Frank N. Martin

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire

BackgroundPythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species.ResultsThe P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans.ConclusionsAccess to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.

Soilborne Plant Diseases Caused by Pythium spp.: Ecology, Epidemiology, and Prospects for Biological Control

Soilborne root diseases caused by plant pathogenic Pythium species cause serious losses in a number of agricultural production systems, which has led to a considerable effort devoted to the development of biological agents for disease control. In this article we review information on the ecology and biological control of these pathogens with the premise that a clear understanding of the ecology of the pathogen will assist in the development of efficacious biocontrol agents. The lifecycles of the pathogens and etiology of host infection also are reviewed, as are epidemiological concepts of inoculum-disease relationships and the influence of environmental factors on pathogen aggressiveness and host susceptibility. A number of fungal and bacterial biocontrol agents are discussed and parallels between their ecology and that of the target pathogens highlighted. The mechanisms by which these microbial agents suppress diseases caused by Pythium spp., such as interference with pathogen survival, disruption of the...

Phylogenetic relationships among Phytophthora species inferred from sequence analysis of mitochondrially encoded cytochrome oxidase I and II genes

The phylogenetic relationships of 51 isolates representing 27 species of Phytophthora were assessed by sequence alignment of 568 bp of the mitochondrially encoded cytochrome oxidase II gene. A total of 1299 bp of the cytochrome oxidase I gene also were examined for a subset of 13 species. The cox II gene trees constructed by a heuristic search, based on maximum parsimony for a bootstrap 50% majority-rule consensus tree, revealed 18 species grouping into seven clades and nine species unaffiliated with a specific clade. The phylogenetic relationships among species observed on cox II gene trees did not exhibit consistent similarities in groupings for morphology, pathogenicity, host range or temperature optima. The topology of cox I gene trees, constructed by a heuristic search based on maximum parsimony for a bootstrap 50% majority-rule consensus tree for 13 species of Phytophthora, revealed 10 species grouping into three clades and three species unaffiliated with a specific clade. The groupings in general agreed with what was observed in the cox II tree. Species relationships observed for the cox II gene tree were in agreement with those based on ITS regions, with several notable exceptions. Some of these differences were noted in species in which the same isolates were used for both ITS and cox II analysis, suggesting either a differential rate of evolutionary divergence for these two regions or incorrect assumptions about alignment of ITS sequences. Analysis of combined data sets of ITS and cox II sequences generated a tree that did not differ substantially from analysis of ITS data alone, however, the results of a partition homogeneity test suggest that combining data sets may not be valid.

Phylogenetic relationships among some Pythium species inferred from sequence analysis of the mitochondrially encoded cytochrome oxidase II gene

The phylogenetic relationships of 67 iso- lates representing 24 species of Pythium were assessed by sequence alignment of 684 bp of the mitochon- drially-encoded cytochrome oxidase II gene. Se- quence differences among species ranged 1.6-14.7% substitutions. The species grouped into three major clades that were, in a general sense, reflective of zoos- porangial or hyphal swelling morphology. Clade I contained species with globose to spherical zoospo- rangia or spherical hyphal swellings. Clade II was comprised of four species, only one of which pro- duced zoosporangia (P. ultimum var. sporangiiferum) with the remaining species producing only spherical hyphal swellings. Species with filamentous to lobulate zoosporangia were in clade III. Pythium oligandrum, a species that produces subglobose zoosporangia with interconnecting filamentous parts was intermediate between species with inflated to lobulate filamentous zoosporangia and species that produced spherical to globose zoosporangia (clades I and II). Two species that produced globose zoosporangia (P pulchrum and P rostratum) grouped together separately from the other clades, as did P. nunn. The evolutionary relationships among species obtained by analysis of cox II DNA sequence data corresponds well with the genomic location of this mitochondrially encoded gene as well as the location of the nuclear encoded 5S rRNA gene for a subset of species examined. Char- acteristics such as heterothallism, oogonial ornamen- tation, mycoparasitism and the presence of linear mi- tochondrial genomes were polyphyletic. The only species that contained isolates that did not group to- gether were P ultimum and P irregulare, possible rea- sons for this are discussed.

Identification and Detection of Phytophthora: Reviewing Our Progress, Identifying Our Needs

With the increased attention given to the genus Phytophthora in the last decade in response to the ecological and economic impact of several invasive species (such as P. ramorum, P. kernoviae, and P. alni), there has been a significant increase in the number of described species. In part, this is due to the extensive surveys in historically underexplored ecosystems (e.g., forest and stream ecosystems) undertaken to determine the spread of invasive species and the involvement of Phytophthora species in forest decline worldwide (e.g., oak decline). The past decade has seen an approximate doubling in the number of described species within the genus Phytophthora, and the number will likely continue to increase as more surveys are completed and greater attention is devoted to clarifying phylogenetic relationships and delineating boundaries in species complexes. The development of molecular resources, the availability of credible sequence databases to simplify identification of new species, and the sequencing of several genomes have provided a solid framework to gain a better understanding of the biology, diversity, and taxonomic relationships within the genus. This information is much needed considering the impact invasive or exotic Phytophthora species have had on natural ecosystems and the regulatory issues associated with their management. While this work is improving our ability to identify species based on phylogenetic grouping, it has also revealed that the genus has a much greater diversity than previously appreciated.

One stop shop: backbones trees for important phytopathogenic genera: I (2014)

Many fungi are pathogenic on plants and cause significant damage in agriculture and forestry. They are also part of the natural ecosystem and may play a role in regulating plant numbers/density. Morphological identification and analysis of plant pathogenic fungi, while important, is often hampered by the scarcity of discriminatory taxonomic characters and the endophytic or inconspicuous nature of these fungi. Molecular (DNA sequence) data for plant pathogenic fungi have emerged as key information for diagnostic and classification studies, although hampered in part by non-standard laboratory practices and analytical methods. To facilitate current and future research, this study provides phylogenetic synopses for 25 groups of plant pathogenic fungi in the Ascomycota, Basidiomycota, Mucormycotina (Fungi), and Oomycota, using recent molecular data, up-to-date names, and the latest taxonomic insights. Lineage-specific laboratory protocols together with advice on their application, as well as general observations, are also provided. We hope to maintain updated backbone trees of these fungal lineages over time and to publish them jointly as new data emerge. Researchers of plant pathogenic fungi not covered by the present study are invited to join this future effort. Bipolaris, Botryosphaeriaceae, Botryosphaeria, Botrytis, Choanephora, Colletotrichum, Curvularia, Diaporthe, Diplodia, Dothiorella, Fusarium, Gilbertella, Lasiodiplodia, Mucor, Neofusicoccum, Pestalotiopsis, Phyllosticta, Phytophthora, Puccinia, Pyrenophora, Pythium, Rhizopus, Stagonosporopsis, Ustilago and Verticillium are dealt with in this paper.

Molecular Detection of Phytophthora ramorum, the Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered from Diseased Plant Material.

ABSTRACT Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is Phy-tophthora ramorum, although species such as P. nemorosa and P. pseudo-syringae often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of the cox I and II genes for detection of Phytophthora spp. in general, and P. ramorum, P. nemorosa, and P. pseudosyringae in particular. The first-round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for polymerase chain reaction (PCR) amplification and the other specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures, the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species, the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first-round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa, or P. pseudo-syringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA, the limit of detection for P. ramorum using this marker system was approximately 2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure, the sensitivity of detection was reduced by 100- to 1,000-fold, depending on the plant species. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for P. ramorum, amplification with a previously published rDNA internal transcribed spacer species-specific primer pair. Results were compared and validated with three different brands of thermal cyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests that they will have utility for pathogen detection in other Phytophthora pathosystems.

Phylogenetic relationships of Phytophthora ramorum, P. nemorosa, and P. pseudosyringae, three species recovered from areas in California with sudden oak death

Sudden oak death has been an emerging disease problem in coastal California and has caused significant losses in forest ecosystems in some regions of the state. The causal agent of this disease has been described as Phytophthora ramorum with two other less aggressive species, P. nemorosa and P. pseudosyringae, recovered from some symptomatic plants. The phylogenetic relationship of these species with other members of the genus was examined by sequence alignment of 667 bp of the mitochondrially-encoded cytochrome oxidase II gene and the nuclear encoded rDNA internal transcribed spacer region. P. ramorum was most closely related to P. hibernalis and P. lateralis in trees from both regions, although the specific relationship among species differed depending on the tree. In the cox II tree these species were on a single clade with P. lateralis basal to a group containing P. ramorum and P. hibernalis. On the maximum parsimony ITS tree P. ramorum was most closely affiliated with P. lateralis and in the same clade as P. hibernalis, but with maximum likelihood analysis P. ramorum was basal to a grouping of P. hibernalis and P. lateralis. While bootstrap support was strong for the grouping of these species together, it was not for determining the relationship among them. In contrast to the cox II tree, the clade containing these three species grouped with P. cryptogea, P. drechsleri, P. erythroseptica, and P. syringae in the ITS tree. Since the same isolates of these species were used for both the cox II and ITS sequence analysis, this difference in species grouping suggests either a differential rate of evolutionary divergence for these two regions, incorrect assumptions about alignment of ITS sequences, or different evolutionary histories of the regions under study. Analysis of combined cox II and ITS data sets gave trees where the relationships among these species were the same as for the ITS tree alone, although the results of the partition homogeneity test (P=0.072) suggest caution should be used in interpretation of this data. All analyses supported a close relationship between P. ilicis, P. nemorosa and P. pseudosyringae, although the analysis did not clarify the evolutionary relationships among these three species. Interestingly, these three species had a unique 6 bp deletion in the cox II gene just before the termination codon. While there was some similarity in phylogenetic grouping of these species and morphological characteristics, this was not consistent across all comparisons in the genus. Data would suggest that P. ramorum, P. nemorosa and P. pseudosyringae are phylogenetically distinct new species and not the result of interspecific hybridization.

Development of an Assay for Rapid Detection and Quantification of Verticillium dahliae in Soil

ABSTRACT Verticillium dahliae is responsible for Verticillium wilt on a wide range of hosts, including strawberry, on which low soil inoculum densities can cause significant crop loss. Determination of inoculum density is currently done by soil plating but this can take 6 to 8 weeks to complete and delay the growers ability to make planting decisions. To provide a faster means for estimating pathogen populations in the soil, a multiplexed TaqMan real-time polymerase chain reaction (PCR) assay based on the ribosomal DNA (rDNA) intergenic spacer (IGS) was developed for V. dahliae. The assay was specific for V. dahliae and included an internal control for evaluation of inhibition due to the presence of PCR inhibitors in DNA extracted from soil samples. An excellent correlation was observed in regression analysis (R(2) = 0.96) between real-time PCR results and inoculum densities determined by soil plating in a range of field soils with pathogen densities as low as 1 to 2 microsclerotia/g of soil. Variation in copy number of the rDNA was also evaluated among isolates by SYBR Green real-time PCR amplification of the V. dahliae-specific amplicon compared with amplification of several single-copy genes and was estimated to range from ≈24 to 73 copies per haploid genome, which translated into possible differences in results among isolates of ≈1.8 cycle thresholds. Analysis of the variation in results of V. dahliae quantification among extractions of the same soil sample indicated that assaying four replicate DNA extractions for each field sample would provide accurate results. A TaqMan assay also was developed to help identify colonies of V. tricorpus on soil plates.

Standardizing the Nomenclature for Clonal Lineages of the Sudden Oak Death Pathogen, Phytophthora ramorum

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.