Global invasion history of the emerging plant pathogen Phytophthora multivora.

BACKGROUND: global trade in living plants and plant material has significantly increased the geographic distribution of many plant pathogens. As a consequence, several pathogens have been first found and described in their introduced range where they may cause severe damage on naïve host species. Knowing the center of origin and the pathways of spread of a pathogen is of importance for several reasons, including identifying natural enemies and reducing further spread. Several Phytophthora species are well-known invasive pathogens of natural ecosystems, including Phytophthora multivora. Following the description of P. multivora from dying native vegetation in Australia in 2009, the species was subsequently found to be common in South Africa where it does not cause any remarkable disease. There are now reports of P. multivora from many other countries worldwide, but not as a commonly encountered species in natural environments. RESULTS: a global collection of 335 isolates from North America, Europe, Africa, Australia, the Canary Islands, and New Zealand was used to unravel the worldwide invasion history of P. multivora, using 10 microsatellite markers for all isolates and sequence data from five loci from 94 representative isolates. Our population genetic analysis revealed an extremely low heterozygosity, significant non-random association of loci and substantial genotypic diversity suggesting the spread of P. multivora readily by both asexual and sexual propagules. The P. multivora populations in South Africa, Australia, and New Zealand show the most complex genetic structure, are well established and evolutionary older than those in Europe, North America and the Canary Islands. CONCLUSIONS: according to the conducted analyses, the world invasion of P. multivora most likely commenced from South Africa, which can be considered the center of origin of the species. The pathogen was then introduced to Australia, which acted as bridgehead population for Europe and North America. Our study highlights a complex global invasion pattern of P. multivora, including both direct introductions from the native population and secondary spread/introductions from bridgehead populations.

Population genetic analysis of a parasitic mycovirus to infer the invasion history of its fungal host.

Hymenoscyphus fraxineus mitovirus 1 (HfMV1) occurs in the fungus Hymenoscyphus fraxineus, an introduced plant pathogen responsible for the devastating ash dieback epidemic in Europe. Here, we explored the prevalence and genetic structure of HfMV1 to elucidate the invasion history of both the virus and the fungal host. A total of 1298 H. fraxineus isolates (181 from Japan and 1117 from Europe) were screened for the presence of this RNA virus and 301 virus-positive isolates subjected to partial sequence analysis of the viral RNA polymerase gene. Our results indicate a high mean prevalence (78.7%) of HfMV1 across European H. fraxineus isolates, which is supported by the observed high transmission rate (average 83.8%) of the mitovirus into sexual spores of its host. In accordance with an expected founder effect in the introduced population in Europe, only 1.1% of the Japanese isolates were tested virus positive. In Europe, HfMV1 shows low nucleotide diversity but a high number of haplotypes, which seem to be subject to strong purifying selection. Phylogenetic and clustering analysis detected two genetically distinct HfMV1 groups, both present throughout Europe. This pattern supports the hypothesis that only two (mitovirus-carrying) H. fraxineus individuals were introduced into Europe as previously suggested from the bi-allelic nature of the fungus. Moreover, our data points to reciprocal mating events between the two introduced individuals, which presumably initiated the ash dieback epidemic in Europe.

Development of new polymorphic microsatellite markers for three closely related plant-pathogenic Phytophthora species using 454-pyrosequencing and their potential applications.

Phytophthora spp. (oomycetes) are causal agents of devastating diseases on a high number of crops, ornamentals, and native plants worldwide. Neutral molecular markers are increasingly being used to investigate the genetic population structure and possible pathways of spread of different plant pathogens, including Phytophthora spp. In this study, polymorphic microsatellite markers were developed for three species of the former Phytophthora citricola species complex-namely, P. multivora, P. plurivora, and P. pini (P. citricola I)-using the 454-pyrosequencing technique. In total, 35 polymorphic microsatellite loci were found and further characterized: 11 for P. plurivora, 16 for P. multivora, and 8 for P. pini. Microsatellites with dinucleotide motifs repeated 6 to 10 times were the most common for all three species. On average, 65 alleles per species and 5.3 alleles per locus were detected. Most loci were characterized by a low observed heterozygosity, which might be due to the homothallic mating system of the three Phytophthora spp. targeted. Cross amplification of the newly developed markers was tested on 17 Phytophthora spp. belonging to five different internal transcribed spacer clades. Transferability success was generally low and decreased with increasing genetic distance from the species to the three target species. A set of four loci was selected to easily discriminate P. plurivora, P. multivora, and P. pini on the basis of presence or absence of a polymerase chain reaction amplicon on an agarose gel.

Temperature effects on parasite prevalence in a natural hybrid complex.

Both host susceptibility and parasite infectivity commonly have a genetic basis, and can therefore be shaped by coevolution. However, these traits are often sensitive to environmental variation, resulting in genotype-by-environment interactions. We tested the influence of temperature on host-parasite genetic specificity in the Daphnia longispina hybrid complex, exposed to the protozoan parasite Caullerya mesnili. Infection rates were higher at low temperature. Furthermore, significant differences between host clones, but not between host taxa, and a host genotype-by-temperature interaction were observed.

Detection of a Conspecific Mycovirus in Two Closely Related Native and Introduced Fungal Hosts and Evidence for Interspecific Virus Transmission.

Hymenoscyphus albidus is a native fungus in Europe where it behaves as a harmless decomposer of leaves of common ash. Its close relative Hymenoscyphus fraxineus was introduced into Europe from Asia and currently threatens ash (Fraxinus sp.) stands all across the continent causing ash dieback. H. fraxineus isolates from Europe were previously shown to harbor a mycovirus named Hymenoscyphus fraxineus Mitovirus 1 (HfMV1). In the present study, we describe a conspecific mycovirus that we detected in H. albidus. HfMV1 was consistently identified in H. albidus isolates (mean prevalence: 49.3%) which were collected in the sampling areas before the arrival of ash dieback. HfMV1 strains in both fungal hosts contain a single ORF of identical length (717 AA) for which a mean pairwise identity of 94.5% was revealed. The occurrence of a conspecific mitovirus in H. albidus and H. fraxineus is most likely the result of parallel virus evolution in the two fungal hosts. HfMV1 sequences from H. albidus showed a higher nucleotide diversity and a higher number of mutations compared to those from H. fraxineus, probably due to a bottleneck caused by the introduction of H. fraxineus in Europe. Our data also points to multiple interspecific virus transfers from H. albidus to H. fraxineus, which could have contributed to the intraspecific virus diversity found in H. fraxineus.

Detection and genetic characterisation of a novel mycovirus in Hymenoscyphus fraxineus, the causal agent of ash dieback.

The ascomycete fungus Hymenoscyphus fraxineus (synonym: Hymenoscyphuspseudoalbidus, basionym: Chalara fraxinea) is a new invasive pathogen causing severe dieback of ash trees (Fraxinus spp.) in Europe. The disease was first recorded in the 1990s in Eastern Poland and Lithuania and has meanwhile spread across the continent and to Great Britain. Mycoviruses are commonly found in all major groups of plant pathogenic fungi. Some of these viruses can cause debilitating disease or reduce virulence in their fungal host and thereby can act as biological control agents (e.g. hypoviruses in the chestnut blight fungus). The main aim of our study is to identify mycoviruses in the ash dieback pathogen and to describe their phylogenetic position. Using RNAseq we could identify a viral sequence in two fungal isolates. Amino acid sequence comparison suggests that the detected mycovirus is a putative new member of the genus Mitovirus (family Narnaviridae), and we suggest naming it Hymenoscyphus fraxineus mitovirus 1 (HfMV1). The novel mitovirus is highly variable amongst European isolates and seems to diverge rapidly. This is very promising for the future search of a strain adequate for biological control measures.

Effects of juvenile host density and food availability on adult immune response, parasite resistance and virulence in a Daphnia-parasite system.

Host density can increase infection rates and reduce host fitness as increasing population density enhances the risk of becoming infected either through increased encounter rate or because host condition may decline. Conceivably, potential hosts could take high host density as a cue to up-regulate their defence systems. However, as host density usually covaries with food availability, it is difficult to examine the importance of host density in isolation. Thus, we performed two full-factorial experiments that varied juvenile densities of Daphnia magna (a freshwater crustacean) and food availability independently. We also included a simulated high-density treatment, where juvenile experimental animals were kept in filtered media that previously maintained Daphnia at high-density. Upon reaching adulthood, we exposed the Daphnia to their sterilizing bacterial parasite, Pasteuria ramosa, and examined how the juvenile treatments influenced the likelihood and severity of infection (Experiment I) and host immune investment (Experiment II). Neither juvenile density nor food treatments affected the likelihood of infection; however, well-fed hosts that were well-fed as juveniles produced more offspring prior to sterilization than their less well-fed counterparts. By contrast, parasite growth was independent of host juvenile resources or host density. Parasite-exposed hosts had a greater number of circulating haemocytes than controls (i.e., there was a cellular immune response), but the magnitude of immune response was not mediated by food availability or host density. These results suggest that density dependent effects on disease arise primarily through correlated changes in food availability: low food could limit parasitism and potentially curtail epidemics by reducing both the host's and parasite's reproduction as both depend on the same food.

Population history and pathways of spread of the plant pathogen Phytophthora plurivora.

Human activity has been shown to considerably affect the spread of dangerous pests and pathogens worldwide. Therefore, strict regulations of international trade exist for particularly harmful pathogenic organisms. Phytophthora plurivora, which is not subject to regulations, is a plant pathogen frequently found on a broad range of host species, both in natural and artificial environments. It is supposed to be native to Europe while resident populations are also present in the US. We characterized a hierarchical sample of isolates from Europe and the US and conducted coalescent-, migration, and population genetic analysis of sequence and microsatellite data, to determine the pathways of spread and the demographic history of this pathogen. We found P. plurivora populations to be moderately diverse but not geographically structured. High levels of gene flow were observed within Europe and unidirectional from Europe to the US. Coalescent analyses revealed a signal of a recent expansion of the global P. plurivora population. Our study shows that P. plurivora has most likely been spread around the world by nursery trade of diseased plant material. In particular, P. plurivora was introduced into the US from Europe. International trade has allowed the pathogen to colonize new environments and/or hosts, resulting in population growth.