Carol L. Groves

Identification and Characterization of Isolates of Phytophthora infestans Using Fatty Acid Methyl Ester (FAME) Profiles

The utility of fatty acid profiles for characterization and differentiation of isolates of P. infestans was investigated. Two libraries of fatty acid methyl ester (FAME) profiles (one representing average genotype characteristics and one representing individual isolate characteristics) were established from at least eight replicate samples of each of 25 different isolates of P. infestans, including representative isolates of US-1, US-6, US-7, US-8, US-11, US-14, and US-17 genotypes. These libraries then were used to identify and characterize additional unknown isolates. Fatty acid profile characteristics also were compared with cultural and genetic characteristics of the isolates. FAME profiles for isolates of P. infestans were consistent over multiple extractions and distinctly different from profiles for isolates of other Phytophthora species, such as P. capsici and P. erythroseptica, as well as isolates of Pythium spp. and various other fungal groups. Overall, profiles from different isolates within the same genotype shared similar characteristics, although there was overlap among some genotypes. Incubation temperature, growth medium, and prolonged storage on agar media all significantly affected fatty acid profiles; however, when these conditions were kept constant, profiles were distinct, consistent, and reproducible over time. Isolate profiles were sufficiently specific that individual isolates could be distinctly identified by FAME profiles. In general, individual isolate characteristics were more determinant than genotype group characteristics, although genotype could be determined for most isolates tested. Results indicated that FAME profiles can be an additional tool useful for characterizing isolates and populations of P. infestans.

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

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.

Variable concentration of soil-applied insecticides in potato over time: implications for management of Leptinotarsa decemlineata

BACKGROUND Select populations of Colorado potato beetle, Leptinotarsa decemlineata, in Wisconsin have recently become resistant to soil-applied neonicotinoids in potato. Sublethal insecticide concentrations persisting in foliage through the growing season may select for resistance over successive years of use. Over the 2 years of this study, the aim was to document the in-plant insecticide concentrations over time that result from four different types of soil-applied insecticide delivery for thiamethoxam and imidacloprid in potato, and to measure the impact upon L. decemlineata populations following treatments. After plant emergence, insect life stages were counted and plant tissue was assayed weekly for nine consecutive weeks using ELISA. RESULTS Peak concentration of both imidacloprid and thiamethoxam occurred in the first sample week following plant emergence. The average concentration of both insecticides dissipated sharply over time as the plant canopy expanded 50 days after planting in all delivery treatments. Both insecticides were detected at low levels during the later weeks of the study. Among-plant concentrations of both neonicotinoids were highly variable throughout the season. Populations of L. decemlineata continued to develop and reproduce throughout the period of declining insecticide concentrations. CONCLUSION Sublethal, chronic exposure to soil-applied systemic insecticides resulting from these delivery methods may accelerate selection for resistant insects in potato.

Development and Evaluation of Glycine max Germplasm Lines with Quantitative Resistance to Sclerotinia sclerotiorum

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating fungal pathogen of soybean that can cause significant yield losses to growers when environmental conditions are favorable for the disease. The development of resistant varieties has proven difficult. However, poor resistance in commercial cultivars can be improved through additional breeding efforts and understanding the genetic basis of resistance. The objective of this project was to develop soybean germplasm lines that have a high level of Sclerotinia stem rot resistance to be used directly as cultivars or in breeding programs as a source of improved Sclerotinia stem rot resistance. Sclerotinia stem rot-resistant soybean germplasm was developed by crossing two sources of resistance, W04-1002 and AxN-1-55, with lines exhibiting resistance to Heterodera glycines and Cadophora gregata in addition to favorable agronomic traits. Following greenhouse evaluations of 1,076 inbred lines derived from these crosses, 31 lines were evaluated for resistance in field tests during the 2014 field season. Subsequently, 11 Sclerotinia stem rot resistant breeding lines were moved forward for field evaluation in 2015, and seven elite breeding lines were selected and evaluated in the 2016 field season. To better understand resistance mechanisms, a marker analysis was conducted to identify quantitative trait loci linked to resistance. Thirteen markers associated with Sclerotinia stem rot resistance were identified on chromosomes 15, 16, 17, 18, and 19. Our markers confirm previously reported chromosomal regions associated with Sclerotinia stem rot resistance as well as a novel region of chromosome 16. The seven elite germplasm lines were also re-evaluated within a greenhouse setting using a cut petiole technique with multiple S. sclerotiorum isolates to test the durability of physiological resistance of the lines in a controlled environment. This work presents a novel and comprehensive classical breeding method for selecting lines with physiological resistance to Sclerotinia stem rot and a range of agronomic traits. In these studies, we identify four germplasm lines; 91–38, 51–23, SSR51–70, and 52–82B exhibiting a high level of Sclerotinia stem rot resistance combined with desirable agronomic traits, including high protein and oil contents. The germplasm identified in this study will serve as a valuable source of physiological resistance to Sclerotinia stem rot that could be improved through further breeding to generate high-yielding commercial soybean cultivars.

Molecular Identification of Sclerotinia trifoliorum and Sclerotinia sclerotiorum Isolates from the United States and Poland

Symptoms of clover rot caused by Sclerotinia trifoliorum or S. sclerotiorum are identical, making differentiation and identification of the causal species difficult and time consuming. Polymerase chain reaction (PCR) amplification and nucleotide sequencing were used to examine 40 isolates of S. trifoliorum (29 from Poland, 11 from the United States) and 55 isolates of S. sclerotiorum (26 from Poland, 29 from the United States). We determined that amplification of the β-tubulin and calmodulin genes with TU1/TU2/TU3 and SscadF1/SscadR1 PCR primers and the presence of introns and single-nucleotide polymorphisms (SNP) within the ribosomal DNA (rDNA) as detected with NS1/NS8 and internal transcribed spacer (ITS)1/ITS4 PCR primers are effective for rapidly and accurately differentiating between the two species of Sclerotinia. In addition, our research revealed a lack of intraspecies variation within S. sclerotiorum isolates from the United States and Poland using these same molecular markers. We detected a relatively high degree of intraspecies variability among isolates of S. trifoliorum from the United States and Poland using the presence of introns and SNP within the rDNA. SNP and nuclear small-subunit rDNA analyses revealed distinct groups of S. trifoliorum among the isolates used in this study. The results of this study provide useful information for clover breeders and pathologists looking to develop clover varieties with durable resistance.

First Report of Fusarium culmorum Causing Fusarium Head Blight of Wheat in Wisconsin

Fusarium Head Blight (FHB) of wheat is economically important as it causes significant yield loss, reduces seed quality, and often contaminates grain with mycotoxins, threatening both animal and human health. Fusarium head blight is caused by several Fusarium species, known as the Fusarium head blight disease complex (Xu and Nicholson, 2009). In June 2016, as part of an FHB survey across Wisconsin, we observed symptoms typical of FHB in a winter wheat field (Triticum aestivum L.) located in Door County, Wisconsin. Temperatures were cooler than average and wet during anthesis at this location. Ten symptomatic heads were collected from one production field in Door County. On average, diseased heads showed 40% symptomatic spikelets. Individual heads exhibited bleached kernels and pinkish-orange sporulation typical of that of FHB. Symptomatic kernels were surface-disinfested in 95% ethanol and 0.06 % sodium hypochlorite. Five seeds per head from the ten heads sampled were chosen randomly and placed in a Petri...

Re-emergence of Tobacco streak virus Infecting Soybean in the United States and Canada

Melissa D. Irizarry, Department of Plant Pathology, Iowa State University, Ames 50011; Carol L. Groves, Department of Plant Pathology, University of Wisconsin-Madison 53706; Manjula G. Elmore, Department of Plant Pathology, Iowa State University, Ames 50011; Carl A. Bradley, Department of Crop Sciences, University of Illinois, Urbana 61801 (current address: Department of Plant Pathology, University of Kentucky, Princeton 42445); Ranjit Dasgupta and Thomas L. German, Department of Entomology, University of WisconsinMadison 53706; Douglas J. Jardine, Department of Plant Pathology, Kansas State University, Manhattan 66506; Erika Saalau Rojas, Department of Plant Pathology, Iowa State University, Ames 50011 (current address: UMass Cranberry Station, University of MassachusettsAmherst, East Wareham 02538); Damon L. Smith, Department of Plant Pathology, University of Wisconsin-Madison 53706; Albert U. Tenuta, Ontario Ministry of Agriculture, Food, and Rural Affairs; University of Guelph, Ridgetown N0P 2C0, Canada; Steven A. Whitham and Daren S. Mueller, Department of Plant Pathology, Iowa State University, Ames 50011