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.

Deep injection and the potential of biochar to reduce fumigant emissions and effects on nematode control.

Reducing fumigant emissions is essential for minimizing the environmental impacts of pre-plant soil fumigation. Low permeability plastic films are effective at reducing emissions but have high initial purchase, installation, and disposal costs. The objective of this study was to evaluate if deep fumigant injection and biochar soil amendments can reduce emissions, improve fumigant distribution in soil, and provide acceptable control of plant parasitic nematodes. A pre-plant soil fumigation trial was conducted in a commercial orchard in the San Joaquin Valley, CA, USA. Treatments included two rates of Telone® C-35 (a mixture of 1,3-dichloropropene and chloropicrin) under totally impermeable film or with no surface seal, two injection depths (45 or 65 cm), and two biochar rates (20 or 40 ton ha-1). Emission rates were generally low due to rain events encountered during the trial, but data clearly showed that the deep injection enhanced fumigant delivery to depths below 60 cm and resulted in significantly lower peak emission compared to the standard injection depth. Biochar applied at 40 ton ha-1 had the lowest emission rates during 1-month monitoring period. Although variability in nematode survival was high, tarped, deep injection, and biochar treatment showed lower survival of nematodes at various depths. Increase in fumigant persistence, especially chloropicrin, was observed in this study, likely due to the high soil moisture and low temperature. All data indicate that biochar amendments can help reduce fumigant emissions without reducing nematode control; however, additional research is needed to optimize treatments, determine the affordability of various biochar materials, and validate results under a range of field conditions.

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.

Emission and transport of 1,3-dichloropropene and chloropicrin in a large field tarped with VaporSafe TIF.

Tarping fumigated fields with low permeability films such as commercial Totally Impermeable Film (TIF) can significantly reduce emissions, but it can also increase fumigant residence time in the soil such that extended tarp-covering durations may be required to address potential exposure risks during tarp-cutting and removal. In an effort to develop safe practices for using TIF, a large field study was conducted in the San Joaquin Valley of California. Comprehensive data on emissions (measured with dynamic flux chambers), fate, and transport of 1,3-dichloropropene and chloropicrin were collected in a 3.3 ha field fumigated with Pic-Clor 60 via broadcast shank application. Low emission flux (below 15 µg m(-2) s(-1)) was observed from the tarped field throughout the tarp-covering period of 16 days with total emission loss of <8% of total applied for both chemicals. Although substantially higher flux was measured at tarp edges (up to 440 µg m(-2) s(-1)), the flux was reduced to below 0.5 µg m(-2) s(-1) beyond 2 m of tarp edge where total mass loss was estimated to be ≤ 1% of total applied to the field. Emission flux increased following tarp-cutting, but was much lower compared to 5 or 6 d tarp-covering periods determined in other fields. This study demonstrated the ability of TIF to significantly reduce fumigant emissions with supporting data on fumigant movement in soil. Proper management on use of the tarp, such as extending tarp-covering period, can reduce negative impact on the environment and help maintain the beneficial use of soil fumigants for agricultural productions.

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.