Phylogeography, origin and population structure of the self-fertile emerging plant pathogen Phytophthora pseudosyringae.

Phytophthora pseudosyringae is a self-fertile pathogen of woody plants, particularly associated with tree species from the genera Fagus, Notholithocarpus, Nothofagus and Quercus, which is found across Europe and in parts of North America and Chile. It can behave as a soil pathogen infecting roots and the stem collar region, as well as an aerial pathogen infecting leaves, twigs and stem barks, causing particular damage in the United Kingdom and western North America. The population structure, migration and potential outcrossing of a worldwide collection of isolates were investigated using genotyping-by-sequencing. Coalescent-based migration analysis revealed that the North American population originated from Europe. Historical gene flow has occurred between the continents in both directions to some extent, yet contemporary migration is overwhelmingly from Europe to North America. Two broad population clusters dominate the global population of the pathogen, with a subgroup derived from one of the main clusters found only in western North America. Index of association and network analyses indicate an influential level of outcrossing has occurred in this preferentially inbreeding, homothallic oomycete. Outcrossing between the two main population clusters has created distinct subgroups of admixed individuals that are, however, less common than the main population clusters. Differences in life history traits between the two main population clusters should be further investigated together with virulence and host range tests to evaluate the risk each population poses to natural environments worldwide.

Can the Seed Trade Provide a Potential Pathway for the Global Distribution of Foliar Pathogens? An Investigation into the Use of Heat Treatments to Reduce Risk of Dothistroma septosporum Transmission via Seed Stock.

The international plant trade results in the accidental movement of invasive pests and pathogens, and has contributed significantly to recent range expansion of pathogens including Dothistroma septosporum. Seeds are usually thought to present a lower biosecurity risk than plants, but the importation of Pinus contorta seeds from North America to Britain in the mid-1900s, and similarities between British and Canadian D. septosporum populations suggests seeds could be a pathway. Dothistroma septosporum has not been isolated from seeds, but inadequately cleaned seed material could contain infected needle fragments. This case study investigated whether cone kilning, and wet and dry heat treatments could reduce D. septosporum transmission without damaging seed viability. Pinus needles infected with D. septosporum were incubated alongside cones undergoing three commercial seed extraction processes. Additional needles were exposed to temperatures ranging from 10 to 67 °C dry heat for up to 48 h, or incubated in water heated to between 20 and 60 °C for up to one hour. Pinus sylvestris seeds were exposed to 60 and 65 dry heat °C for 48 h, and further seed samples incubated in water heated to between 20 and 60 °C for up to one hour. Dothistroma septosporum survived the three kilning processes and while seeds were not damaged by dry heat exceeding 63.5 °C, at this temperature no D. septosporum survived. Wet heat treatments resulted in less than 10% pathogen survival following incubation at 40 °C, while at this temperature the seeds suffered no significant impacts, even when submerged for one hour. Thus, commercial seed kilning could allow D. septosporum transmission, but elevated wet and dry heat treatments could be applied to seed stock to minimise pathogen risk without significantly damaging seed viability.

Population structure and diversity of the needle pathogen Dothistroma pini suggests human-mediated movement in Europe.

Dothistroma needle blight (DNB) is an important disease of Pinus species that can be caused by one of two distinct but closely related pathogens; Dothistroma septosporum and Dothistroma pini. Dothistroma septosporum has a wide geographic distribution and is relatively well-known. In contrast, D. pini is known only from the United States and Europe, and there is a distinct lack of knowledge regarding its population structure and genetic diversity. The recent development of 16 microsatellite markers for D. pini provided an opportunity to investigate the diversity, structure, and mode of reproduction for populations collected over a period of 12 years, on eight different hosts in Europe. In total, 345 isolates from Belgium, the Czech Republic, France, Hungary, Romania, Western Russia, Serbia, Slovakia, Slovenia, Spain, Switzerland, and Ukraine were screened using microsatellite and species-specific mating type markers. A total of 109 unique multilocus haplotypes were identified and structure analyses suggested that the populations are influenced by location rather than host species. Populations from France and Spain displayed the highest levels of genetic diversity followed by the population in Ukraine. Both mating types were detected in most countries, with the exception of Hungary, Russia and Slovenia. Evidence for sexual recombination was supported only in the population from Spain. The observed population structure and several shared haplotypes between non-bordering countries provides good evidence that the movement of D. pini in Europe has been strongly influenced by human activity in Europe.

Phylogeography and population structure of the global, wide host-range hybrid pathogen Phytophthora × cambivora.

Invasive, exotic plant pathogens pose a major threat to native and agricultural ecosystems. Phytophthora × cambivora is an invasive, destructive pathogen of forest and fruit trees causing severe damage worldwide to chestnuts (Castanea), apricots, peaches, plums, almonds and cherries (Prunus), apples (Malus), oaks (Quercus), and beech (Fagus). It was one of the first damaging invasive Phytophthora species to be introduced to Europe and North America, although its origin is unknown. We determined its population genetic history in Europe, North and South America, Australia and East Asia (mainly Japan) using genotyping-by-sequencing. Populations in Europe and Australia appear clonal, those in North America are highly clonal yet show some degree of sexual reproduction, and those in East Asia are partially sexual. Two clonal lineages, each of opposite mating type, and a hybrid lineage derived from these two lineages, dominated the populations in Europe and were predominantly found on fagaceous forest hosts (Castanea, Quercus, Fagus). Isolates from fruit trees (Prunus and Malus) belonged to a separate lineage found in Australia, North America, Europe and East Asia, indicating the disease on fruit trees could be caused by a distinct lineage of P. × cambivora, which may potentially be a separate sister species and has likely been moved with live plants. The highest genetic diversity was found in Japan, suggesting that East Asia is the centre of origin of the pathogen. Further surveys in unsampled, temperate regions of East Asia are needed to more precisely identify the location and range of the centre of diversity.

Dermacentor reticulatus - a tick on its way from glacial refugia to a panmictic Eurasian population.

The ornate dog tick (Dermacentor reticulatus) shows a recently expanding geographic distribution. Knowledge on its intraspecific variability, population structure, rate of genetic diversity and divergence, including its evolution and geographic distribution, is crucial to understand its dispersal capacity. All such information would help to evaluate the potential risk of future spread of associated pathogens of medical and veterinary concern. A set of 865 D. reticulatus ticks was collected from 65 localities across 21 countries, from Portugal in the west to Kazakhstan and southern Russia in the east. Cluster analyses of 16 microsatellite loci were combined with nuclear (ITS2, 18S) and mitochondrial (12S, 16S, COI) sequence data to uncover the ticks' population structures and geographical patterns. Approximate Bayesian computation was applied to model evolutionary relationships among the found clusters. Low variability and a weak phylogenetic signal showing an east-west cline were detected both for mitochondrial and nuclear sequence markers. Microsatellite analyses revealed three genetic clusters, where the eastern and western cluster gradient was supplemented by a third, northern cluster. Alternative scenarios could explain such a tripartite population structure by independent formation of clusters in separate refugia, limited gene flow connected with isolation by distance causing a "bipolar pattern", and the northern cluster deriving from admixture between the eastern and western populations. The best supported demographic scenario of this tick species indicates that the northern cluster derived from admixture between the eastern and western populations 441 (median) to 224 (mode) generations ago, suggesting a possible link with the end of the Little Ice Age in Europe.

Diversity, migration routes, and worldwide population genetic structure of Lecanosticta acicola, the causal agent of brown spot needle blight.

Lecanosticta acicola is a pine needle pathogen causing brown spot needle blight that results in premature needle shedding with considerable damage described in North America, Europe, and Asia. Microsatellite and mating type markers were used to study the population genetics, migration history, and reproduction mode of the pathogen, based on a collection of 650 isolates from 27 countries and 26 hosts across the range of L. acicola. The presence of L. acicola in Georgia was confirmed in this study. Migration analyses indicate there have been several introduction events from North America into Europe. However, some of the source populations still appear to remain unknown. The populations in Croatia and western Asia appear to originate from genetically similar populations in North America. Intercontinental movement of the pathogen was reflected in an identical haplotype occurring on two continents, in North America (Canada) and Europe (Germany). Several shared haplotypes between European populations further suggests more local pathogen movement between countries. Moreover, migration analyses indicate that the populations in northern Europe originate from more established populations in central Europe. Overall, the highest genetic diversity was observed in south-eastern USA. In Europe, the highest diversity was observed in France, where the presence of both known pathogen lineages was recorded. Less than half of the observed populations contained mating types in equal proportions. Although there is evidence of some sexual reproduction taking place, the pathogen spreads predominantly asexually and through anthropogenic activity.

Highly Clonal Structure and Abundance of One Haplotype Characterise the Diplodia sapinea Populations in Europe and Western Asia.

Diplodia sapinea is a cosmopolitan endophyte and opportunistic pathogen having occurred on several conifer species in Europe for at least 200 years. In Europe, disease outbreaks have increased on several Pinus spp. in the last few decades. In this study, the genetic structure of the European and western Asian D. sapinea population were investigated using 13 microsatellite markers. In total, 425 isolates from 15 countries were analysed. A high clonal fraction and low genetic distance between most subpopulations was found. One single haplotype dominates the European population, being represented by 45.3% of all isolates and found in nearly all investigated countries. Three genetically distinct subpopulations were found: Central/North European, Italian and Georgian. The recently detected subpopulations of D. sapinea in northern Europe (Estonia) share several haplotypes with the German subpopulation. The northern European subpopulations (Latvia, Estonia and Finland) show relatively high genetic diversity compared to those in central Europe suggesting either that the fungus has existed in the North in an asymptomatic/endophytic mode for a long time or that it has spread recently by multiple introductions. Considerable genetic diversity was found even among isolates of a single tree as 16 isolates from a single tree resulted in lower clonal fraction index than most subpopulations in Europe, which might reflect cryptic sexual proliferation. According to currently published allelic patterns, D. sapinea most likely originates from North America or from some unsampled population in Asia or central America. In order to enable the detection of endophytic or latent infections of planting stock by D. sapinea, new species-specific PCR primers (DiSapi-F and Diplo-R) were designed. During the search for Diplodia isolates across the world for species specific primer development, we identified D. africana in California, USA, and in the Canary Islands, which are the first records of this species in North America and in Spain.

Worldwide Genetic Structure Elucidates the Eurasian Origin and Invasion Pathways of Dothistroma septosporum, Causal Agent of Dothistroma Needle Blight.

Dothistroma septosporum, the primary causal agent of Dothistroma needle blight, is one of the most significant foliar pathogens of pine worldwide. Its wide host and environmental ranges have led to its global success as a pathogen and severe economic damage to pine forests in many regions. This comprehensive global population study elucidated the historical migration pathways of the pathogen to reveal the Eurasian origin of the fungus. When over 3800 isolates were examined, three major population clusters were revealed: North America, Western Europe, and Eastern Europe, with distinct subclusters in the highly diverse Eastern European cluster. Modeling of historical scenarios using approximate Bayesian computation revealed the North American cluster was derived from an ancestral population in Eurasia. The Northeastern European subcluster was shown to be ancestral to all other European clusters and subclusters. The Turkish subcluster diverged first, followed by the Central European subcluster, then the Western European cluster, which has subsequently spread to much of the Southern Hemisphere. All clusters and subclusters contained both mating-types of the fungus, indicating the potential for sexual reproduction, although asexual reproduction remained the primary mode of reproduction. The study strongly suggests the native range of D. septosporum to be in Eastern Europe (i.e., the Baltic and Western Russia) and Western Asia.

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum.

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

Looking for relationships between the populations of Dothistroma septosporum in northern Europe and Asia.

Dothistroma septosporum, a notorious pine needle pathogen with an unknown historical geographic origin and poorly known distribution pathways, is nowadays found almost in all areas inhabited by pines (Pinus spp.). The main aim of this study was to determine the relationship between North European and East Asian populations. In total, 238 Eurasian D. septosporum isolates from 11 countries, including 211 isolates from northern Europe, 16 isolates from Russian Far East and 11 isolates from Bhutan were analysed using 11 species-specific microsatellite and mating type markers. The most diverse populations were found in northern Europe, including the Baltic countries, Finland and European Russia. Notably, D. septosporum has not caused heavy damage to P. sylvestris in northern Europe, which may suggest a long co-existence of the host and the pathogen. No indication was obtained that the Russian Far East or Bhutan could be the indigenous area of D. septosporum, as the genetic diversity of the fungus there was low and evidence suggests gene flow from northern Europe to Russian Far East. On the western coast of Norway, a unique genetic pattern was observed, which differed from haplotypes dominating other Fennoscandian populations. As an agent of dothistroma needle blight, only D. septosporum was documented in northern Europe and Asia, while D. pini was found in Ukraine and Serbia.

Multilocus sequence typing provides insights into the population structure and evolutionary potential of Brenneria goodwinii, associated with acute oak decline.

Brenneria goodwinii is one of the most frequently isolated Gram-negative bacteria from native oak species, Quercus robur and Q. petraea, affected by acute oak decline (AOD) in the UK. We investigated the population biology of this bacterial species using a multilocus sequence analysis to determine the population structure and evolutionary potential. Seven partial housekeeping genes were used in the analyses. Amongst 44 bacterial strains from seven different locations, we identified 22 unique sequence types [STs]; only one ST was found at two separate locations. Phylogenetic and cluster-based analyses suggested that B. goodwinii STs form two main distinct groups; however, no geographical pattern of their distribution could be observed. Clonality and recombination tests demonstrated that the studied population is primarily clonal, however both mutation and recombination processes play a role in shaping the genetic structure and evolution of the population. Our study suggests that the B. goodwinii population on oak in the UK has an endemic form, with background recombination appearing to generate new alleles more frequently than mutation, despite the introduction of nucleotide substitutions being approximately twice less likely than mutation. The newly emerged STs subsequently undergo clonal expansion to become dominant genotypes within their specific geographical locations and even within the individual host oak trees.

Persistence of Dothistroma septosporum on Abscised Pine Needles and its Implications for Disease Management.

Dothistroma septosporum is a serious foliar pathogen of pines in Britain. To determine the contribution of spores produced on abscised needles to the D. septosporum inoculum load in a forest environment, survival of the fungus was investigated in commercially grown plantations of Corsican pine (Pinus nigra subsp. laricio) in southern England. The fungus persisted for longer in abscised needles retained in the canopy compared with needles on the litter layer of the forest floor. High relative humidity had a significant negative effect on persistence in the canopy needles, highlighting the effect of moisture on rates of microbial decay. The median lethal time 50 (LT50) of infective propagules from needles in the litter layer was 20 to 28 days, whereas for needles suspended in the canopy it was 34 to 48 days. Microsatellite markers revealed that the population of D. septosporum deemed persistent (i.e., recovered from abscised needles after exposure for 28 to 32 weeks) was similar to the general population of D. septosporum in the forest stand (i.e., isolates recovered from attached needles and after exposure for only 4 weeks). Overall, abscised needles with acervuli of D. septosporum contribute only moderately and for a limited time to inoculum levels of the pathogen in an infected forest stand.