Damon L. Smith

Discriminatory simplex and multiplex PCR for four species of the genus Sclerotinia.

Sclerotinia sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Eriks, and S. homoeocarpa F.T. Benn are the most relevant plant pathogenic species within the genus Sclerotinia because of their large range of economically important hosts, including tomato, peanut, alfalfa, and turfgrass, among others. Species identification based on morphological characteristics is challenging and time demanding, especially when one crop hosts multiple species. The objective of this study was to design specific primers compatible with multiplexing, for rapid, sensitive and accurate detection and discrimination among four Sclerotinia species. Specific primers were designed for the aspartyl protease gene of S. sclerotiorum, the calmodulin gene of S. trifoliorum, the elongation factor-1 alpha gene of S. homoeocarpa, and the laccase 2 gene of S. minor. The specificity and sensitivity of each primer set was tested individually and in multiplex against isolates of each species and validated using genomic DNA from infected plants. Each primer set consistently amplified DNA of its target gene only. DNA fragments of different sizes were amplified: a 264 bp PCR product for S. minor, a 218 bp product for S. homoeocarpa, a 171 bp product for S. sclerotiorum, and a 97 bp product for S. trifoliorum. These primer sets can be used individually or in multiplex for identification of Sclerotinia spp. in pure culture or from infected plants. The multiplex assay had a lower sensitivity limit than the simplex assays (0.0001 pg/μL DNA of each species). The multiplex assay developed is an accurate and rapid tool to differentiate between the most relevant plant pathogenic Sclerotinia species in a single PCR reaction.

Corn yield loss estimates due to diseases in the United States and Ontario, Canada from 2012 to 2015.

Annual decreases in corn yield caused by diseases were estimated by surveying members of the Corn Disease Working Group in 22 corn-producing states in the United States and in Ontario, Canada, from 2012 through 2015. Estimated loss from each disease varied greatly by state and year. In general, foliar diseases such as northern corn leaf blight, gray leaf spot, and Goss’s wilt commonly caused the largest estimated yield loss in the northern United States and Ontario during nondrought years. Fusarium stalk rot and plant-parasitic nematodes caused the most estimated loss in the southernmost United States. The estimated mean economic loss due to yield loss by corn diseases in the United States and Ontario from 2012 to 2015 was

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

76.51 USD per acre. The cost of disease-mitigating strategies is another potential source of profit loss. Results from this survey will provide scientists, breeders, government, and educators with data to help inform and prioritize research, policy, and educational efforts in corn pathology and disease management. M U E L L E R E T A L . , P L A N T H E A L T H P R O G R E S S 1 7 (2 0 1 6 )

Plant-derived antifungal agent poacic acid targets β-1,3-glucan

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.

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

Significance The search for new antifungal compounds is struggling to keep pace with emerging fungicide resistance. Through chemoprospecting of an untapped reservoir of inhibitory compounds, lignocellulosic hydrolysates, we have identified a previously undescribed antifungal agent, poacic acid. Using both chemical genomics and morphological analysis together for the first time, to our knowledge, we identified the cellular target of poacic acid as β-1,3-glucan. Through its action on the glucan layer of fungal cell walls, poacic acid is a natural antifungal agent against economically significant fungi and oomycete plant pathogens. This work highlights the chemical diversity within lignocellulosic hydrolysates as a source of potentially valuable chemicals. A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides. We report the discovery of poacic acid, a potent antifungal compound found in lignocellulosic hydrolysates of grasses. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. Morphological analysis revealed that cells treated with poacic acid behaved similarly to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Poacic acid causes rapid cell lysis and is synergistic with caspofungin and fluconazole. The cellular target was identified; poacic acid localized to the cell wall and inhibited β-1,3-glucan synthesis in vivo and in vitro, apparently by directly binding β-1,3-glucan. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development. The discovery of poacic acid as a natural antifungal agent targeting β-1,3-glucan highlights the potential side use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds and further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates.

Effect of glyphosate application on sudden death syndrome of glyphosate-resistant soybean under field conditions

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.

Sclerotinia homoeocarpa Overwinters in Turfgrass and Is Present in Commercial Seed

Sudden death syndrome (SDS), caused by Fusarium virguliforme, is an important yield limiting disease of soybean. Glyphosate is used to control weeds in soybean; however, its effect on SDS is not clearly understood. The objective of this study was to examine the impact of glyphosate on SDS, yield, and plant nutrition under field conditions. Fourteen field experiments were conducted in Iowa, Illinois, Indiana, Michigan, Wisconsin, and Ontario, Canada during 2011 to 2013. The experiment consisted of six treatment combinations of glyphosate and herbicides not containing glyphosate. Disease index was significantly different across the location-years, ranging from 0 to 65. The highest disease was noted in locations with irrigation, indicating that high soil moisture favors development of SDS. There were no effects of herbicide treatments or interactions on disease. The foliar disease index among the treatments over all years ranged from 9 to 13. Glyphosate-treatments also tended to yield more than treatments of herbicides not containing glyphosate. There were no interactions between glyphosate-treatments and total manganese in plant tissue. The interaction of glyphosate with other nutrients in plant tissue was inconclusive. This 14 location-year study demonstrated that glyphosate application did not increase SDS severity or adversely affect soybean yield under field conditions.

A Site-Specific, Weather-Based Disease Regression Model for Sclerotinia Blight of Peanut

Dollar spot is the most economically important disease of amenity turfgrasses in the United States, yet little is known about the source of primary inoculum for this disease. With the exception of a few isolates from the United Kingdom, Sclerotinia homoeocarpa, the causal agent of dollar spot, does not produce spores. Consequently, it was assumed that overwintering of this organism in soil, thatch, and plant debris provides primary inoculum for dollar spot epidemics. Overwintering of S. homoeocarpa in roots and shoots of symptomatic and asymptomatic creeping bentgrass turfgrass was quantified over the course of a three-year field experiment. Roots did not consistently harbor S. homoeocarpa, whereas S. homoeocarpa was isolated from 30% of symptomatic shoots and 10% of asymptomatic shoots in the spring of two out of three years. The presence of stroma-like pathogen material on leaf blades was associated with an increase in S. homoeocarpa isolation and colony diameter at 48 hpi. Commercial seed has also been hypothesized to be a potential source of initial inoculum for S. homoeocarpa. Two or more commercial seed lots of six creeping bentgrass cultivars were tested for contamination with S. homoeocarpa using culture-based and molecular detection methods. A viable, pathogenic isolate of S. homoeocarpa was isolated from one commercial seed lot and contamination of this lot was confirmed with nested PCR using S. homoeocarpa specific primers. A sensitive nested PCR assay detected S. homoeocarpa contamination in eight of twelve (75%) commercial seed lots. Seed source, but not cultivar or resistance to dollar spot, influenced contamination by S. homoeocarpa. Overall, this research suggests that seeds are a potential source of initial inoculum for dollar spot epidemics and presents the need for further research in this area.

Seed Transmission of Soybean vein necrosis virus: The First Tospovirus Implicated in Seed Transmission

In North Carolina, losses due to Sclerotinia blight of peanut, caused by the fungus Sclerotinia minor, are an estimated 1 to 4 million dollars annually. In general, peanut (Arachis hypogaea) is very susceptible to Sclerotinia blight, but some partially resistant virginia-type cultivars are available. Up to three fungicide applications per season are necessary to maintain a healthy crop in years highly favorable for disease development. Improved prediction of epidemic initiation and identification of periods when fungicides are not required would increase fungicide efficiency and reduce production costs on resistant and susceptible cultivars. A Sclerotinia blight disease model was developed using regression strategies in an effort to describe the relationships between modeled environmental variables and disease increase. Changes in incremental disease incidence (% of newly infected plants of the total plant population per plot) for the 2002-2005 growing seasons were statistically transformed and described using 5-day moving averages of modeled site-specific weather variables (localized, mathematical estimations of weather data derived at a remote location) obtained from SkyBit (ZedX, Inc.). Variables in the regression to describe the Sclerotinia blight disease index included: mean relative humidity (linear and quadratic), mean soil temperature (quadratic), maximum air temperature (linear and quadratic), maximum relative humidity (linear and quadratic), minimum air temperature (linear and quadratic), minimum relative humidity (linear and quadratic), and minimum soil temperature (linear and quadratic). The model explained approximately 50% of the variability in Sclerotinia blight index over 4 years of field research in eight environments. The relationships between weather variables and Sclerotinia blight index were independent of host partial resistance. Linear regression models were used to describe progress of Sclerotinia blight on cultivars and breeding lines with varying levels of partial resistance. Resistance affected the rate of disease progress, but not disease onset. The results of this study will be used to develop site- and cultivar-specific spray advisories for Sclerotinia blight.

Analysis of Factors That Influence the Epidemiology of Sclerotinia minor on Peanut

Soybean vein necrosis virus (SVNV; genus Tospovirus; Family Bunyaviridae) is a negative-sense single-stranded RNA virus that has been detected across the United States and in Ontario, Canada. In 2013, a seed lot of a commercial soybean variety (Glycine max) with a high percentage of discolored, deformed and undersized seed was obtained. A random sample of this seed was planted in a growth room under standard conditions. Germination was greater than 90% and the resulting seedlings looked normal. Four composite samples of six plants each were tested by reverse transcription polymerase chain reaction (RT-PCR) using published primers complimentary to the S genomic segment of SVNV. Two composite leaflet samples retrieved from seedlings yielded amplicons with a size and sequence predictive of SVNV. Additional testing of twelve arbitrarily selected individual plants resulted in the identification of two SVNV positive plants. Experiments were repeated by growing seedlings from the same seed lot in an isolated room inside a thrips-proof cage to further eliminate any external source of infection. Also, increased care was taken to reduce any possible PCR contamination. Three positive plants out of forty-eight were found using these measures. Published and newly designed primers for the L and M RNAs of SVNV were also used to test the extracted RNA and strengthen the diagnosis of viral infection. In experiments, by three scientists, in two different labs all three genomic RNAs of SVNV were amplified in these plant materials. RNA-seq analysis was also conducted using RNA extracted from a composite seedling sample found to be SVNV-positive and a symptomatic sample collected from the field. This analysis revealed both sense and anti-sense reads from all three gene segments in both samples. We have shown that SVNV can be transmitted in seed to seedlings from an infected seed lot at a rate of 6%. To our knowledge this is the first report of seed-transmission of a Tospovirus.