Phytophthora foliorum sp. nov., a new species causing leaf blight of azalea

a b s t r a c t A previously unknown Phytophthora was recovered more than 60 times from evergreen hybrid azalea leaves collected during surveys for the sudden oak death pathogen Phytophthora ramorum in California and Tennessee. The novel Phytophthora was discovered when genomic DNA from this species cross-reacted with the ITS-based diagnostic PCR primers used to screen plants for the presence of P. ramorum. This species had caducous, semi-papillate sporangia, was homothallic with both paragynous and amphigynous antheridia, and was pathogenic on both wounded and intact azalea leaves. Nuclear and mitochondrial sequence data indicate that this species is related to, but distinct from, P. ramorum. AFLP analysis indicates that the isolates of this species have limited genotypic diversity and share no markers with P. ramorum. This paper presents the formal description of P. foliorum as a new species and underscores the need for caution when relying solely on DNA-based diagnostic tools

Multiple origins of downy mildews and mito-nuclear discordance within the paraphyletic genus Phytophthora.

Phylogenetic relationships between thirteen species of downy mildew and 103 species of Phytophthora (plant-pathogenic oomycetes) were investigated with two nuclear and four mitochondrial loci, using several likelihood-based approaches. Three Phytophthora taxa and all downy mildew taxa were excluded from the previously recognized subgeneric clades of Phytophthora, though all were strongly supported within the paraphyletic genus. Downy mildews appear to be polyphyletic, with graminicolous downy mildews (GDM), brassicolous downy mildews (BDM) and downy mildews with colored conidia (DMCC) forming a clade with the previously unplaced Phytophthora taxon totara; downy mildews with pyriform haustoria (DMPH) were placed in their own clade with affinities to the obligate biotrophic P. cyperi. Results suggest the recognition of four additional clades within Phytophthora, but few relationships between clades could be resolved. Trees containing all twenty extant downy mildew genera were produced by adding partial coverage of seventeen additional downy mildew taxa; these trees supported the monophyly of the BDMs, DMCCs and DMPHs but suggested that the GDMs are paraphyletic in respect to the BDMs or polyphyletic. Incongruence between nuclear-only and mitochondrial-only trees suggests introgression may have occurred between several clades, particularly those containing biotrophs, questioning whether obligate biotrophic parasitism and other traits with polyphyletic distributions arose independently or were horizontally transferred. Phylogenetic approaches may be limited in their ability to resolve some of the complex relationships between the "subgeneric" clades of Phytophthora, which include twenty downy mildew genera and hundreds of species

Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species

Recombinase polymerase amplification (RPA) assays are valuable molecular diagnostic tools that can detect and identify plant pathogens in the field without time-consuming DNA extractions. Historically, RPA assay reagents were commercially available as a lyophilized pellet in microfuge strip tubes, but have become available in liquid form more recently—both require the addition of primers and probes prior to use, which can be challenging to handle in a field setting. Lyophilization of primers and probes, along with RPA reagents, contained within a single tube limits the risk of contamination, eliminates the need for refrigeration, as the lyophilized reagents are stable at ambient temperatures, and simplifies field use of the assays. This study investigates the potential effect of preformulation on assay performance using a previously validated Phytophthora genus-specific RPA assay, lyophilized with primers and probes included with the RPA reagents. The preformulated lyophilized Phytophthora RPA assay was compared with a quantitative polymerase chain reaction (qPCR) assay and commercially available RPA kits using three qPCR platforms (BioRad CFX96, QuantStudio 6 and Applied Biosystems ViiA7) and one isothermal platform (Axxin T16-ISO RPA), with experiments run in four separate labs. The assay was tested for sensitivity (ranging from 500 to 0.33 pg of DNA) and specificity using purified oomycete DNA, as well as crude extracts of Phytophthora-infected and non-infected plants. The limit of detection (LOD) using purified DNA was 33 pg in the CFX96 and ViiA7 qPCR platforms using the preformulated kits, while the Axxin T16-ISO RPA chamber and the QuantStudio 6 platform could detect down to 3.3 pg with or without added plant extract. The LOD using a crude plant extract for the BioRad CFX96 was 330 pg, whereas the LOD for the ViiA7 system was 33 pg. These trials demonstrate the consistency and uniformity of pathogen detection with preformulated RPA kits for Phytophthora detection when conducted by different labs using different instruments for measuring results

Multiple origins of downy mildews and mito-nuclear discordance within the paraphyletic genus <i>Phytophthora</i>

<div><p>Phylogenetic relationships between thirteen species of downy mildew and 103 species of <i>Phytophthora</i> (plant-pathogenic oomycetes) were investigated with two nuclear and four mitochondrial loci, using several likelihood-based approaches. Three <i>Phytophthora</i> taxa and all downy mildew taxa were excluded from the previously recognized subgeneric clades of <i>Phytophthora</i>, though all were strongly supported within the paraphyletic genus. Downy mildews appear to be polyphyletic, with graminicolous downy mildews (GDM), brassicolous downy mildews (BDM) and downy mildews with colored conidia (DMCC) forming a clade with the previously unplaced <i>Phytophthora</i> taxon totara; downy mildews with pyriform haustoria (DMPH) were placed in their own clade with affinities to the obligate biotrophic <i>P</i>. <i>cyperi</i>. Results suggest the recognition of four additional clades within <i>Phytophthora</i>, but few relationships between clades could be resolved. Trees containing all twenty extant downy mildew genera were produced by adding partial coverage of seventeen additional downy mildew taxa; these trees supported the monophyly of the BDMs, DMCCs and DMPHs but suggested that the GDMs are paraphyletic in respect to the BDMs or polyphyletic. Incongruence between nuclear-only and mitochondrial-only trees suggests introgression may have occurred between several clades, particularly those containing biotrophs, questioning whether obligate biotrophic parasitism and other traits with polyphyletic distributions arose independently or were horizontally transferred. Phylogenetic approaches may be limited in their ability to resolve some of the complex relationships between the “subgeneric” clades of <i>Phytophthora</i>, which include twenty downy mildew genera and hundreds of species.</p></div

Cladogram with proposed relationships between the genus <i>Phytophthora</i>, the four downy mildew groups, and outgroups.

<p>Dotted lines indicate possible incongruence between subgeneric clades suggested by nuclear-only and mitochondrial-only trees (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192502#pone.0192502.s008" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192502#pone.0192502.s009" target="_blank">S3</a> Figs). Clade 11, containing <i>P</i>. <i>lilii</i> could not be included in the current study, but appears related to clades 6–8 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192502#pone.0192502.ref024" target="_blank">24</a>]. <i>Phytophthora stricta</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192502#pone.0192502.ref038" target="_blank">38</a>] appears to occupy an additional subgeneric clade. Figure created with TreeGraph2 and Inkscape.</p

Asexual sporulation in <i>Phytophthora</i> taxon mugwort strain SCVWD302.

<p>Sporangia are non-papillate, non-caducous and variable in shape, borne in tight sympodial clusters on long stalks. The distal tips of the sporangia may swell with cytoplasm, producing ovoid to obpyriform shapes or sometimes forming a beak-like protuberance. A, ovoid sporangium; B, external proliferation with evacuated sporangium and obpyriform sporangium; C, ellipsoid sporangium with protuberance. DIC micrographs with Leica DM5000B at 1000X. Scale bar = 10 μm.</p

Most likely tree from a 118-taxon alignment of <i>Phytophthora</i> and downy mildew species.

<p>Tree generated with 200 heuristic best tree searches using Garli 2.01. Bipartitions receiving significant support in Garli, MrBayes or PhyloBayes analyses are thickened and bipartitions receiving significant support in all three analyses are greatly thickened. Significant support was defined as bootstrap value ≥ 0.70 or posterior probability ≥ 0.95. Slashes indicate branches shortened for display purposes. The subgeneric clades of <i>Phytophthora</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192502#pone.0192502.ref012" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192502#pone.0192502.ref024" target="_blank">24</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192502#pone.0192502.ref025" target="_blank">25</a>] are numbered in bold; additional clades appearing in the tree are numbered 13–16 and denoted with an asterisk. Clade 11, containing <i>P</i>. <i>lilii</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192502#pone.0192502.ref024" target="_blank">24</a>] could not be included in the current study. DM, downy mildew; BDM, brassicolous DM; DMCC, DM with colored conidia; DMPH, DM with pyriform haustoria; GDM, graminicolous DM. <i>Phytophthora</i> strain numbers beginning with “P” refer to the World Oomycete Genetic Resource Collection. Figure created with TreeGraph2 and Inkscape.</p

Summary of loci used for phylogenetic inference.

<p>Summary of loci used for phylogenetic inference.</p