Factors underlying the prevalence of Pythium infection of corn seeds following seed treatment application of tebuconazole

Microbial communities are essential for soil health, but fungicide application may have significant effects on their structure. It is difficult to predict whether non-target pathogens of applied fungicides in the soil will cause crop damage. Tebuconazole is a triazole fungicide that can be used as a seed treatment and thereby introduced to the soil. However, seed-applied tebuconazole has a potential risk of causing poor emergence of corn (Zea mays) seedlings. Using soil with a history of poor corn seedling emergence, we demonstrate through TA-cloning and isolation that the poor emergence of corn seedlings from tebuconazole-coated corn seeds was primarily due to infection by surviving soil pathogens, specifically Pythium species that are not targeted by tebuconazole, rather than the phytotoxic effects of tebuconazole. Bioassay tests on tebuconazole amended media showed that tebuconazole can suppress soil fungi while allowing Pythium to grow. Pythium species primarily contributing to the corn seed rot were more pathogenic at cooler temperatures. Furthermore, the non-target biocontrol agent of Trichoderma spp. was strongly inhibited by tebuconazole. Taken together, the non-target effects of tebuconazole are likely not significant under favorable plant growing conditions, but are considerable due to low-temperature stress.

Detection of Pythium spp. in golf course irrigation systems

Many Pythium spp. are causal agents of diseases of turfgrasses. Pythium spp. disseminate through irrigation systems in agricultural settings, and this study provides evidence that Pythium spp. also disseminate through golf course irrigation. Water samples were collected from irrigation heads and water sources at ten golf courses in Missouri and Kansas. Samples were collected from 2018 to 2019 in April, July, and October. Phosphorus, nitrogen, and chloride concentrations were measured from irrigation head samples to determine if these parameters influence frequency of Pythium spp. detected. Pythium spp. were detected in samples through baiting and membrane filtration. Cultures were isolated on PARP media and DNA was extracted from putative Pythium isolates. The ITS region was PCR amplified and sequenced. Phylogenetic trees were constructed using representative sample sequences, sequences from seven morphologically identified reference isolates of Pythium, and similar Genbank accessions. Detected Oomycete species include Lagenidium giganteum, Pythium biforme, P. insidiosum, P. marsipium, P. plurisporium, and Saprolegnia hypogyna. Twenty-one clades lacked species-level resolution, and fourteen of these clades were associated with Pythium species. Clades A, C, D, E, I, and M contain Pythium species that cause root and crown rot on creeping bentgrass. Detected Pythium communities were dependent on the detection method used and sampling source. Pythium frequency and diversity were highest in April 2019. Sample temperature, sampling site, chloride, and nutrient concentrations did not influence Pythium frequency in samples. Irrigation systems using surface water sources contained at least three Pythium spp. over the course of two years.

Rapid detection of red rot disease pathogens (Pythium chondricola/P. porphyrae) in Pyropia yezoensis (Rhodophyta) using PCR-RFLP method

Red rot disease is one of the most well-known algal diseases infecting red algae Pyropia species. This disease seriously decreases the quality and quantity of Pyropia aquaculture products in Korea, Japan, and China. Recently we first found that Pythium chondricola (Oomycetes) infecting blades of Pyropia yezoensis. Therefore two Pythium species (P. chondricola/P. porphyrae) have been reported as red rot disease pathogens. In this study, we developed a species-specific molecular marker for discriminating the two red rot disease pathogens. Using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method based on the mitochondrial cytochrome c oxidase subunit 2 (cox2) and nuclear ribosomal RNA large subunit (LSU) regions, these two Pythium species were successfully classified without a sequencing step. This new method showed high specificity and efficiency for detecting red rot disease pathogens at the species level for both of the cultured samples and field samples. Therefore the molecular markers developed in this study are effective for long-term monitoring on the infection and distribution pattern of each Pythium species in Pyropia aquaculture farms. Moreover, the molecular monitoring can provide useful information for predicting infection and preventing mass mortality of Pyropia species by red rot disease.

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.

Review of Pythium species Causing Damping-off in Corn

Seedling blights and root rots caused by Pythium species account for almost twenty-five million dollars in annual losses to corn (Zea mays) production in the United States and Ontario. Variations in annual rainfall and increasing use of no-till agriculture can favor soilborne pathogens like Pythium. To date, forty-four species have been reported as pathogenic to corn in the United States. The average annual corn planting date in the United States has shifted approximately one week earlier in the past decade, exposing young corn plants to longer germination periods of generally cooler temperatures, favoring attack by Pythium. Optimal temperatures, aggressiveness, and response to chemical and biological treatment options vary by species. This review consolidates the species of Pythium reported as corn pathogens in literature to date. It also provides an insight into management strategies and discusses variations in fungicide sensitivity observed in corn-related Pythium species.

Pythium spp. associated with root rot and stunting of winter wheat in North Carolina

In eastern North Carolina, mild to severe stunting and root rot have reduced yields of winter wheat, especially during years with abundant rainfall. Causal agents of root rot of wheat in this region were previously identified as Pythium irregulare, P. vanterpoolii, and P. spinosum. To investigate species prevalence, 114 isolates of Pythium were obtained from symptomatic wheat plants collected in 8 counties. Twelve species were recovered, with P. irregulare (32%), P. vanterpoolii (17%), and P. spinosum (16%) the most common. Pathogenicity screens were performed with selected isolates of each species, and slight to severe necrosis of young roots was observed. The aggressiveness of five isolates each of P. irregulare, P. vanterpoolii, and P. spinosum was compared on a single cultivar of wheat at 14°C, and very aggressive isolates were found within all species. In vitro growth of these isolates was measured at 14°C and 20°C, and all isolates grew faster at the warmer temperature. The effects of varying temperatures and rates of nitrogen on root rot caused by Pythium species alone or in combination were investigated. All inoculation treatments caused severe root rot under all conditions tested, and disease was more severe at 12/14°C compared to 18/20°C, but there was no effect of nitrogen application.

Characterization of Pythium Species Collected from a Multiple Time-Point Sampling of Cucurbits in South Carolina

Species of Pythium cause root and stem rot in cucurbits, but no formal surveys have been conducted in the United States to identify which species are responsible. The cucurbit hosts bottle gourd, cucumber, Hubbard squash, and watermelon were transplanted in May, July, September, and November into sentinel plots in four and five different fields in 2017 and 2018, respectively, in South Carolina. Eight of the nine fields were replanted in March 2019. Isolates (600) were collected and identified by sequencing DNA of the mitochondrial cytochrome oxidase I region. The four most common species were P. spinosum (45.6% of all isolates), P. myriotylum (20.0%), P. irregulare (15.3%), and P. aphanidermatum (12.8%). P. myriotylum and P. aphanidermatum were predominantly isolated in May, July, and September, whereas P. spinosum and P. irregulare were predominantly isolated in November and March. Isolates of P. ultimum, P. irregulare, and P. spinosum were more virulent than isolates of P. myriotylum and P. aphanidermatum at 25°C. Representative isolates were screened in vitro for sensitivity to three fungicides: mefenoxam, propamocarb, and oxathiapiprolin. All isolates were sensitive to mefenoxam and propamocarb, but these same isolates were insensitive to oxathiapiprolin, except those classified taxonomically in Pythium clade I.