Emergence of the sudden oak death pathogen Phytophthora ramorum

The recently emerged plant pathogen Phytophthora ramorum is responsible for causing the sudden oak death epidemic. This review documents the emergence of P. ramorum based on evolutionary and population genetic analyses. Currently infection by P. ramorum occurs only in Europe and North America and three clonal lineages are distinguished: EU1, NA1 and NA2. Ancient divergence of these lineages supports a scenario in which P. ramorum originated from reproductively isolated populations and underwent at least four global migration events. This recent work sheds new light on mechanisms of emergence of exotic pathogens and provides crucial insights into migration pathways.     

Spatio-temporal analysis of an invasive plant pathogen (Phytophthora ramorum) in England and Wales

Phytophthora ramorum is a damaging invasive plant pathogen and was first discovered in the UK in 2002. Spatial point analyses were applied to the occurrence of this disease in England and Wales during the period of 2003–2006 in order to assess its spatio-temporal spread. Out of the 4301 garden centres and nurseries (GCN) surveyed, there were 164, 105, 123 and 41 sites with P. ramorum in 2003, 2004, 2005 and 2006, respectively. Spatial analysis of the observed point patterns of GCN outbreaks suggested that these sites were significantly clumped within a radius of ca 60 km in 2003, but not in later years. Further analyses were conducted to determine the relationship of GCN outbreak sites over two consecutive years and thus to infer possible disease spread over time. This analysis suggested that disease spread among GCN sites was most likely to have occurred within a distance of 60 km for 2003–2004, but not for the later years. There were 35, 63, 81 and 58 sites with P. ramorum in the semi-natural environment (SNE). Analyses were carried out to assess whether infected GCN sites could act as an inoculum source of infected SNE plants or vice versa. In all years, there was a significant spatial closeness among GCN and SNE outbreak sites within a distance of 1 km. But a significant relationship over a longer distance (within 60 km) was only observed between cases in 2003 and 2004. These analyses suggest that statutory actions taken so far appear to have reduced the extent of long-distance spread of P. ramorum among garden centres and nurseries, but not the disease spread at a shorter distance between GCN and SNE sites.     

Phytophthora ramorum: a pathogen with a remarkably wide host range causing sudden oak death on oaks and ramorum blight on woody ornamentals

Phytophthora ramorum is an oomycete plant pathogen classified in the kingdom Stramenopila. P. ramorum is the causal agent of sudden oak death on coast live oak and tanoak as well as ramorum blight on woody ornamental and forest understorey plants. It causes stem cankers on trees, and leaf blight or stem dieback on ornamentals and understorey forest species. This pathogen is managed in the USA and Europe by eradication where feasible, by containment elsewhere and by quarantine in many parts of the world. Genomic resources provide information on genes of interest to disease management and have improved tremendously since sequencing the genome in 2004. This review provides a current overview of the pathogenicity, population genetics, evolution and genomics of P. ramorum. Taxonomy: Phytophthora ramorum (Werres, De Cock & Man in't Veld): kingdom Stramenopila; phylum Oomycota; class Peronosporomycetidae; order Pythiales; family Pythiaceae; genus Phytophthora. Host range: The host range is very large and the list of known hosts continues to expand at the time of writing. Coast live oak and tanoak are ecologically, economically and culturally important forest hosts in the USA. Rhododendron, Viburnum, Pieris, Syringa and Camellia are key ornamental hosts on which P. ramorum has been found repeatedly, some of which have been involved in moving the pathogen via nursery shipments. Disease symptoms: P. ramorum causes two different diseases with differing symptoms: sudden oak death (bleeding lesions, stem cankers) on oaks and ramorum blight (twig dieback and/or foliar lesions) on tree and woody ornamental hosts. Useful websites: http://nature.berkeley.edu/comtf/ , http://rapra.csl.gov.uk/ , http://www.aphis.usda.gov/plant_health/plant_pest_info/pram/index.shtml , http://genome.jgi‐psf.org/Phyra1_1/Phyra1_1.home.html , http://pamgo.vbi.vt.edu/ , http://pmgn.vbi.vt.edu/ , http://vmd.vbi.vt.edu./ , http://web.science.oregonstate.edu/bpp/labs/grunwald/resources.htm , http://www.defra.gov.uk/planth/pramorum.htm , http://www.invasive.org/browse/subject.cfm?sub=4603 , http://www.forestry.gov.uk/forestry/WCAS‐4Z5JLL     

Genetic epidemiology of the Sudden Oak Death pathogen Phytophthora ramorum in California

A total of 669 isolates of Phytophthora ramorum, the pathogen responsible for Sudden Oak Death, were collected from 34 Californian forests and from the ornamental plant-trade. Seven microsatellite markers revealed 82 multilocus genotypes (MGs) of which only three were abundant (>10%). Iteratively collapsing based upon minimum Φ_ST, yielded five meta-samples and five singleton populations. Populations in the same meta-sample were geographically contiguous, with one exception, possibly explained by the trade of infected plants from the same source into different locations. Multidimensional scaling corroborated this clustering and identified nursery populations as genetically most distant from the most recent outbreaks. A minimum-spanning network illustrated the evolutionary relationships among MGs, with common genotypes at the centre and singletons at the extremities; consistent with colonization by a few common genotypes followed by local evolution. Coalescent migration analyses used the original data set and a data set in which local genotypes were collapsed into common ancestral genotypes. Both analyses suggested that meta-samples 1 (Santa Cruz County) and 3 (Sonoma and Marin Counties), act as sources for most of the other forests. The untransformed data set best explains the first phases of the invasion, when the role of novel genotypes may have been minimal, whereas the second analysis best explains migration patterns in later phases of the invasion, when prevalence of novel genotypes was likely to have become more significant. Using this combined approach, we discuss possible migration routes based on our analyses, and compare them to historical and field observations from several case studies.     

Population dynamics of the sudden oak death pathogen Phytophthora ramorum in Oregon from 2001 to 2004

Phytophthora ramorum (Oomycetes) is an emerging plant pathogen in forests in southwestern Oregon (Curry County). Moreover, since 2003 it has been repeatedly isolated from plants in Oregon nurseries. In this study, we analysed the genetic diversity of the P. ramorum population in Oregon from 2001 to 2004 by using microsatellites. A total of 323 isolates (272 from the infested forest; 51 from nurseries) were screened at 10 loci. The overall P. ramorum population in Oregon is characterized by low genetic diversity and has all the hallmarks of an introduced organism. All isolates within the A2 mating type belonged to the same clonal lineage and no recombinant genotypes were found. The forest population (24 genotypes) was dominated by a single multilocus genotype which persisted over years, indicating that eradication efforts in the forest have not completely eliminated inoculum sources. In contrast, genotypic evidence suggests that eradication was effective in nurseries. In 2003 and 2004, a total of 11 genotypes were found in the nurseries (one belonged to the European lineage of P. ramorum) but no genotype was recovered in both sampling years. Significant differentiation and low gene flow were detected between nursery and forest populations. Only two nursery genotypes were also found in the forest, and then at low frequency. Thus, the nursery infestation is not caused by the genotypes observed in Curry County, but likely resulted through introduction of novel genotypes from nurseries out-of-state. This highlights the continued importance of sanitation and quarantine in nurseries to prevent further introduction and spread of P. ramorum.     

Reconstruction of the Sudden Oak Death epidemic in California through microsatellite analysis of the pathogen Phytophthora ramorum

The genetic structure of the clonally reproducing Sudden Oak Death (SOD) pathogen in California was investigated using seven variable microsatellites. A total of 35 multilocus genotypes were identified among 292 samples representative of populations from 14 forest sites and of the nursery trade. amova indicated significant genetic variability both within (44.34%) and among populations (55.66%). Spatial autocorrelation analyses indicated that Moran's index of similarity reached a minimum of 0.1 at 350 m, increased to 0.4 at 1500 m and then decreased to zero at 10 km. These results suggest a bimodal pattern of spread, with medium range dispersal (1500–10 000 m) putatively attributed to the presence of strong winds. Lack of genetic structure was identified for three groups of populations. One group notably included the nurseries' population and two forest populations, both linked to early reports of the pathogen. A neighbour-joining analysis based on pairwise Φ_ST values indicated that the clade inclusive of the nurseries' populations is basal to all California populations. A network analysis identified three common genotypes as the likely founders of the California infestation and proposes a stepwise model for local evolution of novel genotypes. This was supported by the identification in the same locations of novel genotypes and of their 1- or 2-step parents. We hypothesize that the few undifferentiated population groups indicate historical human spread of the pathogen, while the general presence of genetically structured populations indicates that new infestations are currently generated by rare medium or long-range natural movement of the pathogen, followed by local generation of new genotypes.     

Ancient isolation and independent evolution of the three clonal lineages of the exotic sudden oak death pathogen Phytophthora ramorum

The genus Phytophthora includes some of the most destructive plant pathogens affecting agricultural and native ecosystems and is responsible for a number of recent emerging and re-emerging infectious diseases of plants. Sudden oak death, caused by the exotic pathogen P. ramorum , has caused extensive mortality of oaks and tanoaks in Northern California, and has brought economic losses to US and European nurseries as well due to its infection of common ornamental plants. In its known range, P. ramorum occurs as three distinct clonal lineages. We inferred the evolutionary history of P. ramorum from nuclear sequence data using coalescent-based approaches. We found that the three lineages have been diverging for at least 11% of their history, an evolutionarily significant amount of time estimated to be on the order of 165 000 to 500 000 years. There was also strong evidence for historical recombination between the lineages, indicating that the ancestors of the P. ramorum lineages were members of a sexually reproducing population. Due to this recombination, the ages of the lineages varied within and between loci, but coalescent analyses suggested that the European lineage may be older than the North American lineages. The divergence of the three clonal lineages of P. ramorum supports a scenario in which the three lineages originated from different geographic locations that were sufficiently isolated from each other to allow independent evolution prior to introduction to North America and Europe. It is thus probable that the emergence of P. ramorum in North America and Europe was the result of three independent migration events.     

Susceptibility to Phytophthora ramorum in a key infectious host: landscape variation in host genotype, host phenotype, and environmental factors

Sudden oak death is an emerging forest disease caused by the invasive pathogen Phytophthora ramorum. Genetic and environmental factors affecting susceptibility to P. ramorum in the key inoculum-producing host tree Umbellularia californica (bay laurel) were examined across a heterogeneous landscape in California, USA. Laboratory susceptibility trials were conducted on detached leaves and assessed field disease levels for 97 host trees from 12 225-m(2) plots. Genotype and phenotype characteristics were assessed for each tree. Effects of plot-level environmental conditions (understory microclimate, amount of solar radiation and topographic moisture potential) on disease expression were also evaluated. Susceptibility varied significantly among U. californica trees, with a fivefold difference in leaf lesion size. Lesion size was positively related to leaf area, but not to other phenotypic traits or to field disease level. Genetic diversity was structured at three spatial scales, but primarily among individuals within plots. Lesion size was significantly related to amplified fragment length polymorphism (AFLP) markers, but local environment explained most variation in field disease level. Thus, substantial genetic variation in susceptibility to P. ramorum occurs in its principal foliar host U. californica, but local environment mediates expression of susceptibility in nature.     

Identification of cell wall-associated proteins from Phytophthora ramorum

The oomycete genus Phytophthora comprises a large group of fungal-like plant pathogens. Two Phytophthora genomes recently have been sequenced; one of them is the genome of Phytophthora ramorum, the causal agent of sudden oak death. During plant infection, extracellular proteins, either soluble secreted proteins or proteins associated with the cell wall, play important roles in the interaction with host plants. Cell walls of P. ramorum contain 1 to 1.5% proteins, the remainder almost exclusively being accounted for by glucan polymers. Here, we present an inventory of cell-wall-associated proteins based on mass spectrometric sequence analysis of tryptic peptides obtained by proteolytic digestion of sodium dodecyl sulfate-treated mycelial cell walls. In total, 17 proteins were identified, all of which are authentic secretory proteins. Functional classification based on homology searches revealed six putative mucins or mucin-like proteins, five putative glycoside hydrolases, two transglutaminases, one annexin-like protein, the elicitin protein RAM5, one protein of unknown function, and one Kazal-type protease inhibitor. We propose that the cell wall proteins thus identified are important for pathogenicity.     

The plant pathogen Phytophthora andina emerged via hybridization of an unknown Phytophthora species and the Irish potato famine pathogen, P. infestans

Emerging plant pathogens have largely been a consequence of the movement of pathogens to new geographic regions. Another documented mechanism for the emergence of plant pathogens is hybridization between individuals of different species or subspecies, which may allow rapid evolution and adaptation to new hosts or environments. Hybrid plant pathogens have traditionally been difficult to detect or confirm, but the increasing ease of cloning and sequencing PCR products now makes the identification of species that consistently have genes or alleles with phylogenetically divergent origins relatively straightforward. We investigated the genetic origin of Phytophthora andina, an increasingly common pathogen of Andean crops Solanum betaceum, S. muricatum, S. quitoense, and several wild Solanum spp. It has been hypothesized that P. andina is a hybrid between the potato late blight pathogen P. infestans and another Phytophthora species. We tested this hypothesis by cloning four nuclear loci to obtain haplotypes and using these loci to infer the phylogenetic relationships of P. andina to P. infestans and other related species. Sequencing of cloned PCR products in every case revealed two distinct haplotypes for each locus in P. andina, such that each isolate had one allele derived from a P. infestans parent and a second divergent allele derived from an unknown species that is closely related but distinct from P. infestans, P. mirabilis, and P. ipomoeae. To the best of our knowledge, the unknown parent has not yet been collected. We also observed sequence polymorphism among P. andina isolates at three of the four loci, many of which segregate between previously described P. andina clonal lineages. These results provide strong support that P. andina emerged via hybridization between P. infestans and another unknown Phytophthora species also belonging to Phytophthora clade 1c.     

Extensive variation in nuclear mitochondrial DNA content between the genomes of Phytophthora sojae and Phytophthora ramorum

Fragments of mitochondrial DNA (mtDNA) transferred to the nuclear genome are called nuclear mitochondrial DNAs (NUMTs). We report here a comparison of NUMT content between genomes from two species of the same genus. Analysis of the genomes of Phytophthora sojae and P. ramorum revealed large differences in the NUMT content of the two genomes: 16.27 x 10(-3) and 2.28 x 10(-3)% of each genome, respectively. Substantial differences also exist between the two species in the sizes of the NUMTs found in each genome, with ranges of 20 to 405 bp for P. sojae and 19 to 137 bp for P. ramorum. Furthermore, in P. sojae, fragments from the mitochondrial genes rns, rnl, coxl, and nad (various subunits) are found most frequently, whereas P. ramorum NUMTs most often originate from the cox3, rpsl4, nad4, and nad5 genes. The large differences in the presumptive mtDNA insertions suggest that the insertions occurred subsequent to the divergence of the two species, and this is supported by sequence comparisons among the NUMTs and the mtDNA sequences of the two species. P. sojae mtDNA sequences inserted in the nuclear genome appear to have been altered as a result of insertions, deletions, inversions, and translocations and provide insights into active mechanisms of sequence divergence in this plant pathogen. No clear examples were found of NUMTs forming functional nuclear genes or of NUMTs inserted into exons or introns of any nuclear gene.     

Aberrant genome size and instability of Phytophthora ramorum oospore progenies

The functionality of the sexual cycle in the heterothallic pathogen Phytophthora ramorum, causal agent of Sudden Oak Death, has recently been demonstrated. Sexual reproduction could create genotypic variation and increase the pathogen's ability to adapt to other host plants or changing environments. Genetic characterization using co-dominant microsatellite markers and flow cytometry of single-oospore progeny of crosses between a European A1 isolate and North American or European A2 isolates revealed a considerable number of non-Mendelian inheritance events. This includes inheritance of more than two alleles at a locus and non-inheritance of alleles from one parent at another locus. The progenies were mitotically unstable: zoospore and hyphal tip derivatives of the progenies showed genotypic rearrangements and phenotypic variation. Flow cytometry confirmed variation and instability in DNA content of the single-oospore progenies. This indicates that single-oospore progenies not only display aberrant genomic and phenotypic variation due to meiotic irregularities, but also extra variation as a result of post-meiotic genomic rearrangements.     

Challenges in predicting invasive reservoir hosts of emerging pathogens: mapping Rhododendron ponticum as a foliar host for Phytophthora ramorum and Phytophthora kernoviae in the UK

Invasive species can increase the susceptibility of ecosystems to disease by acting as reservoir hosts for pathogens. Invasive hosts are often sparsely recorded and not in equilibrium, so predicting their spatial distributions and overlap with other hosts is problematic. We applied newly developed methods for modelling the distribution of invasive species to the invasive shrub Rhododendron ponticum—a foliar reservoir host for the Phytophthora oomycete plant pathogens, P. ramorum and P. kernoviae, that threaten woodland and heathland habitat in Scotland. We compiled eleven datasets of biological records for R. ponticum (1,691 points, 8,455 polygons) and developed Maximum Entropy (MaxEnt) models incorporating landscape, soil and climate predictors. Our models produced accurate predictions of current suitable R. ponticum habitat (training AUC = 0.838; test AUC = 0.838) that corresponded well with population performance (areal cover). Continuous broad-leaved woodland cover, low elevation (<400 m a.s.l.) and intermediate levels of soil moisture (or Enhanced Vegetation Index) favoured presence of R. ponticum. The high coincidence of suitable habitat with both core native woodlands (54 % of woodlands) and plantations of another sporulation host, Larix kaempferi (64 % of plantations) suggests a high potential for spread of Phytophthora infection to woodland mediated by R. ponticum. Incorporating non-equilibrium modelling methods did not improve habitat suitability predictions of this invasive host, possibly because, as a long-standing invader, R. ponticum has filled more of its available habitat at this national scale than previously suspected.     

Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host.

Infection by pathogenic fungi involves breaching the outer layer of the host by either mechanical or enzymatic means. Subtilisin-like proteinases are considered to be important in the infection process of entomopathogenic, nematophagous, and mycoparasitic fungi. Little is known regarding the expression of such proteinases by plant pathogenic fungi. Magnaporthe poae, a fungal pathogen of Kentucky bluegrass, expressed a subtilisin-like proteinase, proteinase Mp1, in the infected roots. Antibody was produced against the purified enzyme. From immunoblot analysis, expression of the proteinase in infected roots correlated with increasing severity of disease symptoms. Sequence analysis of a genomic clone indicated proteinase Mp1 was homologous to other fungal subtilisin-like proteinases. DNA gel blot analysis indicated proteinase Mp1 was encoded by a small gene family.