conducted as previously described (Pule et al. 2013). SSR genotyping revealed that all 14 P. infestans samples belonged to clonal lineage EU_23_A1 (EU23), which has a phenotype (A1 and metalaxyl sensitive) and SSR genotype matching the US-23 lineage (Saville et al., 2021). As expected, minor polymorphisms were detected among the samples at loci Pi02, G11, D13 and SSR4. Mefenoxam sensitivity testing of seven potato isolates from the Free State (n = 3) and Western Cape (n = 4), and one tomato isolate was conducted as previously described (Mcleod et al. 2001). All isolates were sensitive to mefenoxam since no infection and sporulation occurred at 3 µg/ml. This was expected since EU23 has been reported as mefenoxam sensitive in other countries (Kawchuk et al., 2011; McGrath et al., 2015). Replacement of the US-1 clonal lineage by EU23 suggests that the latter lineage is more aggressive or fit than US-1, but this must be verified especially on potatoes. On tomatoes, on the other hand, EU23 is known as a highly aggressive lineage (Kawchuk et al., 2011; McGrath et al., 2015; Saville et al., 2021). Therefore, population displacements may have first occurred on tomatoes from where the lineage spread to potatoes. In the Cape coastal potato production regions, population displacement may have been supported by the withdrawal of mefenoxam/metalaxyl from the region since 1996 because the EU23 lineage is mefenoxam sensitive, as opposed to the previously prevailing US-1 mefenoxam-resistant lineage. More severe potato late blight epidemics has not been observed in recent years in South Africa. However, tomato late blight has increased and is more prevalent in the Limpopo province. The source of the introduction of EU23 into South Africa is unknown. Only test-tube plants and/or greenhouse tubers may be imported into South Africa since 1997. Therefore, the illegal importation of planting material may have introduced the new genotype. Whether this could have occurred from neighbouring African countries is unknown since P. infestans genotyping has not been conducted in these countries. In Africa, EU23 has been reported in northern African countries (Tunisia, Algeria and Egypt) (Saville et al., 2021; El-Ganainy et al., 2023). Mefenoxam and metalaxyl applications will likely be effective again in the Western Cape, but more samples will have to be tested to confirm this. This will provide growers with a more cost-effective fungicide (metalaxyl) since alternative actives with comparable systemic and curative activity are more expensive.

Examining Phenotypic Traits Contributing to the Spread in Northern European Potato Crops of EU_41_A2, a New Clonal Lineage of Phytophthora infestans.

Until recently, genotypes of Phytophthora infestans were regionally distributed in Europe, with populations in western Europe being dominated by clonal lineages and those in northern Europe being genetically diverse because of frequent sexual reproduction. However, since 2013 a new clonal lineage (EU_41_A2) has successfully established itself and expanded in the sexually recombining P. infestans populations of northern Europe. The objective of this study was to study phenotypic traits of the new clonal lineage of P. infestans, which may explain its successful establishment and expansion within sexually recombining populations. Fungicide sensitivity, aggressiveness, and virulence profiles of isolates of EU_41_A2 were analyzed and compared with those of the local sexual populations from Denmark, Norway, and Estonia. None of the phenotypic data obtained from the isolates collected from Denmark, Estonia, and Norway independently explained the invasive success of EU_41_A2 within sexual Nordic populations. Therefore, we hypothesize that the expansion of this new genotype could result from a combination of fitness traits and more favorable environmental conditions that have emerged in response to climate change.

Characterization of Phytophthora infestans Populations in Cyprus, the Southernmost Potato-Producing European Country.

Cyprus is the southernmost island country of Europe, located in the Mediterranean. Despite its limited area, potato production is considered an integral source of the national agricultural revenue. During 2010-2012, a late blight epidemic period for the country, the population structure of Phytophthora infestans was analyzed via a sample of 539 isolates collected from all of the main potato-cultivating regions of Cyprus. We determined mating type, mefenoxam sensitivity, and genetic polymorphism at 12 simple sequence repeat (SSRs) loci. Although both mating types were detected in the country, a gradual but dynamic shift toward A2 dominance was manifested over time. The pathogen population also demonstrated reduced sensitivity to the phenylamide fungicide, since 96.2% of the tested isolates had high (70.3%) and intermediate (25.9%) resistance to mefenoxam, which suggests that it should be replaced with other active ingredients in local disease management strategies. The genotypic analysis also revealed the predominance of the highly aggressive mefenoxam-insensitive EU_13_A2 lineage across the country, with a frequency of 79.2%. Other samples comprised an older lineage EU_2_A1 (19.5%), a very low proportion of EU_23_A1 (0.37%), and others that did not match any known lineage (0.92%). SSRs data supported triploid genomes among the dominant lineages, and patterns of their asexual population history were also apparent. A high subclonal variation of the 13_A2 population was detected, which suggested introduction events of this widespread genotype to Cyprus from major tuber-exporting countries. Present data indicate the severe impact of inoculum migration to the structure of the local population; thus, current phytosanitary procedures should be reconsidered and possibly attuned. This is the first comprehensive study to elucidate the diversity of P. infestans in Cyprus and could serve as a baseline for future monitoring of this highly adaptive plant pathogen, given that late blight management strategies should be constantly refined according to the traits of the dominant genotypes of P. infestans.

The Irish potato famine pathogen Phytophthora infestans originated in central Mexico rather than the Andes.

Phytophthora infestans is a destructive plant pathogen best known for causing the disease that triggered the Irish potato famine and remains the most costly potato pathogen to manage worldwide. Identification of P. infestan's elusive center of origin is critical to understanding the mechanisms of repeated global emergence of this pathogen. There are two competing theories, placing the origin in either South America or in central Mexico, both of which are centers of diversity of Solanum host plants. To test these competing hypotheses, we conducted detailed phylogeographic and approximate Bayesian computation analyses, which are suitable approaches to unraveling complex demographic histories. Our analyses used microsatellite markers and sequences of four nuclear genes sampled from populations in the Andes, Mexico, and elsewhere. To infer the ancestral state, we included the closest known relatives Phytophthora phaseoli, Phytophthora mirabilis, and Phytophthora ipomoeae, as well as the interspecific hybrid Phytophthora andina. We did not find support for an Andean origin of P. infestans; rather, the sequence data suggest a Mexican origin. Our findings support the hypothesis that populations found in the Andes are descendants of the Mexican populations and reconcile previous findings of ancestral variation in the Andes. Although centers of origin are well documented as centers of evolution and diversity for numerous crop plants, the number of plant pathogens with a known geographic origin are limited. This work has important implications for our understanding of the coevolution of hosts and pathogens, as well as the harnessing of plant disease resistance to manage late blight.

Crop connectivity under climate change: future environmental and geographic risks of potato late blight in Scotland.

The impact of climate change on dispersal processes is largely ignored in risk assessments for crop diseases, as inoculum is generally assumed to be ubiquitous and nonlimiting. We suggest that consideration of the impact of climate change on the connectivity of crops for inoculum transmission may provide additional explanatory and predictive power in disease risk assessments, leading to improved recommendations for agricultural adaptation to climate change. In this study, a crop-growth model was combined with aerobiological models and a newly developed infection risk model to provide a framework for quantifying the impact of future climates on the risk of disease occurrence and spread. The integrated model uses standard meteorological variables and can be easily adapted to various crop pathosystems characterized by airborne inoculum. In a case study, the framework was used with data defining the spatial distribution of potato crops in Scotland and spatially coherent, probabilistic climate change data to project the future connectivity of crop distributions for Phytophthora infestans (causal agent of potato late blight) inoculum and the subsequent risk of infection. Projections and control recommendations are provided for multiple combinations of potato cultivar and CO2 emissions scenario, and temporal and spatial averaging schemes. Overall, we found that relative to current climatic conditions, the risk of late blight will increase in Scotland during the first half of the potato growing season and decrease during the second half. To guide adaptation strategies, we also investigated the potential impact of climate change-driven shifts in the cropping season. Advancing the start of the potato growing season by 1 month proved to be an effective strategy from both an agronomic and late blight management perspective.

Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing.

A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.

Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen.

Pest and pathogen losses jeopardise global food security and ever since the 19(th) century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.

THAPBI PICT-a fast, cautious, and accurate metabarcoding analysis pipeline.

THAPBI PICT is an open source software pipeline for metabarcoding analysis of Illumina paired-end reads, including cases of multiplexing where more than one amplicon is amplified per DNA sample. Initially a Phytophthora ITS1 Classification Tool (PICT), we demonstrate using worked examples with our own and public data sets how, with appropriate primer settings and a custom database, it can be applied to other amplicons and organisms, and used for reanalysis of existing datasets. The core dataflow of the implementation is (i) data reduction to unique marker sequences, often called amplicon sequence variants (ASVs), (ii) dynamic thresholds for discarding low abundance sequences to remove noise and artifacts (rather than error correction by default), before (iii) classification using a curated reference database. The default classifier assigns a label to each query sequence based on a database match that is either perfect, or a single base pair edit away (substitution, deletion or insertion). Abundance thresholds for inclusion can be set by the user or automatically using per-batch negative or synthetic control samples. Output is designed for practical interpretation by non-specialists and includes a read report (ASVs with classification and counts per sample), sample report (samples with counts per species classification), and a topological graph of ASVs as nodes with short edit distances as edges. Source code available from https://github.com/peterjc/thapbi-pict/ with documentation including installation instructions.

Population Dynamics of Phytophthora infestans in Egypt Reveals Clonal Dominance of 23_A1 and Displacement of 13_A2 Clonal Lineage.

Potato (Solanum tuberosum L.) and tomato (S. lycopersicum L.) are the most economically important vegetable crops in Egypt and worldwide. The winter crop in Egypt is particularly prone to late blight caused by Phytophthora infestans. A total of 152 P. infestans isolates were isolated from the 2013, 2014, 2016 and 2018 winter crops with 82 isolates from potato, 69 from tomato and one isolate from eggplant (S. melongena L.). All isolates belonged to the A1 mating type with no evidence of A2 or self-fertile strains. The majority of isolates (53%) were sensitive to metalaxyl, 32% were intermediate and 15% were resistant. Variation in aggressiveness between three P. infestans isolates EG-005 (13_A2) and EG-276 (23_A1) from potato, and EG-237 (23_A1) from eggplant was determined on tuber slices and leaflets of 10 potato cultivars. The eggplant isolate EG-237 showed higher sporulation capacity compared with the other tested isolates and was able to infect potato (Lady Rosetta cv) and tomato (Super Strain B cv). The simple sequence repeat (SSR) genotyping data showed that in contrast to our previous work (3-year period 2010-12) in which the proportion of 13_A2 lineage was 35%, all isolates belonged to the 23_A1 lineage. There was no evidence for the existence of the A2 mating type or 13_A2 lineage even in the destroyed field crops of some cultivars (Cara, Bellini and Valor) that had been reported as resistant to 23_A1. The data have been submitted into the Euroblight database to allow temporal and spatial genetic diversity to be examined in comparison with other regional P. infestans populations. The AVR2 and AVR2-like RXLR effector genes were amplified and sequenced. In the avirulent AVR2 gene, only one heterozygous SNP was detected at position 31 in the N terminus in six isolates out of eleven, whereas two heterozygous SNPs were detected at position 29 in the N-terminus and ninety-two in the C- terminus of the AVR2-like gene. This suggests that changes in the previously reported virulence profile of 23_A1 are not related to commercial cultivars carrying the R2 gene. In addition, this is the first report of P. infestans on eggplant in Egypt.

Climate Change and Defense against Pathogens in Plants.

Most reviews of climate change are epidemiological, focusing on impact assessment and risk mapping. However, there are many reports of the effects of environmental stress factors on defense mechanisms in plants against pathogens. We review those representative of key climate change-related stresses to determine whether there are any patterns or trends in adaptation responses. We recognize the complexity of climate change itself and the multitrophic nature of the complex biological interactions of plants, microbes, soil, and the environment and, therefore, the difficulty of reductionist dissection approaches to resolving the problems. We review host defense genes, germplasm, and environmental interactions in different types of organisms but find no significant group-specific trends. Similarly, we review by host defense mechanism type and by host-pathogen trophic relationship but identify no dominating mechanism for stress response. However, we do identify core stress response mechanisms playing key roles in multiple response pathways whether to biotic or abiotic stress. We suggest that these should be central to mechanistic climate change plant defense research. We also recognize biodiversity, heterogeneity, and the need for understanding stress in a true systems biology approach as being essential components of progressing our understanding of and response to climate change.

DNA Metabarcoding and Isolation by Baiting Complement Each Other in Revealing Phytophthora Diversity in Anthropized and Natural Ecosystems.

Isolation techniques supplemented by sequencing of DNA from axenic cultures have provided a robust methodology for the study of Phytophthora communities in agricultural and natural ecosystems. Recently, metabarcoding approaches have emerged as new paradigms for the detection of Phytophthora species in environmental samples. In this study, Illumina DNA metabarcoding and a conventional leaf baiting isolation technique were compared to unravel the variability of Phytophthora communities in different environments. Overall, 39 rhizosphere soil samples from a natural, a semi-natural and a horticultural small-scale ecosystem, respectively, were processed by both baiting and metabarcoding. Using both detection techniques, 28 out of 39 samples tested positive for Phytophthora. Overall, 1,406,613 Phytophthora internal transcribed spacer 1 (ITS1) sequences and 155 Phytophthora isolates were obtained, which grouped into 21 taxa, five retrieved exclusively by baiting (P. bilorbang; P. cryptogea; P. gonapodyides; P. parvispora and P. pseudocryptogea), 12 exclusively by metabarcoding (P. asparagi; P. occultans; P. psycrophila; P. syringae; P. aleatoria/P. cactorum; P. castanetorum/P. quercina; P. iranica-like; P. unknown sp. 1; P. unknown sp. 2; P. unknown sp. 3; P. unknown sp. 4; P. unknown sp. 5) and four with both techniques (P. citrophthora, P. multivora, P. nicotianae and P. plurivora). Both techniques complemented each other in describing the variability of Phytophthora communities from natural and managed ecosystems and revealing the presence of rare or undescribed Phytophthora taxa.

Highly Diverse Phytophthora infestans Populations Infecting Potato Crops in Pskov Region, North-West Russia.

There is limited understanding of the genetic variability in Phytophthora infestans in the major potato cultivation region of north-western Russia, where potato is grown primarily by small households with limited chemical treatment of late blight. In this study, the mating type, sensitivity to metalaxyl, and genotype and population genetic diversity (based on 12 simple sequence repeat (SSR) markers) of 238 isolates of P. infestans from the Pskov region during the years 2010-2013 were characterized. The aim was to examine the population structure, phenotypic and genotypic diversity, and the prevalent reproductive mode of P. infestans, as well as the influence of the location, time, and agricultural management practices on the pathogen population. The frequency of the A2 mating was stable over the four seasons and ranged from 33 to 48% of the sampled population. Both mating types occurred simultaneously in 90% of studied fields, suggesting the presence of sexual reproduction and oospore production in P. infestans in the Pskov region. Metalaxyl-sensitive isolates prevailed in all four years (72%), however, significantly fewer sensitive isolates were found in samples from large-scale conventional fields. A total of 50 alleles were detected in the 141 P. infestans isolates analyzed for genetic diversity. Amongst the 83 SSR multilocus genotypes (MLGs) detected, 65% were unique and the number of MLGs varied between locations from 3 to 20. These results, together with the high genotypic diversity observed in all the locations and the lack of significance of linkage disequilibrium, suggest that sexual recombination is likely responsible for the unique MLGs and the high genetic diversity found in the Pskov region population, resembling those of north-eastern European populations.

Genotypic and Phenotypic Structure of the Population of Phytophthora infestans in Egypt Revealed the Presence of European Genotypes.

Late blight disease of potato and tomato, caused by Phytophthora infestans, results in serious losses to Egyptian and global potato and tomato production. To understand the structure and dynamics of the Egyptian population of P. infestans, 205 isolates were collected from potato and tomato plants during three growing seasons in 2010-2012. The characterization was achieved by mating-type assay, metalaxyl sensitivity assay, and virulence pattern. Additionally, genotyping of 85 Egyptian isolates and 15 reference UK isolates was performed using 12 highly informative microsatellite (SSR) markers David E. L. Cooke and five effector (RxLR) genes. Mating-type testing showed that 58% (118 of 205) of the isolates belonged to mating type A1, 35% (71 isolates) to mating type A2, and the rest 8% (16 isolates) were self-fertile. The phenotype of metalaxyl response was represented as 45% resistant, 43% sensitive, and 12% as intermediate. Structure analysis grouped the 85 identified genotypes into two main clonal lineages. The first clonal lineage comprised 21 isolates belonging to A2 mating type and 8 self-fertile isolates. This clonal lineage was identified as Blue_13 or EU_13_A2. The second main clonal lineage comprised 55 isolates and was identified as EU_23_A1. A single isolate with a novel SSR genotype that formed a distinct genetic grouping was also identified. The effector sequencing showed good correspondence with the virulence data and highlighted differences in the presence and absence of loci as well as nucleotide polymorphism that affect gene function. This study indicated a changing population of P. infestans in Egypt and discusses the findings in the context of late blight management.

Presence/absence, differential expression and sequence polymorphisms between PiAVR2 and PiAVR2-like in Phytophthora infestans determine virulence on R2 plants.

• A detailed molecular understanding of how oomycete plant pathogens evade disease resistance is essential to inform the deployment of durable resistance (R) genes. • Map-based cloning, transient expression in planta, pathogen transformation and DNA sequence variation across diverse isolates were used to identify and characterize PiAVR2 from potato late blight pathogen Phytophthora infestans. • PiAVR2 is an RXLR-EER effector that is up-regulated during infection, accumulates at the site of haustoria formation, and is recognized inside host cells by potato protein R2. Expression of PiAVR2 in a virulent P. infestans isolate conveys a gain-of-avirulence phenotype, indicating that this is a dominant gene triggering R2-dependent disease resistance. PiAVR2 presence/absence polymorphisms and differential transcription explain virulence on R2 plants. Isolates infecting R2 plants express PiAVR2-like, which evades recognition by R2. PiAVR2 and PiAVR2-like differ in 13 amino acids, eight of which are in the C-terminal effector domain; one or more of these determines recognition by R2. Nevertheless, few polymorphisms were observed within each gene in pathogen isolates, suggesting limited selection pressure for change within PiAVR2 and PiAVR2-like. • Our results direct a search for R genes recognizing PiAVR2-like, which, deployed with R2, may exert strong selection pressure against the P. infestans population.

Evolutionary trait-based approaches for predicting future global impacts of plant pathogens in the genus Phytophthora.

Plant pathogens are introduced to new geographical regions ever more frequently as global connectivity increases. Predicting the threat they pose to plant health can be difficult without in-depth knowledge of behaviour, distribution and spread. Here, we evaluate the potential for using biological traits and phylogeny to predict global threats from emerging pathogens.We use a species-level trait database and phylogeny for 179 Phytophthora species: oomycete pathogens impacting natural, agricultural, horticultural and forestry settings. We compile host and distribution reports for Phytophthora species across 178 countries and evaluate the power of traits, phylogeny and time since description (reflecting species-level knowledge) to explain and predict their international transport, maximum latitude and host breadth using Bayesian phylogenetic generalised linear mixed models.In the best-performing models, traits, phylogeny and time since description together explained up to 90%, 97% and 87% of variance in number of countries reached, latitudinal limits and host range, respectively. Traits and phylogeny together explained up to 26%, 41% and 34% of variance in the number of countries reached, maximum latitude and host plant families affected, respectively, but time since description had the strongest effect.Root-attacking species were reported in more countries, and on more host plant families than foliar-attacking species. Host generalist pathogens had thicker-walled resting structures (stress-tolerant oospores) and faster growth rates at their optima. Cold-tolerant species are reported in more countries and at higher latitudes, though more accurate interspecific empirical data are needed to confirm this finding. Policy implications. We evaluate the potential of an evolutionary trait-based framework to support horizon-scanning approaches for identifying pathogens with greater potential for global-scale impacts. Potential future threats from Phytophthora include Phytophthora x heterohybrida, P. lactucae, P. glovera, P. x incrassata, P. amnicola and P. aquimorbida, which are recently described, possibly under-reported species, with similar traits and/or phylogenetic proximity to other high-impact species. Priority traits to measure for emerging species may be thermal minima, oospore wall index and growth rate at optimum temperature. Trait-based horizon-scanning approaches would benefit from the development of international and cross-sectoral collaborations to deliver centralised databases incorporating pathogen distributions, traits and phylogeny.

Insights into evolving global populations of Phytophthora infestans via new complementary mtDNA haplotype markers and nuclear SSRs.

In many parts of the world the damaging potato late blight pathogen, Phytophthora infestans, is spread as a succession of clonal lineages. The discrimination of genetic diversity within such evolving populations provides insights into the processes generating novel lineages and the pathways and drivers of pathogen evolution and dissemination at local and global scales. This knowledge, in turn, helps optimise management practices. Here we combine two key methods for dissecting mitochondrial and nuclear diversity and resolve intra and inter-lineage diversity of over 100 P. infestans isolates representative of key clonal lineages found globally. A novel set of PCR primers that amplify five target regions are provided for mitochondrial DNA sequence analysis. These five loci increased the number of mtDNA haplotypes resolved from four with the PCR RFLP method to 37 (17, 6, 8 and 4 for Ia, Ib, IIa, and IIb haplotypes, respectively, plus 2 Herb-1 haplotypes). As with the PCR RFLP method, two main lineages, I and II were defined. Group I contained 25 mtDNA haplotypes that grouped broadly according to the Ia and Ib types and resolved several sub-clades amongst the global sample. Group II comprised two distinct clusters with four haplotypes corresponding to the RFLP type IIb and eight haplotypes resolved within type IIa. The 12-plex SSR assay revealed 90 multilocus genotypes providing accurate discrimination of dominant clonal lineages and other genetically diverse isolates. Some association of genetic diversity and geographic region of contemporary isolates was observed; US and Mexican isolates formed a loose grouping, distinct from isolates from Europe, South America and other regions. Diversity within clonal lineages was observed that varied according to the age of the clone. In combination, these fine-scale nuclear and maternally inherited mitochondrial markers enabled a greater level of discrimination among isolates than previously available and provided complementary perspectives on evolutionary questions relating to the diversity, phylogeography and the origins and spread of clonal lineages of P. infestans.

Use of genome sequence data in the design and testing of SSR markers for Phytophthora species.

BACKGROUND: Microsatellites or single sequence repeats (SSRs) are a powerful choice of marker in the study of Phytophthora population biology, epidemiology, ecology, genetics and evolution. A strategy was tested in which the publicly available unigene datasets extracted from genome sequences of P. infestans, P. sojae and P. ramorum were mined for candidate SSR markers that could be applied to a wide range of Phytophthora species. RESULTS: A first approach, aimed at the identification of polymorphic SSR loci common to many Phytophthora species, yielded 171 reliable sequences containing 211 SSRs. Microsatellites were identified from 16 target species representing the breadth of diversity across the genus. Repeat number ranged from 3 to 16 with most having seven repeats or less and four being the most commonly found. Trinucleotide repeats such as (AAG)n, (AGG)n and (AGC)n were the most common followed by pentanucleotide, tetranucleotide and dinucleotide repeats. A second approach was aimed at the identification of useful loci common to a restricted number of species more closely related to P. sojae (P. alni, P. cambivora, P. europaea and P. fragariae). This analysis yielded 10 trinucleotide and 2 tetranucleotide SSRs which were repeated 4, 5 or 6 times. CONCLUSION: Key studies on inter- and intra-specific variation of selected microsatellites remain. Despite the screening of conserved gene coding regions, the sequence diversity between species was high and the identification of useful SSR loci applicable to anything other than the most closely related pairs of Phytophthora species was challenging. That said, many novel SSR loci for species other than the three 'source species' (P. infestans, P. sojae and P. ramorum) are reported, offering great potential for the investigation of Phytophthora populations. In addition to the presence of microsatellites, many of the amplified regions may represent useful molecular marker regions for other studies as they are highly variable and easily amplifiable from different Phytophthora species.

Large sub-clonal variation in Phytophthora infestans from recent severe late blight epidemics in India.

The population structure of the Phytophthora infestans populations that caused the recent 2013-14 late blight epidemic in eastern India (EI) and northeastern India (NEI) was examined. The data provide new baseline information for populations of P. infestans in India. A migrant European 13_A2 genotype was responsible for the 2013-14 epidemic, replacing the existing populations. Mutations have generated substantial sub-clonal variation with 24 multi-locus genotypes (MLGs) found, of which 19 were unique variants not yet reported elsewhere globally. Samples from West Bengal were the most diverse and grouped alongside MLGs found in Europe, the UK and from neighbouring Bangladesh but were not linked directly to most samples from south India. The pathogen population was broadly more aggressive on potato than on tomato and resistant to the fungicide metalaxyl. Pathogen population diversity was higher in regions around the international borders with Bangladesh and Nepal. Overall, the multiple shared MLGs suggested genetic contributions from UK and Europe in addition to a sub-structure based on the geographical location within India. Our data indicate the need for improved phytosanitary procedures and continuous surveillance to prevent the further introduction of aggressive lineages of P. infestans into the country.