T. J. Gulya

Phomopsis Stem Canker: A Reemerging Threat to Sunflower (Helianthus annuus) in the United States.

Phomopsis stem canker causes yield reductions on sunflower (Helianthus annuus L.) on several continents, including Australia, Europe, and North America. In the United States, Phomopsis stem canker incidence has increased 16-fold in the Northern Great Plains between 2001 and 2012. Although Diaporthe helianthi was assumed to be the sole causal agent in the United States, a newly described species, D. gulyae, was found to be the primary cause of Phomopsis stem canker in Australia. To determine the identity of Diaporthe spp. causing Phomopsis stem canker in the Northern Great Plains, 275 infected stems were collected between 2010 and 2012. Phylogenetic analyses of sequences of the ribosomal DNA internal transcribed spacer region, elongation factor subunit 1-α, and actin gene regions of representative isolates, in comparison with those of type specimens, confirmed two species (D. helianthi and D. gulyae) in the United States. Differences in aggressiveness between the two species were determined using the stem-wound method in the greenhouse; overall, D. helianthi and D. gulyae did not vary significantly (P≤0.05) in their aggressiveness at 10 and 14 days after inoculation. These findings indicate that both Diaporthe spp. have emerged as sunflower pathogens in the United States, and have implications on the management of this disease.

Effect of Fungicide and Timing of Application on Management of Sunflower Rust

Sunflower rust is an important yield-limiting disease in sunflower production in the Great Plains of the United States. Rust severity and incidence have increased between 2002 and 2011, and genetic resistance is limited in most commercial hybrids, particularly the high-value confectionary market type. Although fungicides are available for rust management in the United States, management recommendations are insufficient. Specifically, efficacy and timing data are very limited for fungicides in FRAC groups 7 and 11. Seventeen fungicide efficacy and timing trials were conducted between 2008 and 2011 in North Dakota. Timings evaluated across the four years included single or multiple applications at growth stages (GS): GS V8-V12 (late vegetative), GS R1 (terminal bud formation), GS R3-4 (elongation of bud), GS R5 (flowering), and GS R6 (completion of flowering). With few exceptions, fungicide applications of DMIs and QoIs controlled disease greater than SDHI fungicides. Fungicide applications made at R5, either singly or in combination, consistently resulted in greater disease control. A negative correlation (r = -0.7756) between disease control and yield was observed, resulting in a yield reduction of 6.6% for every 1% increase in disease severity.

Genotyping-by-Sequencing Uncovers the Introgression Alien Segments Associated with Sclerotinia Basal Stalk Rot Resistance from Wild Species—I. Helianthus argophyllus and H. petiolaris

Basal stalk rot (BSR), caused by Sclerotinia sclerotiorum, is a devastating disease in sunflower worldwide. The progress of breeding for Sclerotinia BSR resistance has been hampered due to the lack of effective sources of resistance for cultivated sunflower. Our objective was to transfer BSR resistance from wild annual Helianthus species into cultivated sunflower and identify the introgressed alien segments associated with BSR resistance using a genotyping-by-sequencing (GBS) approach. The initial crosses were made between the nuclear male sterile HA 89 with the BSR resistant plants selected from wild Helianthus argophyllus and H. petiolaris populations in 2009. The selected resistant F1 plants were backcrossed to HA 458 and HA 89, respectively. Early generation evaluations of BSR resistance were conducted in the greenhouse, while the BC2F3 and subsequent generations were evaluated in the inoculated field nurseries. Eight introgression lines; six from H. argophyllus (H.arg 1 to H.arg 6), and two from H. petiolaris (H.pet 1 and H.pet 2), were selected. These lines consistently showed high levels of BSR resistance across seven environments from 2012 to 2015 in North Dakota and Minnesota, USA. The mean BSR disease incidence (DI) for H.arg 1 to H.arg 6, H.pet 1, and H.pet 2 was 3.0, 3.2, 0.8, 7.2, 7.7, 1.9, 2.5, and 4.4%, compared to a mean DI of 36.1% for Cargill 270 (susceptible hybrid), 31.0% for HA 89 (recurrent parent), 19.5% for HA 441 (resistant inbred), and 11.6% for Croplan 305 (resistant hybrid). Genotyping of the highly BSR resistant introgression lines using GBS revealed the presence of the H. argophyllus segments in linkage groups (LGs) 3, 8, 9, 10, and 11 of the sunflower genome, and the H. petiolaris segments only in LG8. The shared polymorphic SNP loci in the introgression lines were detected in LGs 8, 9, 10, and 11, indicating the common introgression regions potentially associated with BSR resistance. Additionally, a downy mildew resistance gene, Pl17, derived from one of the parents, HA 458, was integrated into five introgression lines. Germplasms combining resistance to Sclerotinia BSR and downy mildew represent a valuable genetic source for sunflower breeding to combat these two destructive diseases.

Phenotypic Diversity of Puccinia helianthi (Sunflower Rust) in the United States from 2011 and 2012

Puccinia helianthi, causal agent of sunflower rust, is a macrocyclic and autoecious pathogen. Widespread sexual reproduction of P. helianthi was documented in North Dakota and Nebraska for the first time in 2008 and has since frequently occurred. Concurrently, an increase in sunflower rust incidence, severity, and subsequent yield loss on sunflower has occurred since 2008. Rust can be managed with resistance genes but determination of virulence phenotypes is important for effective gene deployment and hybrid selection. However, the only P. helianthi virulence data available in the United States was generated prior to 2009 and consisted of aggregate virulence phenotypes determined on bulk field collections. The objective of this study was to determine the phenotypic diversity of P. helianthi in the United States. P. helianthi collections were made from cultivated, volunteer, and wild Helianthus spp. at 104 locations across seven U.S. states and one Canadian province in 2011 and 2012. Virulence phenotypes of 238 single-pustule isolates were determined on the internationally accepted differential set. In total, 29 races were identified, with races 300 and 304 occurring most frequently in 2011 and races 304 and 324 occurring most frequently in 2012. Differences in race prevalence occurred between survey years and across geography but were similar among host types. Four isolates virulent to all genes in the differential set (race 777) were identified. The resistance genes found in differential lines HA-R3 (R4b), MC29 (R2 and R10), and HA-R2 (R5) conferred resistance to 96.6, 83.6, and 78.6% of the isolates tested, respectively.

Identification of Novel Sources of Resistance to Sclerotinia Basal Stalk Rot in South African Sunflower Germplasm

Sclerotinia basal stalk rot (BSR) is a serious fungal disease that reduces yield of global sunflower (Helianthus annuus L.) production. Because limited chemical and biological controls of BSR are available and the present-day hybrids lack sufficient resistance, identification of new sources of resistance is needed to manage the disease in the future. A total of 59 cultivated oilseed sunflower accessions from the Agricultural Research Council, Grain Crops Institute, Potchefstroom, South Africa sunflower collection were evaluated for resistance to BSR in artificially inoculated field trials. Nine accessions from the South African sunflower collection were identified with a disease incidence less than or equal to the moderately resistant sunflower oilseed hybrid. These lines can be used in breeding programs to introgress the genes for resistance to Sclerotinia BSR into other adapted lines, providing a more efficient, durable, and environmentally friendly host plant resistance. Cultivated sunflower (Helianthus annuus L.) is one of the most important oilseed crops in the world and is commercially produced in over 68 countries and on all continents except Antarctica (Markell et al. 2015). In the United States, sunflower was planted on approximately 1.6 million acres in 2014, with the highest concentration in the Northern Great Plains states of North and South Dakota (USDA-NASS). Two types of sunflower are grown with 75% oilseed and 25% confectionery (eatable kernels). Diseases are considered the most important biological yield-limiting factor for sunflower production in the region, and diseases caused by Sclerotinia sclerotiorum (Lib.) de Bary, are among the most common and most devastating (Block et al. 2016; Markell et al. 2015). Sclerotinia sclerotiorum is a necrotrophic fungus capable of causing stem diseases (commonly called “white mold” or Sclerotinia wilt) on an extensive host range of more than 400 broadleaf plant species, which includes many weeds and crops (Markell et al. 2015; Schwartz 2005). The life cycle begins and ends with the sclerotia, a long-lived pathogen structure consisting of a hyphal mass covered with a hard black rind that commonly survives for 3 to 7 years in the soil. For the vast majority of these hosts, the infection process begins when the sclerotia germinate carpogenically (producing airborne ascospores released by the mushroom-like apothecia that infect the plants). Ascospores that germinate on an adequate food source, usually flowers or senescent host tissue, will produce mycelium that can invade adjacent healthy tissues, commonly leaves, petioles, and ultimately stems (Block et al. 2016; Schwartz 2005). In sunflower, S. sclerotiorum causes three distinctly different diseases: head rot (HR); midstalk rot (MSR); and basal stalk rot (BSR) (also called Sclerotinia wilt). While the disease cycles of sunflower MSR and HR are the same as those of S. sclerotiorum diseases on other crops, the disease cycle of BSR is unique. Root exudates from sunflowers stimulate myceliogenic germination (producing vegetative hyphae) from the sclerotia, allowing the fungus to penetrate the taproot and causing rotting of the basal stem and root tissues (Block et al. 2016; Bolton et al. 2006; Gulya et al. 1997). The first observation of BSR in a field is a sudden wilting of the entire plant (Fig. 1). Signs and symptoms include a tan to manila-colored, water-soaked, and soft lesion girdling the base of the stalk at the soil line; a white mycelium (Fig. 2); shredding of stalks; abundant small, black sclerotia in and around the lesions; and lodging (Markell et al. 2014). Sclerotinia BSR is a serious problem in humid, temperate sunflower-growing areas, as well as in tropical and subtropical regions of the world (Block at al. 2016). This disease causes serious economic losses in sunflower globally and is one of the most important sunflower diseases in the United States (Gulya et al. 1997). Despite this, management tools for the disease are insufficient; crop rotation is of marginal use owing to the long-lived nature of the sclerotia, foliar fungicide application (commonly used for management of white mold in other crops) is not useful owing to the unique myceliogenic infection process in sunflower, fungicide seed treatments provide insufficient control, and the presentday hybrids and cultivated lines lack sufficient tolerance and resistance. Consequently, identification of new and effective sources of resistance is critical for managing this disease. No major gene(s) have been identified thus far conferring complete resistance against S. sclerotiorum in cultivated sunflower. Resistance to BSR is genetically complex and conditioned by multiple genes, but differences in resistance to BSR have been reported in artificially inoculated field-screening trials involving diverse Corresponding author: Gerald J. Seiler; E-mail: gerald.seiler@ars.usda.gov This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2017. PLANT HEALTH PROGRESS ¿ 2017, Vol. 18, No. 2 ¿ Page 87 cultivated germplasm (Amouzadeh et al. 2013; Davar et al. 2010; Talukder et al. 2014 a, b). Recently, the United States Department of Agriculture, Agricultural Research Service Sunflower and Plant Biology Unit secured a germplasm exchange with the Agricultural Research Council, Grain Crops Institute in Potchefstroom, South Africa. This presented a rare opportunity to evaluate promising germplasm from another continent under different environmental conditions from those in the United States, and in particular, to address one of the most economically important diseases in both countries. This paper reports the response of sunflower germplasm from South Africa to one of the major diseases of sunflower: Sclerotinia BSR. Field Evaluation A total of 59 cultivated oilseed sunflower (H. annuus L.) accessions from the Agricultural Research Council, Grain Crops Institute, Potchefstroom, South Africa sunflower collection were evaluated for resistance to BSR in artificially inoculated field trials. The evaluated germplasm represented cultivars and breeding lines in the primary gene pool (same species as cultivated sunflower) that can be used for sunflower improvement. A randomized complete block design with two replications was used to evaluate germplasm at the North Dakota State University Research and Extension Center, Carrington, ND, and test plots of Cenex Harvest States (CHS, Inver Grove Heights, MN) at Grandin, ND, in 2014, and Carrington, ND, in 2015. All fields were previously planted to cereal grains. Internal controls included a susceptible commercial oilseed hybrid, Mycogen 270 (Dow AgroSciences, Indianapolis, IN), and a moderately resistant oilseed hybrid, Croplan 305 (Land O’Lakes, Arden Hills, MN). Single-row plots 6 m long, with 75 cm between rows, were thinned to 25 plants per row (accessions and checks) and maintained using standard agronomic practices with no confounding fungicide treatments. In 2014, the trial at Grandin was planted on 23 May and inoculated on 3 July, and the trial at Carrington was planted on 4 June and inoculated 10 July, while in 2015, the Carrington trial was planted on 4 June and inoculated on 9 July. The inoculum consisted of dried Proso millet (Panicum miliaceum L.) infected with mycelial S. sclerotiorum isolate NEB-274 (provided by M. Boosalis, Department of Plant Pathology, University of Nebraska, Lincoln) with 90 g of inoculum placed 10 cm from the row at a depth of 5 cm using a GPS-guided, tractor-mounted, four-row, electrically driven Gandy (Model M902EM-R, Owatonna, MN) applicator with disk openers (Gulya 2004). Plots were artificially inoculated at the V-6 growth stage (Schneiter and Miller 1981). The trials were carried out under rainfed conditions. Seasonal precipitation (June through September, 120 days) was adequate with at least 230 mm at all locations in both years. Disease Rating and Statistical Analysis Visual evaluation of the artificially inoculated plots for disease incidence (DI) of Sclerotinia basal stalk rot was conducted on 16 September 2014 at Grandin, and on 2 October 2014 and 1 October 2015 at Carrington. Disease incidence (DI) percent (number of plants infected/number of plants in the plot) was recorded at the R-9 growth stage (Schneiter andMiller 1981), which is considered physiological maturity, prior to a killing frost. Stalks with a tan to manila basal lesion with white mycelium or black sclerotia at the soil line, or wilted plants with stalk shredding or lodging with black sclerotia present, were considered infected (Gulya 2004, Markell et al. 2014). Data were analyzed using the PROC GLM procedure (SAS version 9.4, SAS Institute Inc., Cary, NC) and Fisher’s protected LSD at the 5% probability level for mean comparisons. An analysis of variance of the disease incidence (visual evaluation) determined that there were significant differences among the accessions, but there was no significant accession 3 location interaction indicating that the genotypes were reacting similarly in all environments so the data were combined over the three locations. There was also no significant block effect at any of the locations. Sources of Resistance Basal stalk rot symptom development was typical and uniform with no other significant disease symptoms evident in the plots. FIGURE 1 Typical symptoms of basal Sclerotinia stalk rot on sunflower, starting with terminal wilting of susceptible genotypes (right).