High-throughput genotyping-by-sequencing facilitates molecular tagging of a novel rust resistance gene, R 15 , in sunflower (Helianthus annuus L.).

KEY MESSAGE: A novel rust resistance gene, R 15 , derived from the cultivated sunflower HA-R8 was assigned to linkage group 8 of the sunflower genome using a genotyping-by-sequencing approach. SNP markers closely linked to R 15 were identified, facilitating marker-assisted selection of resistance genes. The rust virulence gene is co-evolving with the resistance gene in sunflower, leading to the emergence of new physiologic pathotypes. This presents a continuous threat to the sunflower crop necessitating the development of resistant sunflower hybrids providing a more efficient, durable, and environmentally friendly host plant resistance. The inbred line HA-R8 carries a gene conferring resistance to all known races of the rust pathogen in North America and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments of 140 F2 individuals derived from a cross of HA 89 with HA-R8, rust resistance in the population was found to be conferred by a single dominant gene (R 15 ) originating from HA-R8. Genotypic analysis with the currently available SSR markers failed to find any association between rust resistance and any markers. Therefore, we used genotyping-by-sequencing (GBS) analysis to achieve better genomic coverage. The GBS data showed that R 15 was located at the top end of linkage group (LG) 8. Saturation with 71 previously mapped SNP markers selected within this region further showed that it was located in a resistance gene cluster on LG8, and mapped to a 1.0-cM region between three co-segregating SNP makers SFW01920, SFW00128, and SFW05824 as well as the NSA_008457 SNP marker. These closely linked markers will facilitate marker-assisted selection and breeding in sunflower.

Genotyping-by-sequencing targeting of a novel downy mildew resistance gene Pl 20 from wild Helianthus argophyllus for sunflower (Helianthus annuus L.).

KEY MESSAGE: Genotyping-by-sequencing revealed a new downy mildew resistance gene, Pl 20 , from wild Helianthus argophyllus located on linkage group 8 of the sunflower genome and closely linked to SNP markers that facilitate the marker-assisted selection of resistance genes. Downy mildew (DM), caused by Plasmopara halstedii, is one of the most devastating and yield-limiting diseases of sunflower. Downy mildew resistance identified in wild Helianthus argophyllus accession PI 494578 was determined to be effective against the predominant and virulent races of P. halstedii occurring in the United States. The evaluation of 114 BC1F2:3 families derived from the cross between HA 89 and PI 494578 against P. halstedii race 734 revealed that single dominant gene controls downy mildew resistance in the population. Genotyping-by-sequencing analysis conducted in the BC1F2 population indicated that the DM resistance gene derived from wild H. argophyllus PI 494578 is located on the upper end of the linkage group (LG) 8 of the sunflower genome, as was determined single nucleotide polymorphism (SNP) markers associated with DM resistance. Analysis of 11 additional SNP markers previously mapped to this region revealed that the resistance gene, named Pl 20 , co-segregated with four markers, SFW02745, SFW09076, S8_11272025, and S8_11272046, and is flanked by SFW04358 and S8_100385559 at an interval of 1.8 cM. The newly discovered P. halstedii resistance gene has been introgressed from wild species into cultivated sunflower to provide a novel gene with DM resistance. The homozygous resistant individuals were selected from BC2F2 progenies with the use of markers linked to the Pl 20 gene, and these lines should benefit the sunflower community for Helianthus improvement.

Discovery and introgression of the wild sunflower-derived novel downy mildew resistance gene Pl 19 in confection sunflower (Helianthus annuus L.).

KEY MESSAGE: A new downy mildew resistance gene, Pl 19 , was identified from wild Helianthus annuus accession PI 435414, introduced to confection sunflower, and genetically mapped to linkage group 4 of the sunflower genome. Wild Helianthus annuus accession PI 435414 exhibited resistance to downy mildew, which is one of the most destructive diseases to sunflower production globally. Evaluation of the 140 BC1F2:3 families derived from the cross of CMS CONFSCLB1 and PI 435414 against Plasmopara halstedii race 734 revealed that a single dominant gene controls downy mildew resistance in the population. Bulked segregant analysis conducted in the BC1F2 population with 860 simple sequence repeat (SSR) markers indicated that the resistance derived from wild H. annuus was associated with SSR markers located on linkage group (LG) 4 of the sunflower genome. To map and tag this resistance locus, designated Pl 19 , 140 BC1F2 individuals were used to construct a linkage map of the gene region. Two SSR markers, ORS963 and HT298, were linked to Pl 19 within a distance of 4.7 cM. After screening 27 additional single nucleotide polymorphism (SNP) markers previously mapped to this region, two flanking SNP markers, NSA_003564 and NSA_006089, were identified as surrounding the Pl 19 gene at a distance of 0.6 cM from each side. Genetic analysis indicated that Pl 19 is different from Pl 17 , which had previously been mapped to LG4, but is closely linked to Pl 17 . This new gene is highly effective against the most predominant and virulent races of P. halstedii currently identified in North America and is the first downy mildew resistance gene that has been transferred to confection sunflower. The selected resistant germplasm derived from homozygous BC2F3 progeny provides a novel gene for use in confection sunflower breeding programs.

Relocation of a rust resistance gene R 2 and its marker-assisted gene pyramiding in confection sunflower (Helianthus annuus L.).

KEY MESSAGE: The rust resistance gene R 2 was reassigned to linkage group 14 of the sunflower genome. DNA markers linked to R 2 were identified and used for marker-assisted gene pyramiding in a confection type genetic background. Due to the frequent evolution of new pathogen races, sunflower rust is a recurring threat to sunflower production worldwide. The inbred line Morden Cross 29 (MC29) carries the rust resistance gene, R 2 , conferring resistance to numerous races of rust fungus in the US, Canada, and Australia, and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments and SSR marker analyses on the 117 F2 individuals derived from a cross of HA 89 with MC29 (USDA), R 2 was mapped to linkage group (LG) 14 of the sunflower, and not to the previously reported location on LG9. The closest SSR marker HT567 was located at 4.3 cM distal to R 2 . Furthermore, 36 selected SNP markers from LG14 were used to saturate the R 2 region. Two SNP markers, NSA_002316 and SFW01272, flanked R 2 at a genetic distance of 2.8 and 1.8 cM, respectively. Of the three closely linked markers, SFW00211 amplified an allele specific for the presence of R 2 in a marker validation set of 46 breeding lines, and SFW01272 was also shown to be diagnostic for R 2 . These newly developed markers, together with the previously identified markers linked to the gene R 13a , were used to screen 524 F2 individuals from a cross of a confection R 2 line and HA-R6 carrying R 13a . Eleven homozygous double-resistant F2 plants with the gene combination of R 2 and R 13a were obtained. This double-resistant line will be extremely useful in confection sunflower, where few rust R genes are available, risking evolution of new virulence phenotypes and further disease epidemics.

First Report of Phomopsis Stem Canker of Sunflower (Helianthus annuus) Caused by Diaporthe gulyae in Canada.

During September 2012, Phomopsis stem canker was observed on sunflowers (Helianthus annuus L.) in a production field during seed filling with an average incidence of 15% in Morden, Manitoba (approximately 49°11'N and 98°09'W). The infected plants had elongated, brown-black lesions surrounding the leaf petiole, with numerous pycnidia, pith damage, and mid-stem lodging. Twenty sunflower plants were randomly sampled from the field. Isolations were made from the margins of the necrotic stems lesions by plating small pieces (5 mm) on potato dextrose agar (PDA) amended with 0.02% streptomycin sulfate. Plates were incubated at 25°C for 14 days under a 12-h photoperiod, and hyphal tips of white to grey colonies were transferred to PDA. Five isolates producing black pycnidia (occasionally with ostiolate beaks) and alpha conidia were tentatively identified as a Diaporthe sp. Alpha conidia were ellipsoidal, hyaline, and 6.5 to 8.5 × 2.5 to 3.5 µm. DNA was extracted from the mycelium of five isolates, and the ITS region was amplified and sequenced using primers ITS5 and ITS4 (4). BLASTn analysis of the 600-bp fragment (GenBank Accession Nos. KM391960 to KM391964) showed that the best match was Phomopsis sp. AJY-2011a strain T12505G (Diaporthe gulyae R.G. Shivas, S.M. Thompson & A.J. Young [3], Accession No. JF431299) from H. annuus with identities = 540/540 (100%) and gaps = 0/540 (0%). The five D. gulyae isolates were tested for pathogenicity on a sunflower confection inbred cv. HA 288 using the stem-wound method (2). Four-week-old sunflower plants (10 plants per isolate) were inoculated by wounding the stems on the second internode with a micropipette tip and placing a Diaporthe-infested mycelial plug on the wound. All plugs were attached to the wound with Parafilm. The pots were placed on the greenhouse benches at 25°C under a 16-h light/dark cycle. At 3 days after inoculation, dark brown lesions were observed on the stems extending upward from the inoculation site. Stem and leaves wilted, causing plant death 14 days after inoculation. Disease severity was calculated as a percentage of stem lesion (lesion length/stem length × 100%) at 14 days after inoculation. Significant differences (P ≤ 0.05) in disease severity were observed among D. gulyae isolates, which ranged from 34.9 to 100.0% (n = 5). Ten control plants similarly treated with sterile PDA plugs did not display symptoms. To complete Koch's postulates, D. gulyae was re-isolated from the inoculated stems, and the pathogen's identity was confirmed via sequencing of the ITS regions using primers ITS5 and ITS4 (4). The pathogen was not isolated from the control plants. D. gulyae was first reported as a pathogen on H. annuus in Australia and United States in 2011 (1,3). The pathogen was determined to be as or more aggressive than the other causal agents of Phomopsis stem canker (2,3), and its identification in both countries was circumstantially associated with increased incidence and yield loss in commercial production fields (1,3). In Canada, Phomopsis stem canker has been observed in sunflower fields over the last 10 years at low incidences, especially in years with above-normal temperatures during the sunflower growing season; however, the causal agent was not confirmed. To the best of our knowledge, this is the first report of D. gulyae causing Phomopsis stem canker on sunflowers in Canada. Since there is currently no known resistance to D. gulyae in sunflower, this newly discovered pathogen may become a threat to sunflower production in Canada. References: (1) F. Mathew et al. Phytopathology 101:S115, 2011. (2) F. Mathew et al. Phytopathology 103:S2.91, 2013. (3) S. M. Thompson et al. Persoonia 27:80, 2011. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

Molecular tagging of a novel rust resistance gene R(12) in sunflower (Helianthus annuus L.).

Sunflower production in North America has recently suffered economic losses in yield and seed quality from sunflower rust (Puccinia helianthi Schwein.) because of the increasing incidence and lack of resistance to new rust races. RHA 464, a newly released sunflower male fertility restorer line, is resistant to both of the most predominant and most virulent rust races identified in the Northern Great Plains of the USA. The gene conditioning rust resistance in RHA 464 originated from wild Helianthus annuus L., but has not been molecularly marked or determined to be independent from other rust loci. The objectives of this study are to identify molecular markers linked to the rust resistance gene and to investigate the allelism of this gene with the unmapped rust resistance genes present in HA-R6, HA-R8 and RHA 397. Virulence phenotypes of seedlings for the F(2) population and F(2:3) families suggested that a single dominant gene confers rust resistance in RHA 464, and this gene was designated as R(12). Bulked segregant analysis identified ten markers polymorphic between resistant and susceptible bulks. In subsequent genetic mapping, the ten markers covered 33.4 cM of genetic distance on linkage group 11 of sunflower. A co-dominant marker CRT275-11 is the closest marker distal to R(12) with a genetic distance of 1.0 cM, while ZVG53, a dominant marker linked in the repulsion phase, is proximal to R(12) with a genetic distance of 9.6 cM. The allelism test demonstrated that R(12) is not allelic to the rust resistance genes in HA-R6, HA-R8 and RHA 397, and it is also not linked to any previously mapped rust resistance genes. Discovery of the R(12) novel rust resistance locus in sunflower and associated markers will potentially support the molecular marker-assisted introgression and pyramiding of R(12) into sunflower breeding lines.

Genetic mapping of rust resistance genes in confection sunflower line HA-R6 and oilseed line RHA 397.

Few widely effective resistance sources to sunflower rust, incited by Puccinia helianthi Schwein., have been identified in confection sunflower (Helianthus annuus L.). The USDA inbred line HA-R6 is one of the few confection sunflower lines resistant to rust. A previous allelism test indicated that rust resistance genes in HA-R6 and RHA 397, an oilseed-type restorer line, are either allelic or closely linked; however, neither have been characterized nor molecularly mapped. The objectives of this study are (1) to locate the rust resistance genes in HA-R6 and RHA 397 on a molecular map, (2) to develop closely linked molecular markers for rust resistance diagnostics, and (3) to determine the resistance spectrum of two lines when compared with other rust-resistant lines. Two populations of 140 F2:3 families each from the crosses of HA 89, as susceptible parent, with HA-R6 and RHA 397 were inoculated with race 336 of P. helianthi in the greenhouse. The resistance genes (R-genes) in HA-R6 and RHA 397 were molecularly mapped to the lower end of linkage group 13, which encompasses a large R-gene cluster, and were designated as R 13a and R 13b, respectively. In the initial maps, SSR (simple sequence repeat) and InDel (insertion and deletion) markers revealed 2.8 and 8.2 cM flanking regions for R 13a and R 13b, respectively, linked with a common marker set of four co-segregating markers, ORS191, ORS316, ORS581, and ZVG61, in the distal side and one marker ORS464 in the proximal side. To identify new markers closer to the genes, sunflower RGC (resistance gene candidate) markers linked to the downy mildew R-gene Pl 8 and located at the same region as R 13a and R 13b were selected to screen the two F2 populations. The RGC markers RGC15/16 and a newly developed marker SUN14 designed from a BAC contig anchored by RGC251 further narrowed down the region flanking R 13a and R 13b to 1.1 and 0.1 cM, respectively. Both R 13a and R 13b are highly effective against all rust races tested so far. Our newly developed molecular markers will facilitate breeding efforts to pyramid the R 13 genes with other rust R-genes and accelerate the development of rust-resistant sunflower hybrids in both confection and oilseed sunflowers.

First Report of Ascochyta Blight Caused by QoI-Resistant Isolates of Ascochyta rabiei in Chickpea Fields of Nebraska and South Dakota.

Ascochyta blight, caused by Ascochyta rabiei, is a serious disease of chickpea (Cicer arietinum) and fungicide applications are used to manage the disease in the North Central plains (4). During the 2010 growing season, a commercial field near Stanley, SD was treated with pyraclostrobin (Headline, BASF, NC) and called a management failure by the grower. Similarly, limited efficacy of pyraclostrobin was observed in an ascochyta research trial near Scott's Bluff, NE. In both locations, symptoms and signs consistent with A. rabiei infection existed on leaves, stems, and pods; namely, circular brown lesions with concentric rings of dark brown pycnidia. Symptomatic samples were collected, disinfected with 95% ethanol for 1 min, rinsed with sterile water, placed in 0.5% NaOCl for 1 min, and rinsed again with sterile water for 1 min (4). Samples were air dried, placed on potato dextrose agar (PDA) plates for 3 to 7 days, and colonies with morphological characteristics typical of A. rabiei were single-spored and transferred to new PDA plates and incubated for 7 to 14 days. Three and six putative A. rabiei isolates were obtained from South Dakota and Nebraska samples, respectively. Morphological characteristics were consistent with A. rabiei; cultures were brown with concentric rings of dark, pear-shaped pycnidia with an ostiole, and conidia were hyaline, single-celled, and oval-shaped (2). Comparison of the internal transcribed spacer (ITS) region amplified from the genomic DNA of 3-day-old liquid cultures using ITS4/ITS5 primers by BLASTN searches using the nr database in GenBank (Accession Number FJ032643) also confirmed isolates to be A. rabiei. Mismatch amplification mutation assay (MAMA) PCR was used for detection of sensitive and resistant isolates to QoI fungicides (1). Confirmation of the presence of the G143A mutation was carried out by cloning an mRNA fragment of the cytochrome b gene using cDNA synthesized from total RNA of A. rabiei and CBF1/CBR2 (1,3). Total RNA was extracted from 3-day-old liquid cultures and it was used instead of genomic DNA for this PCR to avoid large intronic regions commonly present in mitochondrial genes. The G143A mutation has previously been correlated with resistance to QoI fungicides in other fungal plant pathogens (3). Also, these isolates were determined to be QoI-resistant in vitro by PDA amended with a discriminatory dose of 1 µg/ml of azoxystrobin (4). To our knowledge, this is the first report of QoIresistant A. rabiei isolates causing infections on chickpeas in South Dakota and Nebraska. QoI-resistant isolates were reported in North Dakota and Montana in 2005 and 2007, respectively (4). Of nearly 300 isolates collected from these states from 2005 and 2007, approximately 65% were determined to be QoI resistant (4). The widespread occurrence of QoIresistant isolates and reduction of fungicide performance in fields led the North Dakota State University Cooperative Extension Service to actively discourage the use of QoI fungicides on chickpeas in North Dakota and Montana (4). It is likely that similar recommendations will need to be adopted in South Dakota and Nebraska for profitable chickpea production. References: (1) J. A. Delgado, 2012 Ph.D. Diss. Department of Plant Pathology, North Dakota State University. (2) R. M. Harveson et al. 2011. Online. Plant Health Progress doi:10.1094/PHP-2011-0103-01-DG. (3) Z. Ma et al. Pestic. Biochem. Physiol. 77:66, 2003. (4) K. A. Wise et al. Plant Dis. 93:528, 2009.

Characterization and Pathogenicity of Rhizoctonia solani Isolates Affecting Pisum sativum in North Dakota.

Acreage of dry field pea (Pisum sativum) in North Dakota has increased approximately eightfold from the late 1990s to the late 2000s to over 200,000 ha annually. A coincidental increase in losses to root rots has also been observed. Root rot in dry field pea is commonly caused by a complex of pathogens which included Fusarium spp. and Rhizoctonia solani. R. solani isolates were obtained from roots sampled at the three- to five-node growth stage from North Dakota pea fields and from symptomatic samples received at the Plant Diagnostic Lab at North Dakota State University in 2008 and 2009. Using Bayesian inference and maximum likelihood analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), 17 R. solani pea isolates were determined to belong to anastomosis group (AG)-4 homogenous group (HG)-II and two isolates to AG-5. Pathogenicity of select pea isolates was determined on field pea and two rotation hosts, soybean and dry bean. All isolates caused disease on all hosts; however, the median disease ratings were higher on green pea, dry bean, and soybean cultivars when inoculated with pea isolate AG-4 HG-II. Identification of R. solani AGs and subgroups on field pea and determination of relative pathogenicity on rotational hosts is important for effective resistance breeding and appropriate rotation strategies.

First Report of Ascochyta Blight of Field Pea Caused by Ascochyta pisi in South Dakota.

Tan lesions approximately 1.7 × 0.8 cm with distinct dark brown margins and small pycnidia were observed on leaves of field peas (Pisum sativum L. 'Agassiz') growing in Campbell County, South Dakota (45°45.62'N, 100°9.13'W) in July 2008. Small pieces of symptomatic leaves were surface sterilized (10% NaOCl for 1 min, 70% EtOH for 1 min, and sterile distilled H2O for 2 min) and placed on potato dextrose agar (PDA) for 7 days under fluorescent lights with a 12-h photoperiod to induce sporulation. A pure culture was established by streaking a conidial suspension on PDA and isolating a single germinated spore 3 days later. The culture was grown on clarified V8 media for 10 days. Conidia were 10 to 16 × 3 to 4.5 µm and uniseptate with a slightly constricted septum, similar to those of Ascochyta pisi Lib. The exuding spore mass from pycnidia growing on the medium was carrot red. No chlamydospores or pseudothecia were observed (1,2). To confirm the identity of A. pisi, DNA was extracted from the lyophilized mycelium of the 10-day-old culture with the DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Internal transcribed spacer (ITS) regions I and II were amplified with PCR primers ITS 5 and ITS 4 (3). PCR amplicons were cleaned and directly sequenced in both directions using the primers. A BLASTN search against the NCBI nonredundant nucleotide database was performed using the consensus sequence generated by alignment of the forward and reverse sequences for this region. The consensus sequence (GenBank Accession No. GU722316) most closely matched A. pisi var. pisi strain (GenBank Accession No. EU167557). These observations confirm the identity of the fungus as A. pisi. A suspension of 1 × 106 conidia/ml of the isolate was spray inoculated to runoff on 10 replicate plants of 2-week-old, susceptible green field pea 'Sterling'. Plants were incubated in a dew chamber for 48 h at 18°C and moved to the greenhouse bench where they were maintained at 20 to 25°C with a 12-h photoperiod for 1 week. Tan lesions with dark margins appeared 7 days after inoculation and disease was assessed after 10 days (4). No symptoms were observed on water-treated control plants. A. pisi was reisolated from lesions and confirmed by DNA sequencing of the ITS region, fulfilling Koch's postulates. Currently, states bordering South Dakota (North Dakota and Montana) lead the United States in field pea production. Although acreage is limited in South Dakota, the identification of A. pisi in this region is serious. The disease is yield limiting and foliar fungicides are used for disease management (1). To our knowledge, this is the first report of Ascochyta blight on P. sativum caused by A. pisi occurring in South Dakota and the MonDak production region (the Dakotas and Montana). References: (1) T. W. Bretag et al. Aust. J. Agric. Res. 57:88, 2006. (2) A. S. Lawyer. Page 11 in: The Compendium of Pea Diseases. D. J. Hagedorn, ed. The American Phytopathological Society, St Paul, MN, 1984. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990. (4) J. M. Wroth. Can. J. Bot. 76:1955, 1998.

Emergence of a novel population of Puccinia striiformis f. sp. tritici in Eastern United States.

The geographic range of stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici, has increased dramatically since 2000 in the United States. Yield losses to the disease have been most severe in the eastern United States, where measurable yield loss had been rare prior to 2000. The objective of this study was to examine the phenotypic and genotypic variation among isolates of P. striiformis f. sp. tritici collected from populations in the eastern United States before and since 2000. Virulence phenotype and amplified fragment length polymorphism (AFLP) markers were used to examine 42 isolates collected between 1960 and 2004. In addition, the genetic structure of 59 isolates collected in 2005 using a hierarchical sampling strategy was examined. The data indicated that the contemporary isolates (collected since 2000) were very distinct from older isolates (collected before 2000) based on virulence and AFLP markers, and that the old population prevalent before 2000 may have been replaced by the contemporary population. The old and new populations appear to be genetically distinct and may represent an exotic introduction rather than a mutation in isolates of the old population.

Fusarium Head Blight Inoculum: Species Prevalence and Gibberella zeae Spore Type.

The objectives of this study were to examine the relative abundance of Gibberella zeae ascospores and conidia and other Fusarium species on wheat spikes in a field environment, to relate inoculum counts of G. zeae to airborne spore counts, and to evaluate an inoculum bioassay technique. The inoculum levels of Fusarium species and airborne spores of G. zeae were measured in North Dakota during the 1999, 2000, and 2001 growing seasons. Spores were collected from wheat spikes in a 24-h potted-plant bioassay in a fallowed field and in a spring wheat plot bioassay. Inoculum levels of Fusarium species were assessed by placing a solution recovered from bioassays on selective medium; meanwhile, ascospores and conidia of G. zeae were enumerated microscopically. A Burkard cyclonic sampler measured airborne spore levels in the fallowed field. Wheat spikes were inoculated with known concentrations of conidia or ascospores, and rinsate was put on selective medium at different intervals to compare recovery rates. Known concentrations of both spore types were also applied directly to selective medium to compare with recovery of spore types from inoculated spikes. Fusarium graminearum was the most prevalent Fusarium species on wheat spikes, although F. moniliforme and F. poae counts were highest on some days. Approximately twice as many ascospores were recovered in both the 24-h potted-plant field bioassay and the cyclonic sampler as were conidia. Significantly more colonies were recovered from wheat spikes after conidial inoculation than after ascospore inoculation at an identical concentration regardless of time of rinsate collection. Colony numbers did not differ significantly following application of ascospores and conidia to selective medium. Results confirm the predominance of G. zeae inoculum in North America but indicate conidia play an important role in the primary disease cycle.