Insights into the Host Specificity of a New Oomycete Root Pathogen, Pythium brassicum P1: Whole Genome Sequencing and Comparative Analysis Reveals Contracted Regulation of Metabolism, Protein Families, and Distinct Pathogenicity Repertoire.

Pythium brassicum P1 Stanghellini, Mohammadi, Förster, and Adaskaveg is an oomycete root pathogen that has recently been characterized. It only attacks plant species belonging to Brassicaceae family, causing root necrosis, stunting, and yield loss. Since P. brassicum P1 is limited in its host range, this prompted us to sequence its whole genome and compare it to those of broad host range Pythium spp. such as P. aphanidermatum and P. ultimum var. ultimum. A genomic DNA library was constructed with a total of 374 million reads. The sequencing data were assembled using SOAPdenovo2, yielding a total genome size of 50.3 Mb contained in 5434 scaffolds, N50 of 30.2 Kb, 61.2% G+C content, and 13,232 putative protein-coding genes. Pythium brassicum P1 had 175 species-specific gene families, which is slightly below the normal average. Like P. ultimum, P. brassicum P1 genome did not encode any classical RxLR effectors or cutinases, suggesting a significant difference in virulence mechanisms compared to other oomycetes. Pythium brassicum P1 had a much smaller proportions of the YxSL sequence motif in both secreted and non-secreted proteins, relative to other Pythium species. Similarly, P. brassicum P1 had the fewest Crinkler (CRN) effectors of all the Pythium species. There were 633 proteins predicted to be secreted in the P. brassicum P1 genome, which is, again, slightly below average among Pythium genomes. Pythium brassicum P1 had only one cadherin gene with calcium ion-binding LDRE and DxND motifs, compared to Pythium ultimum having four copies. Pythium brassicum P1 had a reduced number of proteins falling under carbohydrate binding module and hydrolytic enzymes. Pythium brassicum P1 had a reduced complement of cellulase and pectinase genes in contrast to P. ultimum and was deficient in xylan degrading enzymes. The contraction in ABC transporter families in P. brassicum P1 is suggested to be the result of a lack of diversity in nutrient uptake and therefore host range.

Pythium brassicum sp. nov.: A Novel Plant Family-Specific Root Pathogen.

Roots of stunted broccoli plants (Brassica oleracea) from the Palo Verde Valley, CA, were observed at various stages of decay. A species of Pythium with large spiny oogonia was microscopically observed and consistently isolated from decayed roots. Isolates produced spherical, intercalary sporangia (average 34.5 µm in diameter) and aplerotic oospores (average 37.3 µm in diameter) in oogonia (average 47.4 µm in diameter) bearing numerous conical spines (average 8.5 µm in height and 5.5 µm basal width) with blunt apices. A representative broccoli isolate (P1) had a 99% internal transcribed spacer (ITS) sequence similarity with Pythium jasmonium (nom. inval., GenBank AF216654.1), a species which has not been formally described. Three other accessions in GenBank also carry the specific epithet of P. jasmonium and were originally isolated from diseased plants in the family Brassicaceae. In our study, these isolates were pathogenic on broccoli and morphologically similar to P1. P1 was pathogenic to 10 cultivated and 12 wild plants in the family Brassicaceae but not to 18 species of cultivated plants belonging to nine other plant families. Mycelial growth of our isolates occurred between <10 and 35°C, with an optimum of 25°C (maximum growth rate 25 mm/day). Our broccoli isolates are related to other species in subclade B of clade J. Members of subclade B include P. mastophorum, P. uncinulatum, P. buismaniae, P. polymastum, and P. megalacanthum. However, the broccoli isolates, in addition to all those in GenBank that carry the specific epithet of P. jasmonium, possess unique ITS, ß-tubulin, and cox1 sequences that are sufficiently different from other species in subclade B to justify status as a new species. We propose that isolates previously designated as P. jasmonium (nom. inval.) be renamed as P. brassicum sp. nov. based on their apparent plant family-specific pathogenicity.

Olpidium bornovanus-mediated germination of ascospores of Monosporascus cannonballus: a host-specific rhizosphere interaction.

Monosporascus cannonballus, a host-specific root-infecting ascomycete, is the causal agent of a destructive disease of melon (Cucumis melo L.) known as vine decline. Ascospores germinate only in the rhizosphere of melon plants growing in field soil. However, no germination occurs in the rhizosphere of melon plants if the field soil is heated to temperatures >50°C prior to infestation with ascospores. This observation suggested that germination is mediated by one or more heat-sensitive members of the soil microflora. Although bacteria or actinomycetes were heretofore suspected as the germination-inducing microbes, our data demonstrate that Olpidium bornovanus, an obligate, host-specific, root-infecting zoosporic fungus, is responsible. In four experiments conducted in autoclaved field soil amended with various population densities of culturally produced ascospores, significant ascospore germination was recorded only in the rhizosphere of cantaloupe seedlings colonized by O. bornovanus.

Viability of Oomycete Propagules Following Ingestion and Excretion by Fungus Gnats, Shore Flies, and Snails.

Sporangia of Phytophthora capsici and P. nicotianae, as well as hyphal swellings of Pythium splendens, P. sylvaticum, and P. ultimum, were ingested by adult shore flies but none were viable after passing through the digestive tract. Oospores of Pythium aphanidermatum retained their viability following ingestion by adult shore flies. Larval stages of fungus gnats and shore flies ingested sporangia of Phytophthora capsici, P. nicotianae, and P. ramorum, but they were not viable upon excretion. In contrast, hyphal swellings of Pythium splendens, P. sylvaticum, and P. ultimum, chlamydospores of Phytophthora ramorum, and oospores of Pythium aphanidermatum, retained their viability after passage through the digestive tract of these larvae. Snails were capable of ingesting and excreting viable sporangia and chlamydospores of P. ramorum, which upon excretion infected detached leaves. Although the impact of larvae and snails in the rapid dissemination of pathogen propagules is unknown, this work does highlight the possibility that some often-ignored animal-fungus interactions should be considered in long-range dispersal of pathogen propagules via food webs.

Colonization of cantaloupe roots by Monosporascus cannonballus.

Penetration of Monosporascus cannonballus into and growth within cantaloupe roots was studied using light and electron microcopy. Germ tubes penetrated the epidermis, and hyphae grew, without branching, almost directly to the xylem. The hyphae traversed the endodermis into protoxylem cells, and then grew extensively within the lumen of metaxylem vessels. Eventually, the hyphae grew back out into the cortical cells. A relatively low percentage of cells within both the cortex and xylem of lesions contained hyphae. The hyphae were generally localized within the lesion and could rarely be isolated more than 2 mm away from the margin of the lesion. Regardless of tissue type, hyphae were predominately intracellular. M. cannonballus appeared to be most similar to vascular wilt pathogens in its mode of parasitism, but does not spread via the vascular system to above-ground plant tissues.