Isolation of Caribbean Ciguatoxin-5 (C-CTX5) and confirmation of its structure by NMR spectroscopy.

Ciguatera poisoning occurs throughout subtropical and tropical regions globally. The Virgin Islands in the Caribbean Sea is a known hyperendemic region for ciguatera and has been associated with Caribbean ciguatoxin (C-CTX) contamination in fish. An algal C-CTX (C-CTX5) was identified in Gambierdiscus silvae and G. caribeaus isolated from benthic algal samples collected in waters south St. Thomas, US Virgin Islands. The highest CTX-producing isolate, G. silvae 1602 SH-6, was grown at large-scale to isolate sufficient C-CTX5 for structural confirmation by NMR spectroscopy. A series of orthogonal extraction and fractionation procedures resulted in purification of approximately 40 µg of C-CTX5, as estimated by quantitative NMR. A suite of 1D and 2D NMR experiments were acquired that verified the structure originally proposed for C-CTX5. The structural confirmation and successful isolation of C-CTX5 opens the way for work on the stability, toxicology and biotransformation of C-CTXs, as well as for the production of quantitative reference materials for analytical method development and validation. The strategies developed for purification of C-CTX5 may also apply to isolation and purification of CTXs from the Pacific Ocean and other regions.

Gambierone and Sodium Channel Specific Bioactivity Are Associated with the Extracellular Metabolite Pool of the Marine Dinoflagellate Coolia palmyrensis.

Tropical epibenthic dinoflagellate communities produce a plethora of bioactive secondary metabolites, including the toxins ciguatoxins (CTXs) and potentially gambierones, that can contaminate fishes, leading to ciguatera poisoning (CP) when consumed by humans. Many studies have assessed the cellular toxicity of causative dinoflagellate species to better understand the dynamics of CP outbreaks. However, few studies have explored extracellular toxin pools which may also enter the food web, including through alternative and unanticipated routes of exposure. Additionally, the extracellular exhibition of toxins would suggest an ecological function and may prove important to the ecology of the CP-associated dinoflagellate species. In this study, semi-purified extracts obtained from the media of a Coolia palmyrensis strain (DISL57) isolated from the U.S. Virgin Islands were assessed for bioactivity via a sodium channel specific mouse neuroblastoma cell viability assay and associated metabolites evaluated by targeted and non-targeted liquid chromatography tandem and high-resolution mass spectrometry. We found that extracts of C. palmyrensis media exhibit both veratrine enhancing bioactivity and non-specific bioactivity. LC-HR-MS analysis of the same extract fractions identified gambierone and multiple undescribed peaks with mass spectral characteristics suggestive of structural similarities to polyether compounds. These findings implicate C. palmyrensis as a potential contributor to CP and highlight extracellular toxin pools as a potentially significant source of toxins that may enter the food web through multiple exposure pathways.

Effect of Infection Timing by Four Pythium spp. on Soybean Damping-Off Symptoms with and Without Cold Stress.

Pythium spp. cause damping-off of soybean, especially when soil conditions at or shortly after planting are cool and wet. Soybean planting dates continue to shift to earlier dates, so germinating seed and seedlings are exposed to periods of cold stress at a time which favors infection by Pythium, and seedling disease occurs. The objective of this study was to assess infection timing and cold stress on soybean seedling disease severity caused by four Pythium spp. prevalent in Iowa, namely P. lutarium, P. oopapillum, P. sylvaticum, and P. torulosum. Each species was used individually to inoculate soybean cultivar 'Sloan' using a rolled towel assay. Two temperature treatments (continuous 18°C [C18]; a 48-h cold stress period at 10°C [CS]) were applied. Soybean seedling age was divided into five growth stages (GS1 to GS5). Root rot severity and root length were assessed at 2, 4, 7, and 10 days after inoculation (DAI). At C18, root rot was greatest when soybean was inoculated with P. lutarium or P. sylvaticum at GS1 (seed imbibes water) and with P. oopapillum or P. torulosum at GS1, GS2 (radicle elongation), and GS3 (hypocotyl emergence). After CS, soybean susceptibility to P. lutarium and P. sylvaticum was reduced compared to C18 for inoculation at all GSs except GS5 (unifoliate leaf emergence). Conversely, root rot by P. oopapillum and P. torulosum was greater after CS compared to C18. Data from this study demonstrate that greater root rot, and consequently more damping-off, is likely if infection occurs at early germination stages before seedling emergence.

Ecology and diversity of culturable fungal species associated with soybean seedling diseases in the Midwestern United States.

AIMS: To isolate and characterize fungi associated with diseased soybean seedlings in Midwestern soybean production fields and to determine the influence of environmental and edaphic factors on their incidence. METHODS AND RESULTS: Seedlings were collected from fields with seedling disease history in 2012 and 2013 for fungal isolation. Environmental and edaphic data associated with each field was collected. 3036 fungal isolates were obtained and assigned to 76 species. The most abundant genera recovered were Fusarium (73%) and Trichoderma (11.2%). Other genera included Mortierella, Clonostachys, Rhizoctonia, Alternaria, Mucor, Phoma, Macrophomina and Phomopsis. Most recovered species are known soybean pathogens. However, non-pathogenic organisms were also isolated. Crop history, soil density, water source, precipitation and temperature were the main factors influencing the abundance of fungal species. CONCLUSION: Key fungal species associated with soybean seedling diseases occurring in several US production regions were characterized. This work also identified major environment and edaphic factors affecting the abundance and occurrence of these species. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification and characterization of the main pathogens associated with seedling diseases across major soybean-producing areas could help manage those pathogens, and devise more effective and sustainable practices to reduce the damage they cause.

Maize Anthracnose Stalk Rot in the Genomic Era.

Anthracnose stalk rot (ASR) of maize results in millions of dollars in losses annually in the United States. ASR, together with anthracnose leaf blight and anthracnose top dieback, is caused by the fungus Colletotrichum graminicola. Current ASR management recommendations emphasize host resistance and reduction of plant stressors (e.g., drought, heat, low fertility, or soil acidity). Stress reduction may be more difficult to achieve in the future due to more high-intensity production protocols and climate change. Moreover, cultural and chemical management practices may conflict with other important goals, including environmental sustainability and maximization of yield potential. Thus, future ASR management may rely more heavily on host resistance, for which there are relatively few highly effective sources. The last comprehensive review of C. graminicola and maize anthracnose was written over two decades ago. The genomic age has brought important new insights into mechanisms governing the host-pathogen interaction from the application of molecular and cytological technologies. This review provides a summary of our current model of maize anthracnose etiology, including how increased knowledge of molecular and cellular events could contribute to better ASR management. Improved understanding of C. graminicola taxonomy has confirmed that the fungus is specific to Zea mays, and that it colonizes living maize tissues via a critical biotrophic phase. Successful biotrophic establishment relies on an array of secreted protein effectors and secondary metabolites produced at different stages of infection and dispersed to multiple locations. These molecules could provide therapeutic targets for the next generation of transgenic or gene-edited ASR-resistant hybrids.

Reduction of Pythium Damping-Off in Soybean by Biocontrol Seed Treatment.

Pythium spp. is one of the major groups of pathogens that cause seedling diseases on soybean, leading to both preemergence and postemergence damping-off and root rot. More than 100 species have been identified within this genus, with Pythium irregulare, P. sylvaticum, P. ultimum var ultimum, and P. torulosum being particularly important for soybean production given their aggressiveness, prevalence, and abundance in production fields. This study investigated the antagonistic activity of potential biological control agents (BCAs) native to the U.S. Midwest against Pythium spp. First, in vitro screening identified BCAs that inhibit P. ultimum var. ultimum growth. Scanning electron microscopy demonstrated evidence of mycoparasitism of all potential biocontrol isolates against P. ultimum var. ultimum and P. torulosum, with the formation of appressorium-like structures, short hyphal branches around host hyphae, hook-shaped structures, coiling, and parallel growth of the mycoparasite along the host hyphae. Based on these promising results, selected BCAs were tested under field conditions against six different Pythium spp. Trichoderma afroharzianum 26 used alone and a mix of T. hamatum 16 + T. afroharzianum 19 used as seed treatments protected soybean seedlings from Pythium spp. infection, as BCA-treated plots had on average 15 to 20% greater plant stand and vigor than control plots. Our results also indicate that some of these potential BCAs could be added with a fungicide seed treatment with minimum inhibition occurring, depending on the fungicide active ingredient. This research highlights the need to develop tools incorporating biological control as a facet of soybean seedling disease management programs. The harnessing of native BCAs could be integrated with other management strategies to provide efficient control of seedling diseases.

In Vitro Glucuronidation of Caribbean Ciguatoxins in Fish: First Report of Conjugative Ciguatoxin Metabolites.

Ciguatoxins (CTX) are potent marine neurotoxins, which can bioaccumulate in seafood, causing a severe and prevalent human illness known as ciguatera poisoning (CP). Despite the worldwide impact of ciguatera, effective disease management is hindered by a lack of knowledge regarding the movement and biotransformation of CTX congeners in marine food webs, particularly in the Caribbean and Western Atlantic. In this study we investigated the hepatic biotransformation of C-CTX across several fish and mammalian species through a series of in vitro metabolism assays focused on phase I (CYP P450; functionalization) and phase II (UGT; conjugation) reactions. Using liquid chromatography high-resolution mass spectrometry to explore potential C-CTX metabolites, we observed two glucuronide products of C-CTX-1/-2 and provided additional evidence from high-resolution tandem mass spectrometry to support their identification. Chemical reduction experiments confirmed that the metabolites were comprised of four distinct glucuronide products with the sugar attached at two separate sites on C-CTX-1/-2 and excluded the C-56 hydroxyl group as the conjugation site. Glucuronidation is a novel biotransformation pathway not yet reported for CTX or other related polyether phycotoxins, yet its occurrence across all fish species tested suggests that it could be a prevalent and important detoxification mechanism in marine organisms. The absence of glucuronidation observed in this study for both rat and human microsomes suggests that alternate biotransformation pathways may be dominant in higher vertebrates.

Seedling Disease of Corn Caused by Pythium Increases With Proximity of Rye.

Yield loss of corn following a winter rye cover crop (CC) has been associated with increases in seedling disease caused by Pythium spp. We hypothesized that physical separation between the CC and corn could reduce the risk of seedling disease, and benefit corn growth and development. In a growth chamber experiment, corn seedlings were planted at 0 cm and 8 to 10 cm from terminated winter rye plants. Root rot severity was assessed at crop development stage V2, and quantitative PCR was used to estimate the abundance of Pythium clade B and clade F members present in corn roots. Radicle and seminal root rot severity was numerically greater when seedlings were planted 0 cm from terminated rye plants compared with seedlings planted 8 to 10 cm away. Moreover, a greater abundance of Pythium clade B was detected in corn grown within the terminated winter rye compared with corn planted further away (P = 0.0003). No effect of distance between corn and winter rye was detected for Pythium clade F. These data contribute to our understanding of the effect of a winter rye cover crop on corn and will inform field trial management practices for farmers to reduce occasional yield loss of corn following a winter rye cover crop.

Influence of Spatial Planting Arrangement of Winter Rye Cover Crop on Corn Seedling Disease and Corn Productivity.

Despite numerous environmental benefits associated with cover crop (CC) use, some farmers are reluctant to include CCs in their production systems because of reported yield declines in corn. There are numerous potential reasons for this yield decline, including seedling disease. A winter rye CC can serve as a "green bridge" for corn seedling pathogens. We hypothesized that proximity of corn seedling roots to decaying rye CC roots contributes to corn seeding disease. An experimental field plot and an on-farm study were conducted over 2 years to evaluate growth, development, and disease severity of corn seedlings planted at various distances from decaying winter rye CC plants. The experimental field plot study was conducted in a no-till corn-soybean rotation with five replications of a winter rye CC treatment seeded as (i) no-CC control, (ii) broadcast, (iii) 19-cm drilled rows, and (iv) 76-cm drilled rows. The on-farm study was no-till corn-soybean rotation with four replications of a winter rye CC seeded as 38-cm drilled rows, 76-cm drilled rows, and no-CC control. The corn was planted on 76-cm rows shortly after rye was terminated. With multiple seeding arrangements of winter rye, corn was planted at different distances from winter rye. Corn radicle root rot severity and incidence, shoot height, shoot dry weight, corn height and chlorophyll at VT (tasseling), ear parameters, and yield were collected. Soil samples were taken in the corn row and the interrow at winter rye termination, corn planting, and corn growth stage V3 (three leaves with fully developed collars) to estimate the abundance of Pythium clade B members present in soil samples. Our results showed that increased distance between winter rye residue and corn reduced seedling disease and Pythium clade B populations in the radicles and soil and increased shoot dry weight, leaf chlorophyll, plant height, and yield. This suggests that physically distancing the corn crop from the winter rye CC is one way to reduce the negative effects of a winter rye CC on corn.

LC-HRMS and Chemical Derivatization Strategies for the Structure Elucidation of Caribbean Ciguatoxins: Identification of C-CTX-3 and -4.

Ciguatera poisoning is linked to the ingestion of seafood that is contaminated with ciguatoxins (CTXs). The structural variability of these polyether toxins in nature remains poorly understood due to the low concentrations present even in highly toxic fish, which makes isolation and chemical characterization difficult. We studied the mass spectrometric fragmentation of Caribbean CTXs, i.e., the epimers C-CTX-1 and -2 (1 and 2), using a sensitive UHPLC-HRMS/MS approach in order to identify product ions of diagnostic value. We found that the fragmentation of the ladder-frame backbone follows a characteristic pattern and propose a generalized nomenclature for the ions formed. These data were applied to the structural characterization of a pair of so far poorly characterized isomers, C-CTX-3 and -4 (3 and 4), which we found to be reduced at C-56 relative to 1 and 2. Furthermore, we tested and applied reduction and oxidation reactions, monitored by LC-HRMS, in order to confirm the structures of 3 and 4. Reduction of 1 and 2 with NaBH4 afforded 3 and 4, thereby unambiguously confirming the identities of 3 and 4. In summary, this work provides a foundation for mass spectrometry-based characterization of new C-CTXs, including a suite of simple chemical reactions to assist the examination of structural modifications.

Resistance of the SoyNAM Parents to Seed and Root Rot Caused by Four Pythium Species.

Some Pythium spp. cause damping off and root rot in soybeans and other crop species. One of the most effective management tools to reduce disease is host resistance; however, little is known about resistance in soybean to Pythium spp. The soybean nested associated mapping (SoyNAM) parent lines are a set of germplasms that were crossed to a single hub parent to create recombinant inbred line populations for the purpose of mapping agronomic traits. The SoyNAM parents were screened for resistance to Pythium lutarium, Pythium oopapillum, Pythium sylvaticum, and Pythium torulosum in separate assays to evaluate seed and root rot severity. Of the 40 SoyNAM parents, only 'Maverick' was resistant to the four species tested; however, 13 were resistant to three species. Other lines were resistant to two, one, or none of the species tested. Correlations between seed and root rot severity for the lines assessed were weak or insignificant. Results indicate that mechanisms of resistance to seed and root rot caused by Pythium spp. may not necessarily be the same.

Cover Crop Rotation Effects on Growth and Development, Seedling Disease, and Yield of Corn and Soybean.

The effects of winter cover crops on root disease and growth of corn and soybeans are poorly understood. A 3-year field experiment investigated the effect of winter cereal rye (Secale cereale L.) and winter camelina (Camelina sativa [L.] Crantz), used either in all three years or in rotation with each other, on corn (Zea mays L.) and soybean (Glycine max. [L.] Merr.) growth, root disease, and yield. Corn following a cover crop of camelina had reduced root disease, a lower Pythium population in seedling roots, and greater growth and yields compared with corn following a rye cover crop. Camelina and rye cover crops before soybean had either a positive or no effect on soybean growth and development, root disease, and yield. Moreover, Pythium clade B populations were greater in corn seedlings after a rye cover crop compared with those following a camelina cover crop, whereas clade F populations were greater on soybean seedlings following a camelina cover crop compared with seedlings following a rye cover crop. A winter camelina cover crop grown before corn had less-negative effects on corn seedling growth, root disease, and final yield than a winter rye cover crop before corn. Neither cover crop had negative effects on soybean, and the cover crop in the preceding spring had no measurable effects on either corn or soybean.

Li-Fraumeni syndrome: not a straightforward diagnosis anymore-the interpretation of pathogenic variants of low allele frequency and the differences between germline PVs, mosaicism, and clonal hematopoiesis.

The introduction of next-generation sequencing has resulted in testing multiple genes simultaneously to identify inherited pathogenic variants (PVs) in cancer susceptibility genes. PVs with low minor allele frequencies (MAFs) (< 25-35%) are highlighted on germline genetic test reports. In this review, we focus on the challenges of interpreting PVs with low MAF in breast cancer patients undergoing germline testing and the implications for management.The clinical implications of a germline PV are substantial. For PV carriers in high-penetrance genes like BRCA1, BRCA2, and TP53, prophylactic mastectomy is often recommended and radiation therapy avoided when possible for those with Li-Fraumeni syndrome (LFS). For germline PV carriers in more moderate-risk genes such as PALB2, ATM, and CHEK2, annual breast MRI is recommended and prophylactic mastectomies considered for those with significant family histories. Detection of PVs in cancer susceptibility genes can also lead to recommendations for other prophylactic surgeries (e.g., salpingo-oophorectomy) and increased surveillance for other cancers. Therefore, recognizing when a PV is somatic rather than germline and distinguishing somatic mosaicism from clonal hematopoiesis (CH) is essential. Mutational events that occur at a post-zygotic stage are somatic and will only be present in tissues derived from the mutated cell, characterizing classic mosaicism. Clonal hematopoiesis is a form of mosaicism restricted to the hematopoietic compartment.Among the genes in multi-gene panels used for germline testing of breast cancer patients, the detection of a PV with low MAF occurs most often in TP53, though has been reported in other breast cancer susceptibility genes. Distinguishing a germline TP53 PV (LFS) from a somatic PV (TP53 mosaicism or CH) has enormous implications for breast cancer patients and their relatives.We review how to evaluate a PV with low MAF. The identification of the PV in another tissue confirms mosaicism. Older age, exposure to chemotherapy, radiation, and tobacco are known risk factors for CH, as is the absence of a LFS-related cancer in the setting of a TP53 PV with low MAF. The ability to recognize and understand the implications of somatic PVs, including somatic mosaicism and CH, enables optimal personalized care of breast cancer patients.

Effect of Salinity on DMSP Production in Gambierdiscus belizeanus (Dinophyceae).

Dimethylsulfoniopropionate (DMSP) is produced by many species of marine phytoplankton and has been reported to provide a variety of beneficial functions including osmoregulation. Dinoflagellates are recognized as major DMSP producers; however, accumulation has been shown to be highly variable in this group. We explored the effect of hyposaline transfer in Gambierdiscus belizeanus between ecologically relevant salinities (36 and 31) on DMSP accumulation, Chl a, cell growth, and cell volume, over 12 d. Our results showed that G. belizeanus maintained an intracellular DMSP content of 16.3 pmol cell-1 and concentration of 139 mM in both salinities. Although this intracellular concentration was near the median reported for other dinoflagellates, the cellular content achieved by G. belizeanus was the highest reported of any dinoflagellate thus far, owing mainly to its large size. DMSP levels were not significantly affected by salinity treatment but did change over time during the experiment. Salinity, however, did have a significant effect on the ratio of DMSP:Chl a, suggesting that salinity transfer of G. belizeanus induced a physiological response other than DMSP adjustment. A survey of DMSP content in a variety of Gambierdiscus species and strains revealed relatively high DMSP concentrations (1.0-16.4 pmol cell-1 ) as well as high intrageneric and intraspecific variation. We conclude that, although DMSP may not be involved in long-term (3-12 d) osmoregulation in this species, G. belizeanus and other Gambierdiscus species may be important contributors to DMSP production in tropical benthic microalgal communities due to their large size and high cellular content.

Re-evaluation of Seed Transmission of Clavibacter michiganensis subsp. nebraskensis in Zea mays.

The spread of Goss's bacterial wilt and leaf blight of corn (Zea mays), caused by Clavibacter michiganensis subsp. nebraskensis, to a wider geographic range in the early 2000s compared with the late 1960s has generated concern about the possible role of seed transmission in long-distance spread. The objectives of this research were: (1) to determine the percentage of seed infection found in seed harvested from inoculated and noninoculated plants of hybrids that varied in resistance to Goss's wilt; and (2) to estimate the seed transmission rate from these infected seed lots. The greatest percent seed infection was detected in seed from inoculated plants of the most susceptible hybrid and the least in seed from the most resistant hybrid. Seed lots with seed infection that ranged from 3.6 to 37.0% were planted in three field and three greenhouse trials. A total of 12 seed transmission events (Goss's wilt symptomatic seedlings) were identified among 241,850 plants examined, for a seed transmission rate of 0.005%. When the seed transmission rate was recalculated to consider only the infected seed portion of each seed lot, the rate increased to 0.040% (12 events from 30,088 potentially infected plants). Based on the low seed transmission rate observed and previous research on disease spread from a point source, it seems unlikely that seed transmission could introduce enough inoculum to create a serious disease outbreak in a single growing season. However, risk of seed transmission is relevant for phytosanitary restrictions and preventing the introduction of the pathogen to new areas. To date, Goss's wilt has not been detected outside North America, and while the risk of seed transmission is very low, the risk is not zero. Fortunately, the presence of C. michiganensis subsp. nebraskensis in corn seed is readily detectable by established seed health testing methods.

Oomycete Species Associated with Soybean Seedlings in North America-Part II: Diversity and Ecology in Relation to Environmental and Edaphic Factors.

Soybean (Glycine max (L.) Merr.) is produced across a vast swath of North America, with the greatest concentration in the Midwest. Root rot diseases and damping-off are a major concern for production, and the primary causal agents include oomycetes and fungi. In this study, we focused on examination of oomycete species distribution in this soybean production system and how environmental and soil (edaphic) factors correlate with oomycete community composition at early plant growth stages. Using a culture-based approach, 3,418 oomycete isolates were collected from 11 major soybean-producing states and most were identified to genus and species using the internal transcribed spacer region of the ribosomal DNA. Pythium was the predominant genus isolated and investigated in this study. An ecology approach was taken to understand the diversity and distribution of oomycete species across geographical locations of soybean production. Metadata associated with field sample locations were collected using geographical information systems. Operational taxonomic units (OTU) were used in this study to investigate diversity by location, with OTU being defined as isolate sequences with 97% identity to one another. The mean number of OTU ranged from 2.5 to 14 per field at the state level. Most OTU in this study, classified as Pythium clades, were present in each field in every state; however, major differences were observed in the relative abundance of each clade, which resulted in clustering of states in close proximity. Because there was similar community composition (presence or absence) but differences in OTU abundance by state, the ordination analysis did not show strong patterns of aggregation. Incorporation of 37 environmental and edaphic factors using vector-fitting and Mantel tests identified 15 factors that correlate with the community composition in this survey. Further investigation using redundancy analysis identified latitude, longitude, precipitation, and temperature as factors that contribute to the variability observed in community composition. Soil parameters such as clay content and electrical conductivity also affected distribution of oomycete species. The present study suggests that oomycete species composition across geographical locations of soybean production is affected by a combination of environmental and edaphic conditions. This knowledge provides the basis to understand the ecology and distribution of oomycete species, especially those able to cause diseases in soybean, providing cues to develop management strategies.

Oomycete Species Associated with Soybean Seedlings in North America-Part I: Identification and Pathogenicity Characterization.

Oomycete pathogens are commonly associated with soybean root rot and have been estimated to reduce soybean yields in the United States by 1.5 million tons on an annual basis. Limited information exists regarding the frequency and diversity of oomycete species across the major soybean-producing regions in North America. A survey was conducted across 11 major soybean-producing states in the United States and the province of Ontario, Canada. In 2011, 2,378 oomycete cultures were isolated from soybean seedling roots on a semiselective medium (CMA-PARPB) and were identified by sequencing of the internal transcribed spacer region of rDNA. Sequence results distinguished a total of 51 Pythium spp., three Phytophthora spp., three Phytopythium spp., and one Aphanomyces sp. in 2011, with Pythium sylvaticum (16%) and P. oopapillum (13%) being the most prevalent. In 2012, the survey was repeated, but, due to drought conditions across the sampling area, fewer total isolates (n = 1,038) were collected. Additionally, in 2012, a second semiselective medium (V8-RPBH) was included, which increased the Phytophthora spp. isolated from 0.7 to 7% of the total isolates. In 2012, 54 Pythium spp., seven Phytophthora spp., six Phytopythium spp., and one Pythiogeton sp. were recovered, with P. sylvaticum (14%) and P. heterothallicum (12%) being recovered most frequently. Pathogenicity and virulence were evaluated with representative isolates of each of the 84 species on soybean cv. Sloan. A seed-rot assay identified 13 and 11 pathogenic species, respectively, at 13 and 20°C. A seedling-root assay conducted at 20°C identified 43 species as pathogenic, having a significantly detrimental effect on the seedling roots as compared with the noninoculated control. A total of 15 species were pathogenic in both the seed and seedling assays. This study provides a comprehensive characterization of oomycete species present in soybean seedling roots in the major production areas in the United States and Ontario, Canada and provides a basis for disease management and breeding programs.

Factors Affecting the Development and Severity of Goss's Bacterial Wilt and Leaf Blight of Corn, Caused by Clavibacter michiganensis subsp. nebraskensis.

Goss's bacterial wilt and leaf blight, which is caused by Clavibacter michiganensis subsp. nebraskensis, is a disease of corn (Zea mays) that has been increasingly reported across the Midwest since its reemergence in western Nebraska, northeastern Colorado, and southeastern Wyoming during the 2006 growing season. The objective of this study was to identify environmental and agronomic factors contributing to the incidence of the disease across the Corn Belt through a multistate survey conducted during the 2011 growing season. Of the 2,400 surveys distributed throughout nine states, 486 were returned with corn leaf samples, of which 70% tested positive for C. michiganensis subsp. nebraskensis using an enzyme-linked immunosorbent assay. The agronomic data associated with each field were analyzed using classification and regression tree and random forest analyses to identify the factors that contributed most to Goss's bacterial wilt and leaf blight development. A χ2 test of independence was also done to determine relationships between certain variables and disease incidence. The two best predictors of Goss's bacterial wilt and leaf blight were hybrid resistance to Goss's bacterial wilt and leaf blight, as indicated by the seed companies' score and a planting population density >67,500 plants ha-1. Other important predictors included longitude, planting date, crop rotation, percent residue, yield history, tillage, and growth stage. Relationships between glyphosate applications, foliar fungicide applications, and corn rootworm beetle with samples testing positive for C. michiganensis subsp. nebraskensis were also detected. These data contribute to our understanding of factors that increase the risk of Goss's bacterial wilt and leaf blight, and should enable more effective management practices to be adopted or developed.

The Potential for Cereal Rye Cover Crops to Host Corn Seedling Pathogens.

Cover cropping is a prevalent conservation practice that offers substantial benefits to soil and water quality. However, winter cereal cover crops preceding corn may diminish beneficial rotation effects because two grass species are grown in succession. Here, we show that rye cover crops host pathogens capable of causing corn seedling disease. We isolated Fusarium graminearum, F. oxysporum, Pythium sylvaticum, and P. torulosum from roots of rye and demonstrate their pathogenicity on corn seedlings. Over 2 years, we quantified the densities of these organisms in rye roots from several field experiments and at various intervals of time after rye cover crops were terminated. Pathogen load in rye roots differed among fields and among years for particular fields. Each of the four pathogen species increased in density over time on roots of herbicide-terminated rye in at least one field site, suggesting the broad potential for rye cover crops to elevate corn seedling pathogen densities. The radicles of corn seedlings planted following a rye cover crop had higher pathogen densities compared with seedlings following a winter fallow. Management practices that limit seedling disease may be required to allow corn yields to respond positively to improvements in soil quality brought about by cover cropping.

Assessment of sodium channel mutations in Makah Tribal members of the U.S. Pacific Northwest as a potential mechanism of resistance to paralytic shellfish poisoning.

The Makah Tribe of Neah Bay, Washington, has historically relied on the subsistence harvest of coastal seafood, including shellfish, which remains an important cultural and ceremonial resource. Tribal legend describes visitors from other tribes that died from eating shellfish collected on Makah lands. These deaths were believed to be caused by paralytic shellfish poisoning, a human illness caused by ingestion of shellfish contaminated with saxitoxins, which are produced by toxin-producing marine dinoflagellates on which the shellfish feed. These paralytic shellfish toxins include saxitoxin, a potent Na+ channel antagonist that binds to the pore region of voltage gated Na+ channels. Amino acid mutations in the Na+ channel pore have been demonstrated to confer resistance to saxitoxin in softshell clam populations exposed to paralytic shellfish toxins present in their environment. Because of the notion of resistance to paralytic shellfish toxins, we aimed to determine if a resistance strategy was possible in humans with historical exposure to toxins in shellfish. We collected, extracted and purified DNA from buccal swabs of 83 volunteer Makah tribal members and sequenced the skeletal muscle Na+ channel (Nav1.4) at nine loci to characterize potential mutations in the relevant saxitoxin binding regions. No mutations of these specific regions were identified after comparison to a reference sequence. This study suggests that any resistance of Makah Tribal members to saxitoxin is not a function of Nav1.4 modification but may be due to mutations in neuronal or cardiac sodium channels or some other mechanism unrelated to sodium channel function.