The Quest to Identify a New Virus Disease of Sunflower from Nebraska.

Between 2010 and 2018, sunflower plants exhibiting virus-like symptoms, including stunting, mottling, and chlorotic ringspots on leaves, were observed from commercial fields and research plots from four sites within three distinct counties of western Nebraska (Box Butte, Kimball, and Scotts Bluff). Near identical symptoms from field samples were reproduced on seedlings mechanically in the greenhouse on multiple occasions, confirming the presence of a sap-transmissible virus from each site. Symptomatic greenhouse-inoculated plants from the 2010 and 2011 Box Butte samples tested negative for sunflower mosaic virus (SuMV), sunflower chlorotic mottle virus (SuCMoV), and all potyviruses in general by ELISA and RT-PCR. Similar viral-like symptoms were later observed on plants in a commercial sunflower field in Kimball County in 2014, and again from volunteers in research plots in Scotts Bluff County in 2018. Samples from both of these years were again successfully reproduced on seedlings in the greenhouse as before following mechanical transmissions. Symptom expression for all years began 12 to 14 days after inoculation as mild yellow spots followed by the formation of chlorotic ringspots from the mottled pattern. The culture from 2014 tested negatively for three groups of nepoviruses via RT-PCR, ruling this group out. However, transmission electron microscopy assays of greenhouse-infected plants from both 2014 and 2018 revealed the presence of distinct, polyhedral virus particles. With the use of high throughput sequencing and RT-PCR, it was confirmed that the infections from both years were caused by a new virus in the tombusvirus genus and was proposed to be called Sunflower ring spot mottle virus (SuRSMV). Although the major objective of this project was to identify the causal agent of the disease, it became evident that the diagnostic journey itself, with all the barriers encountered on the 10-year trek, was actually more important and impactful than identification.

Genetics and mapping of a novel downy mildew resistance gene, Pl(18), introgressed from wild Helianthus argophyllus into cultivated sunflower (Helianthus annuus L.).

KEY MESSAGE: A novel downy mildew resistance gene, Pl(18), was introgressed from wild Helianthus argophyllus into cultivated sunflower and genetically mapped to linkage group 2 of the sunflower genome. The new germplasm, HA-DM1, carrying Pl(18) has been released to the public. Sunflower downy mildew (DM) is considered to be the most destructive foliar disease that has spread to every major sunflower-growing country of the world, except Australia. A new dominant downy mildew resistance gene (Pl 18) transferred from wild Helianthus argophyllus (PI 494573) into cultivated sunflower was mapped to linkage group (LG) 2 of the sunflower genome using bulked segregant analysis with 869 simple sequence repeat (SSR) markers. Phenotyping 142 BC1F2:3 families derived from the cross of HA 89 and H. argophyllus confirmed the single gene inheritance of resistance. Since no other Pl gene has been mapped to LG2, this gene was novel and designated as Pl (18). SSR markers CRT214 and ORS203 flanked Pl(18) at a genetic distance of 1.1 and 0.4 cM, respectively. Forty-six single nucleotide polymorphism (SNP) markers that cover the Pl(18) region were surveyed for saturation mapping of the region. Six co-segregating SNP markers were 1.2 cM distal to Pl(18), and another four co-segregating SNP markers were 0.9 cM proximal to Pl(18). The new BC2F4-derived germplasm, HA-DM1, carrying Pl(18) has been released to the public. This new line is highly resistant to all Plasmopara halstedii races identified in the USA providing breeders with an effective new source of resistance against downy mildew in sunflower. The molecular markers that were developed will be especially useful in marker-assisted selection and pyramiding of Pl resistance genes because of their close proximity to the gene and the availability of high-throughput SNP detection assays.

Pl(17) is a novel gene independent of known downy mildew resistance genes in the cultivated sunflower (Helianthus annuus L.).

KEY MESSAGE: Pl 17, a novel downy mildew resistance gene independent of known downy mildew resistance genes in sunflowers, was genetically mapped to linkage group 4 of the sunflower genome. Downy mildew (DM), caused by Plasmopara halstedii (Farl.). Berl. et de Toni, is one of the serious sunflower diseases in the world due to its high virulence and the variability of the pathogen. DM resistance in the USDA inbred line, HA 458, has been shown to be effective against all virulent races of P. halstedii currently identified in the USA. To determine the chromosomal location of this resistance, 186 F 2:3 families derived from a cross of HA 458 with HA 234 were phenotyped for their resistance to race 734 of P. halstedii. The segregation ratio of the population supported that the resistance was controlled by a single dominant gene, Pl 17. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) primers were used to identify molecular markers linked to Pl 17. Bulked segregant analysis using 849 SSR markers located Pl 17 to linkage group (LG) 4, which is the first DM gene discovered in this linkage group. An F2 population of 186 individuals was screened with polymorphic SSR and SNP primers from LG4. Two flanking markers, SNP SFW04052 and SSR ORS963, delineated Pl 17 in an interval of 3.0 cM. The markers linked to Pl 17 were validated in a BC3 population. A search for the physical location of flanking markers in sunflower genome sequences revealed that the Pl 17 region had a recombination frequency of 0.59 Mb/cM, which was a fourfold higher recombination rate relative to the genomic average. This region can be considered amenable to molecular manipulation for further map-based cloning of Pl 17.

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.

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.

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.

Genetics and mapping of the R11 gene conferring resistance to recently emerged rust races, tightly linked to male fertility restoration, in sunflower (Helianthus annuus L.).

Sunflower oil is one of the major sources of edible oil. As the second largest hybrid crop in the world, hybrid sunflowers are developed by using the PET1 cytoplasmic male sterility system that contributes to a 20 % yield advantage over the open-pollinated varieties. However, sunflower production in North America has recently been threatened by the evolution of new virulent pathotypes of sunflower rust caused by the fungus Puccinia helianthi Schwein. Rf ANN-1742, an 'HA 89' backcross restorer line derived from wild annual sunflower (Helianthus annuus L.), was identified as resistant to the newly emerged rust races. The aim of this study was to elucidate the inheritance of rust resistance and male fertility restoration and identify the chromosome location of the underlying genes in Rf ANN-1742. Chi-squared analysis of the segregation of rust response and male fertility in F(2) and F(3) populations revealed that both traits are controlled by single dominant genes, and that the rust resistance gene is closely linked to the restorer gene in the coupling phase. The two genes were designated as R ( 11 ) and Rf5, respectively. A set of 723 mapped SSR markers of sunflower was used to screen the polymorphism between HA 89 and the resistant plant. Bulked segregant analysis subsequently located R ( 11 ) on linkage group (LG) 13 of sunflower. Based on the SSR analyses of 192 F(2) individuals, R ( 11 ) and Rf5 both mapped to the lower end of LG13 at a genetic distance of 1.6 cM, and shared a common marker, ORS728, which was mapped 1.3 cM proximal to Rf5 and 0.3 cM distal to R ( 11 ) (Rf5/ORS728/R ( 11 )). Two additional SSRs were linked to Rf5 and R ( 11 ): ORS995 was 4.5 cM distal to Rf5 and ORS45 was 1.0 cM proximal to R ( 11 ). The advantage of such an introduced alien segment harboring two genes is its large phenotypic effect and simple inheritance, thereby facilitating their rapid deployment in sunflower breeding programs. Suppressed recombination was observed in LGs 2, 9, and 11 as it was evident that no recombination occurred in the introgressed regions of LGs 2, 9, and 11 detected by 5, 9, and 22 SSR markers, respectively. R ( 11 ) is genetically independent from the rust R-genes R ( 1 ), R ( 2 ), and R ( 5 ), but may be closely linked to the rust R-gene R ( adv ) derived from wild Helianthus argophyllus, forming a large rust R-gene cluster of R ( adv )/R ( 11 )/R ( 4 ) in the lower end of LG13. The relationship of Rf5 with Rf1 is discussed based on the marker association analysis.

Molecular mapping of the rust resistance gene R ( 4 ) to a large NBS-LRR cluster on linkage group 13 of sunflower.

Rust is a serious fungal disease in the sunflower growing areas worldwide with increasing importance in North America in recent years. Several genes conferring resistance to rust have been identified in sunflower, but few of them have been genetically mapped and linked to molecular markers. The rust resistance gene R ( 4 ) in the germplasm line HA-R3 was derived from an Argentinean open-pollinated variety and is still one of most effective genes. The objectives of this study were to determine the chromosome location of the R ( 4 ) gene and the allelic relationship of R ( 4 ) with the R ( adv ) rust resistance gene. A total of 63 DNA markers previously mapped to linkage group (LG) 13 were used to screen for polymorphisms between two parental lines HA 89 and HA-R3. A genetic map of LG 13 was constructed with 21 markers, resulting in a total map length of 93.8 cM and an average distance of 4.5 cM between markers. Two markers, ZVG61 and ORS581, flanked the R ( 4 ) gene at 2.1 and 0.8 cM, respectively, and were located on the lower end of LG 13 within a large NBS-LRR cluster identified previously. The PCR pattern generated by primer pair ZVG61 was unique in the HA-R3 line, compared to lines HA-R1, HA-R4, and HA-R5, which carry other R ( 4 ) alleles. A SCAR marker linked to the rust resistance gene R ( adv ) mapped to LG 13 at 13.9 cM from the R ( 4 ) locus, indicating that R ( adv ) is not an allele of the R ( 4 ) locus. The markers tightly linked to the R ( 4 ) gene will facilitate gene pyramiding for rust resistance breeding of sunflower.

First Report of Virulence Phenotypes of Puccinia helianthi, Causal Agent of Sunflower Rust in Illinois.

Sunflower rust caused by Puccinia helianthi (Schw.) is an autoecious and macrocyclic rust that occurs on wild perennial, wild annual, and cultivated sunflower species (Helianthus annuus L.). Severe epidemics of sunflower rust can significantly reduce yield and quality of cultivated sunflower (4). Uredinia of P. helianthi were observed in individual sunflower fields in four Illinois counties in 2008, namely; Champaign, LaSalle, McLean, and Livingston. Leaves with uredinia from each field were collected and shipped to the USDA-ARS Sunflower Pathology Laboratory in Fargo, ND. Urediniospores were harvested by rinsing leaves with Soltrol 170 isoparaffin solvent (Chevron Phillips Chemicals, Woodlands, TX). Urediniospores were increased by inoculating the spore suspension onto 21-day-old seedlings of susceptible oilseed hybrid Myc-7350 with a vacuum-pump powered atomizer. After the Soltrol 170 evaporated, plants were placed in a dew chamber for 24 h and then moved to a greenhouse at approximately 20 to 25°C with a 14-h photoperiod. When sporulating uredinia were visible, a bulk collection of urediniospores was made from samples of each location. Additionally, one single-pustule derived isolate from each location was obtained by harvesting urediniospores from a single pustule and increasing as above. To determine the virulence phenotypes, each single-pustule derived isolate and bulk collection were inoculated as above onto at least two replicated sets of the nine international differential lines (each set containing three plants per line) and evaluated after 14 days (1,2). The single-pustule isolates from LaSalle, Livingston, and McLean counties were determined to be virulence phenotype 300. The single-pustule isolate from Champaign produced no infection on any differential lines, including the susceptible, and was therefore considered not viable. The virulence phenotypes of the bulk samples were coded as 330 (Champaign), 324 (McLean), and 336 (Livingston and LaSalle). Virulence of all aforementioned virulence phenotypes was found to be common in a recent survey of 400 Midwestern P. helianthi samples collected in 2007 and 2008 (1). Although sunflower production is limited in Illinois, expansion could occur. This is particularly true in southern Illinois, where double cropping sunflower after winter wheat is a possibility. Urediniospore germination and infection is favored by free moisture and temperatures ranging from 10 to 25°C, while optimum temperature for spore development ranges from 20 to 35°C (3). These environmental conditions are often more common in Illinois than in the major sunflower-producing states in the Great Plains, where sunflower rust is a concern annually. Thus, determination of P. helianthi virulence phenotypes in Illinois may be important for future management of sunflower rust. References: (1) T. J. Gulya and S. G. Markell. National Sunflower Association. Online Publication/Gulya_RustStatus_09, 2009. (2) T. Gulya and S. Masirevic. FAO Eur. Res. Network on Sunflower. Bucarest, Romania. 31, 1995. (3) T. Gulya et al. Sunflower Diseases. Page 263 in: Sunflower Technology and Production. A. A. Schneiter, ed. American Society of Agronomy, Madison, WI, 1997. (4) S. Markell et al. Plant Dis. 93:668, 2009.

Widespread Occurrence of the Aecial Stage of Sunflower Rust Caused by Puccinia helianthi in North Dakota and Minnesota in 2008.

Sunflower rust caused by Puccinia helianthi (Schw.) is widespread in North America and occurs annually on cultivated sunflower (Helianthus annuus L.) and wild annual and perennial Helianthus spp., although severity on the U.S. sunflower crop has been increasing in recent years (2). P. helianthi is a autoecious, macrocyclic rust, but the aecial stage is rarely observed in the field (1,3,4). In most years, the earliest appearance of sunflower rust in North Dakota (ND) and surrounding states usually occurs in early August as the uredinial stage. Initial inoculum can result from urediniospores that overwinter in the Northern Great Plains, urediniospores blown in from areas south of North Dakota, or basidiospores completing the life cycle. However, aecia have been noted very infrequently and never widespread, indicating initial inoculum is usually urediniospores. Aecia of P. helianthi were first observed on 24 June 2008 in a commercial sunflower field (confection hybrid CHS 3126) near Mohall, ND. Aecia cups measuring 0.2 to 0.3 mm in diameter were observed in clusters that were 1 to 7 mm wide in diameter and containing as many as 150 cups. Aecia cups were bright orange but turned brown-black as they senesced. As many as 15 aecial clusters occurred on individual leaves or cotyledons. Aeciospores were ellipsoid, hyaline, and measured approximately 20 µm in diameter. On 4 July 2008, uredinia were first observed in the same Mohall, ND field. At that time, uredinia, aecia, and senesced aecia could all be found on the same leaves. In a non-fungicide-treated strip of the field, pustule coverage on the lower leaves was 10 to 20% by mid-July, pustule coverage on the upper four leaves of plants in the untreated strip was 56% by 27 August, and yield at harvest was less than 200 kg/ha. The rest of the field was sprayed twice with fungicides and yielded 1,571 kg/ha, which is similar to the statewide yield average of 1,573 kg/ha in 2008. To determine the prevalence of aecia in the primary growing regions of ND and Minnesota (MN), surveys were conducted in 75 sunflower fields in 18 counties between 22 and 24 July in ND and 34 fields in 8 counties between 17 and 31 July in MN. Incidence of aecia and uredinia were determined by visual observation of a minimum of 20 plants scouted in a 'W' pattern in the field. Rust was found in 31 and 53% of fields in ND and MN, respectively. In fields where rust was found, both aecia and uredinia were observed in 37% of the fields, aecia only in 29% of the fields, and uredinia only in 34% of the fields. Although it is uncertain why aecia were widespread in 2008, night temperatures in Mohall, ND, where aecia were first observed, reached the dew point temperature on 51 of 61 days in June and July, suggesting that dew or fog likely formed. Thus, favorable conditions for germination and infection early in the growing season indicate widespread occurrence of rust was likely a result of local inoculum sources. The early appearance of aecia is cause for concern for two reasons: significant yield loss can occur when rust appears early in the growing season; and the presence of aecia suggest that the pathogen completed its sexual cycle. When P. helianthi completes its life cycle it is likely that a greater diversity of races will result (4). References: (1) D. L. Bailey. Univ. Minn. Tech. Bull. 16:1, 1923. (2) D. Berglund. Natl. Sunflower Assoc. Online publication. /Berglund_2007_NSA_Survey_08. 2008. (3) H. S. Jackson. Mem. Torrey Bot. Club 18:1, 1931. (4) G. A. Kong et al. Australas. Plant Pathol. 28:320, 1999.

Identifying quantitative trait loci for resistance to Sclerotinia head rot in two USDA sunflower germplasms.

Sclerotinia head rot is a major disease of sunflower in the world, and quantitative trait loci (QTL) mapping could facilitate understanding of the genetic basis of head rot resistance and breeding in sunflower. One hundred twenty-three F2:3 and F2:4 families from a cross between HA 441 and RHA 439 were studied. The mapping population was evaluated for disease resistance in three field experiments in a randomized complete block design with two replicates. Disease incidence (DI) and disease severity (DS) were assessed. A genetic map with 180 target region amplification polymorphism, 32 simple sequence repeats, 11 insertion-deletion, and 2 morphological markers was constructed. Nine DI and seven DS QTL were identified with each QTL explaining 8.4 to 34.5% of phenotypic variance, suggesting the polygenic basis of the resistance to head rot. Five of these QTL were identified in more than one experiment, and each QTL explained more than 12.9% of phenotypic variance. These QTL could be useful in sunflower breeding. Although a positive correlation existed between the two disease indices, most of the respective QTL were located in different chromosomal regions, suggesting a different genetic basis for the two indices.

First Report of Sclerotinia sclerotiorum Infection on Cuphea.

Species of the genus Cuphea (family Lythraceae) are being developed as potential domestic sources of medium length fatty acids (lauric and capric) for use in industrial lubricants and detergents. During September 2004, patches of dead plants were observed in test plots of Cuphea sp. cv. PSR-23 (1) (Cuphea viscosissima Jacq. × C. lanceolata W.T. Aiton) near Morris, MN and Prosper, ND, approximately 200 km apart. Seed yield in the diseased Morris field was 78 kg/ha compared with 516 kg/ha in nearby, nonaffected fields of the same variety, for an 85% yield reduction. Stems were split open to reveal long, cylindrical sclerotia as much as 8 mm long. Isolations from diseased stem tissue and sclerotia were identified as Sclerotinia sclerotiorum (Lib.) de Bary and produced typical sized sclerotia (4 to 6 mm in diameter) after 7 days growth on potato dextrose agar (PDA). Cuphea PSR-23 plants were grown in the greenhouse in individual pots for 5 weeks and then inoculated. Three inoculation methods were used. For the first method, ascospores of a sunflower isolate of S. sclerotiorum were sprayed onto blooming flowers and foliage at a rate of 5,000 spores per ml. The inoculated plants were kept in a dark, 18°C mist chamber for 48 h and then returned to a greenhouse maintained at 24/20°C, day/night temperatures. All 20 inoculated plants were visibly colonized by Sclerotinia sp. after 3 days, and all plants were dead by 7 days. The second inoculation used the petiole inoculation technique employed by canola researchers (2). The blade from the third leaf was excised and a micropipette tip containing an agar disk of mycelia of the Cuphea isolate was placed over the cut end of the petiole. Five days after inoculation, all 30 inoculated plants were dead, while none of the 10 control plants (using sterile agar disks on the cut petiole) were affected. Isolations were made from diseased plants inoculated by all methods, and S. sclerotiorum colonies were observed on PDA medium with typical sclerotia from 4 to 6 mm in diameter. The third inoculation method tested root infection. S. sclerotiorum was grown on autoclaved proso millet (Panicum miliaceum L.) seed for 7 days, and 5 g of colonized millet seed was placed in a hole 6 cm from the base of a Cuphea plant, with one plant per 3.7 liter pot. Sunflower (Helianthus annuus L.; oilseed hybrid Cargill 270) plants served as inoculated controls. None of the 20 Cuphea plants were infected via soil inoculations compared with 70% of 30 sunflower plants that developed basal stalk rot and wilt within 2 weeks after inoculation. To our knowledge, this is the first report of S. sclerotiorum infection on Cuphea sp., and is believed to be the first report of infection on any genus within the Lythraceae (loosestrife family). With over 100 annual and perennial species in the genus Cuphea, the possibility of Sclerotinia spp. resistance needs to be investigated to further develop this potential oilseed crop. References: (1) S. J. Knapp and J. M. Crane. Crop Sci. 40:299, 2000. (2) J. Zhao et al. Plant Dis. 88:1033, 2004.

Molecular variability of sunflower downy mildew, Plasmopara halstedii, from different continents.

Downy mildew of sunflower (Helianthus annuus L.), caused by the pathogen Plasmopara halstedii, is a potentially devastating disease. Seventy-seven isolates of P. halstedii collected in twelve countries from four continents were investigated for RAPD polymorphism with 21 primers. The study led to a binary matrix, which was subjected to various complementary analyses. This is the first report on the international genetic diversity of the pathogen. Similarity indices ranged from 89% to 100%. Neither a consensus unweighted pair group method with arithmetic means (UPGMA) tree constructed after bootstrap resampling of markers nor a principal component analysis based on distance matrix revealed very consistent clusterings of the isolates, and groups did not fit race or geographical origins. Phylogenies were probably obscured by limited diversity. Analysis of molecular variance (AMOVA) and Nei's genetic diversity statistics gave similar conclusions. Most of the genetic diversity was attributable to individual differences. The most differentiated races also had the lowest within-diversity indices, which suggest that they appeared recently with strong bottleneck effects. Our analyses suggest that this pathogen is probably homothallic or has an asexual mode of reproduction and that gene flow among countries can occur through commercial exchanges. Knowledge of the downy mildew populations' structure at the international level will help to devise strategies for controlling this potentially devastating disease.

First Report of Resistance to Metalaxyl in Downy Mildew of Sunflower Caused by Plasmopara halstedii in Spain.

Fifty-two isolates of Plasmopara halstedii Farl. Berl. & de Toni (causal agent of sunflower downy mildew) collected from sunflower (Helianthus annuus L.) in Spain from 1994 to 2000 were evaluated for metalaxyl resistance. The pathogen was identified on the basis of the morphology of the sporangiophores and zoosporangia recovered on the underside of the leaves (2). Metalaxyl (Apron 20% LS) at 2.0 g a.i./kg of seed (labeled European rate) was applied as seed dressing to the susceptible sunflower 'Peredovik'. There were two replications of 40 plants, and the test was repeated three times. Inoculum (sporangia bearing zoospores) was produced on artificially inoculated plants. Seed were germinated in a humidity chamber at 28°C for 24 to 48 h. When the radicle was 0.5 to 1.0 cm long, untreated and treated seedlings were inoculated by dipping the entire plant in an aqueous suspension of 6.0 × 104 sporangia per ml for 4 h, planted in a sand/perlite mixture (2:3 vol/vol), and grown at 16 to 21°C with a 12-h photoperiod. Plants were incubated for 24 to 48 h at 100% relative humidity and 15°C in the dark to enhance sporulation. After 12 days, disease incidence (DI) of inoculated plants was determined as a percentage of plants displaying sporulation of the fungus on the cotyledons and/or true leaves (3). DI was 95 to 100% for the untreated seedlings, but mildew did not develop on seedlings treated with metalaxyl for 51 of the isolates. The remaining isolate caused symptoms on 67% of the treated plants. This isolate was tested in another experiment in which 'Peredovik' seed was treated with metalaxyl at 0, 0.5, 2.0, 3.5, and 5 g a.i./kg of seed. There were four replications of 12 seedlings per treatment, and seedlings were inoculated as described previously. DI in the untreated control was 77%, which was not significantly different from the DI for seed treated with metalaxyl at 0.5, 2.0, and 3.5 g a.i./kg of seed (97, 73, and 96%, respectively). DI for seed treated with metalaxyl at 5.0 g a.i./kg of seed was 37%, which was significantly lower than the other treatments. Although resistance of P. halstedii to metalaxyl has been reported in France (1), to our knowledge, this is the first report of resistance of sunflower downy mildew to metalaxyl in Spain. References: (1) J. M. Albourie et al. Eur. J. Plant Pathol. 104:235, 1998. (2) G. Hall, Mycopathologia 106:205, 1989. (3) M.L. Molinero-Ruiz et al. Plant Disease 86:736, 2002.

Host Range and Characterization of Sunflower mosaic virus.

ABSTRACT Sunflower mosaic is caused by a putative member of the family Potyviridae. Sunflower mosaic virus (SuMV) was characterized in terms of host range, physical and biological characteristics, and partial nucleotide and amino acid sequence. Cells infected with SuMV had cytoplasmic inclusion bodies typical of potyviruses. Of 74 genera tested, only species in Helianthus, Sanvitalia, and Zinnia, all Asteraceae, were systemic hosts. Commercial sunflower hybrids from the United States, Europe, and South Africa were all equally susceptible. The mean length of purified particles is approximately 723 nm. The virus was transmitted by Myzus persicae and Capitphorus elaegni, and also was seedborne in at least one sunflower cultivar. Indirect enzyme-linked immunosorbent assay tests with a broad-spectrum potyvirus monoclonal antibody were strongly positive. SuMV-specific polyclonal antisera recognized SuMV and, to a lesser extent, Tobacco etch virus (TEV). When tested against a panel of 31 potyvirus-differentiating monoclonal antibodies, SuMV was distinct from any potyvirus previously tested. SuMV shared four epitopes with TEV, but had a reaction profile more similar to Tulip breaking virus (TBV). SuMV did not possess epitopes unique only to TBV. The predicted coat protein had a molecular weight of 30.5 kDa. The 3' end of the virus genome was cloned and sequenced. Phylogenetic analysis of the coat protein amino acid sequence revealed that SuMV is a distinct species within the family Potyviridae, most closely related to TEV.

First Report of Cross-Infectivity of Plasmopara halstedii from Marshelder to Sunflower.

In May 1999, marshelder (Iva xanthifolia Nutt.) plants with chlorotic upper leaves and abundant white sporulation on abaxial surfaces were observed in roadside ditches near Fargo, ND. Ovoid to elliptical zoosporangia (20 to 30 µm × 15 to 20 µm) that were borne on sporangiophores 400 to 500 µm long and branched monopodially at right angles were recovered from infected marshelder leaves. Dimensions of the zoosporangia and sporangiophores fall within those reported for Plasmopara halstedii (Farl.) Berl. & de Toni (1). Zoosporangia rinsed from marshelder plants were used to inoculate 3-day-old sunflower seedlings. The seedlings were immersed in a suspension of 2 × 104 zoosporangia per ml for 3 h, and planted in a greenhouse maintained at 18 to 24°C with a 16-h photoperiod. Systemic chlorosis, stunting, and sporulation were observed on sunflower plants 12 days after inoculation. Isolates from two individual marshelder plants were inoculated on a standard set of nine sunflower downy mildew differential lines, and were identified as race 3 (virulence pattern 700) and race 4 (virulence pattern 730). The marshelder isolates were evaluated for metalaxyl sensitivity by a soil drench inoculation method. Seed of an oilseed sunflower hybrid commercially treated with metalaxyl (138 g a.i./100 kg of seed) were planted in flats filled with sand and perlite (1:1, vol/vol). Three days after planting, the flats were drenched with a zoosporangial suspension (2 × 104 zoosporangia per ml) for four consecutive days. Both isolates produced 100% infection on plants grown from metalaxyl-treated seed, indicating the isolates were metalaxyl-insensitive. While over 80 species within 35 Compositae genera are reported to be hosts for P. halstedii, this is the fourth report of pathogenicity on sunflower by zoospores originating from other genera, the other three being Ambrosia (ragweed) (4), Dimorphotheca (cape marigold) (2) and Xanthium (cocklebur) (3). The cross-infectivity of the P. halstedii from Ambrosia, Iva, and Xanthium indicates that Compositae weeds may serve as a reservoir of P. halstedii to infect cultivated sunflower, and these weeds may help to perpetuate the metalaxyl-insensitive strain of P. halstedii. References: (1) G. Hall, Mycopathologia 106:205, 1989. (2) E. E. Leppik, Plant Dis. Rep. 49:940, 1965. (3) F. Viranyi, Helia 7:35, 1985. (4) I. Walcz et al. Helia 33:19, 2000.

First Report of Charcoal Rot (Macrophomina phaseolina) on Sunflower in North and South Dakota.

In September 1998, symptoms suggestive of charcoal rot were observed on oilseed sunflower (Helianthus annuus L.) plants in western North and South Dakota. Symptoms first observed on plants approaching physiological maturity consisted of silver-gray lesions girdling the stem at the soil line, premature plant death, and reduced head diameter. The pith in the lower stem was completely absent or compressed into horizontal layers. Black, spherical microsclerotia were observed in the pith of the lower stem, underneath the epidermis, and on the exterior of the taproot. Confirmation of Macrophomina phaseolina (Tossi) Goid. as the causal agent was based on the size of the microsclerotia, which ranged from 80 to 90 µm in diameter, from both infected sunflowers and pure cultures (1). The only other sunflower pathogen known to form microsclerotia is Verticillium dahliae Kleb., whose microsclerotia are irregular in shape and are 15 to 50 µm in diameter. Some prematurely dead sunflower plants lacked typical charcoal rot stem lesions, but contained Macrophomina microsclerotia. Plants with atypical symptoms were colonized by the sunflower stem weevil (Cylindrocopturus adspersus (LeConte)) and the black sunflower stem weevil (Apion occidentale Fall). This agrees with observations in Texas, where Macrophomina-infected sunflower plants parasitized by stem-feeding insects often displayed atypical charcoal rot symptoms (3). Charcoal rot incidence in 1998 in western North Dakota was 25%, compared with 0% in eastern North Dakota. Charcoal rot was not observed in 1999, the fourth wettest growing season on record, but was observed again in 2000 and 2001. The recent increase in sunflower production in western North and South Dakota, areas typically hotter and drier than the eastern portions of both states, and the potential involvement of stem weevils as vectors of Macrophomina (2) may lead to an increased incidence of charcoal rot in sunflower. References: (1) P. Holliday and E. Punithalingam. Macrophomina phaseolina. No. 275 in: Description of Pathogenic Fungi and Bacteria, CMI, Kew, Surrey, UK, 1970. (2) S. M. Yang et al. Phytopathology 73:1467, 1983. (3) S. M. Yang and D. F. Owen. Phytopathology 72:819, 1982.

First Report of Albugo tragopogonis on Cultivated Sunflower in North America.

White rust, caused by Albugo tragopogonis (Pers.) S.F. Gray, was observed on a few plants of both oilseed and confection sunflowers (Helianthus annuus L.) in northwestern Kansas (Cheyenne County) in 1992. The disease was observed again from 1993 to1995 in nine counties in western Kansas, with incidence per field ranging up to 35%. White rust was found only on late-planted fields in 1996 and 1997 and was not found at all from 1998 to 2001. White rust was also observed on cultivated and wild sunflower (H. annuus) for the first time in eastern Colorado (Kit Carson and Yuma counties) from 1994 to 1997, but was absent from 1998 to 2001. Leaf pustules on both cultivated and wild sunflowers were similar in appearance. Pustules were convex, chlorotic on the upper side of the leaf, and concave and dull white on the under side of the leaf. Pustules on cultivated sunflower were generally limited to three to six leaves in the middle of the plant and affected 10 to 40% of the leaf area. Sporangial dimensions fell within the reported dimensions for A. tragopogonis (2). In 1997, water-soaked lesions 1 to 2 cm long containing oospores of A. tragopogonia were observed on the lower to middle portions of stems of cultivated sunflower in western Kansas and the adjacent area of Colorado. Stem lesions were observed much less frequently than foliar lesions and only in 1997. Sporangia were not observed in stem lesions, nor were any other fungi isolated from these lesions. To our knowledge, this is the first report of white rust occurring on cultivated sunflower in any production area of North America; the disease has not been observed in the major U.S. sunflower production area of North Dakota, South Dakota, and Minnesota. Foliar white rust lesions generally have little economic impact on sunflower, but the presence of stem lesions is significant because stem lesions may lead to lodging (3). Lodging due to A. tragopogonia was not observed in either Kansas or Colorado. White rust has previously only been reported on wild H. annuus in Wisconsin and on perennial Helianthus spp. in Missouri and Illinois (1). References: (1) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN 1989. (2) K. G. Mukeri. Description of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK 1976. (2) P. S. van Wyk et al. Helia 22:83, 1995.

First Report of Puccinia canaliculata on Sunflower.

Puccinia canaliculata (Schwein.) Lagerh. is a macrocyclic, heteroecious rust found on Cyperus spp. (sedges) in North and South America, with the aecial stage reported on Asteraceae (1). In July 1997, aecial pustules of unknown etiology were observed on lower leaves of more than 90% of cultivated sunflower (Helianthus annuus L.) plants at the 6- to 10-leaf stage in a field near Catherine, KS. Individual leaves had one to several large, convex pustules, each measuring 5 to 10 mm in diameter, covering <0.5% of the leaf area. In contrast, the aecial pustules of P. helianthi measure 1 to 2 mm in diameter. Yellow-orange aecial cups occurred on the abaxial leaf surface, with globoid aeciospores averaging 15 × 18.5 µm. Wild common sunflowers (H. annuus) and cocklebur (Xanthium strumarium L.) in the same and two nearby fields had similar pustules. There was a severe infestation of yellow nutsedge (C. esculentus L.) in the sunflower field, but uredia were not found because the nutsedge plants had been killed with glyphosate. Based on the size of aecia, aeciospores, and peridial cells from sunflower and cockelbur (3), the fungus was identified as P. canaliculata. Although sunflower is an alternate host (2), to our knowledge this is the first report of natural infection and is significant because it occurred in a major sunflower production area. Since P. canaliculata is being considered as a bioherbicide for nutsedge control (4), nontarget hosts such as sunflower need to be considered. The possibility of confusing P. xanthii and P. caniculata exists since both rusts occur on cocklebur and sunflower and produce pustules of a similar size. However, since P. xanthii is a microcyclic autoecious rust while P. canaliculata is a full-cycle heteroecious rust, the obvious color difference between the dark telia of P. xanthii and the yellow-orange aecia of P. caniculata should serve to easily differentiate the two species. References: (1) J. C. Arthur. Manual of the Rusts in the United States and Canada. Purdue Research Foundation, Lafayette, IN, 1934. (2) M. B. Callaway et al. Plant Dis. 69:924, 1985. (3) F. D. Kern. Mycologia 11:134, 1919. (4) S. C. Phatak et al. Science. 219:1446, 1983.

First Report of Puccinia xanthii on Sunflower in North America.

Puccinia xanthii Schwein., commonly known as cocklebur rust, is circumglobal on species of Xanthium and Ambrosia. This microcyclic rust has only been observed on oilseed sunflower (Helianthus annuus L.) in Australia (1) and on ornamental sunflowers in South Africa (4). In September 1999, large (4 to 10 mm), raised, chlorotic pustules were observed on the adaxial leaf surface of oilseed sunflower plants (Dekalb 3790) near Hettinger, ND. Telia were associated with the pustules on the abaxial leaf surface. No cocklebur (X. strumarium L.) plants were found in the field, but rust-infected cocklebur plants were collected several kilometers away. Approximately 10% of sunflower plants in the field were affected, and generally only one or two pustules were observed on one or two leaves per plant. In contrast, numerous leaves of cockleur plants were infected with 12 or more pustules. Teliospores from sunflower were brown, two-celled, and averaged 49 × 17 µm, with a distinctly thicker wall at the spore apex and a persistent pedicel averaging 40 µm long. Teliospores from cocklebur were morphologically similar to those from sunflower and averaged 46 × 16 µm. Size and morphology of teliospores from both hosts fit the description of P. xanthii (2). P. xanthii can be distinguished easily from the ubiquitous P. helianthi Schwein. because the latter has smaller telia (1 to 2 mm diameter) and produces wider teliospores (21 to 30 µm diameter). P. xanthii was not found in surveys of 20 other sunflower fields in southwestern North Dakota nor in 45 fields in eastern ND in 1999, nor was P. xanthii found in this or any other sunflower field in 2000 or 2001. To our knowledge, this is the first report of P. xanthii on cultivated or wild sunflower in North America. The relatively few pustules observed on oilseed sunflower agree with the observation that oilseed sunflowers are much less susceptible to P. xanthii (3) than Xanthium spp. References: (1) J. L. Alcorn and J. K. Kochman. Austral. Plant Pathol. Soc. Newsl. 5:33, 1976. (2) G. B. Cummins. Rust Fungi on Legumes and Composites in North America. University of Arizona Press, Tucson, 1978. (3) J. B. Morin et al. Can. J. Bot. 71:959, 1993. (4) Z. A. Pretorius et al. Plant Dis. 84:924, 2000.