Article
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

The objective of this work was to find practical procedures to overcome methodological drawbacks encountered during studies on sunflower downy mildew. Techniques for recovering living isolates of Plasmopara halstedii from the field and for the preservation of infected leaf samples for further molecular analysis were developed. A Polymerase Chain Reaction (PCR)-based test for the detection of P. halstedii in sunflower leaves and a method to remove azoxystrobin from fungicide-treated seeds are proposed. In situ -inoculations of pre-germinated seeds allowed the recovery of living isolates from the field. Three sample-preservation methods were evaluated (silica, heating and lyophilization) resulting in high yield and quality of the DNA extract. It was detected the presence of the pathogen in symptomless leaves through PCR using molecular markers based on expressed sequence tags. A treatment using sodium hypochlorite is recommended for the removal of azoxystrobin from fungicide treated seeds.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

ResearchGate has not been able to resolve any citations for this publication.
Article
Full-text available
Resistance to downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.) is conferred by major resistance genes, denoted Pl. Twenty-two Pl genes have been identified and genetically mapped so far. However, over the past 50 years, wide-scale presence of only a few of them in sunflower crops led to the appearance of new, more virulent pathotypes (races) so it is important for sunflower varieties to carry as wide a range of resistance genes as possible. We analyzed phenotypically 12 novel resistant sources discovered in breeding pools derived from two wild Helianthus species and in eight wild H. annuus ecotypes. All were effective against at least 16 downy mildew pathotypes. We mapped their resistance genes on the sunflower reference genome of 3,600 Mb, in intervals that varied from 75 Kb to 32 Mb using an AXIOM® genotyping array of 49,449 SNP. Ten probably new genes were identified according to resistance spectrum, map position, hypersensitive response to the transient expression of a P. halstedii RXLR effector, or the ecotype/species from which they originated. The resistance source HAS6 was found to carry the first downy mildew resistance gene mapped on chromosome 11, whereas the other resistances were positioned on chromosomes 1, 2, 4, and 13 carrying already published Pl genes that we also mapped physically on the same reference genome. The new genes were designated Pl23–Pl32 according to the current nomenclature. However, since sunflower downy mildew resistance genes have not yet been sequenced, rules for designation are discussed. This is the first large scale physical mapping of both 10 new and 10 already reported downy mildew resistance genes in sunflower.
Article
Full-text available
Genetically homogenous strains of Plasmopara halstedii differing in host specificity and fungicide tolerance were used to test the hypothesis that asexual genetic recombination occurs and may account for the high genotype diversity of this homothallic reproducing oomycete, which causes downy mildew in sunflower. Dual inoculation of sunflower seedlings with single zoospore strains of complementary infection characteristics caused sporulation under conditions where inoculation with each strain alone failed to infect. PCR-based investigation with strain-specific primers proved the presence of genetic traits from both progenitors in single sporangia collected from sporangiophores of such infections. Sister zoospores released from these sporangia revealed the genotype of the one or the other parental strain thus indicating heterokaryology of sporangia. Moreover, some zoospores showed amplification products of both parents, which suggests that the generally mononucleic spores derived from genetic recombination. The possibility of parasexual genetic exchange in the host-independent stage of infection and the evolutionary consequences are discussed.
Article
Full-text available
Sunflower downy mildew is a disease of high global economic impact as well as a causal agent extremely difficult to eradicate. Several approaches for the determination of Plasmopara halstedii (PH) races have been used worldwide during the past decades and are discussed herein. Procedures of isolation, cultivation and maintenance of PH isolates, as well as the screening of sunflower for resistance are also critically reviewed. The predominant, globally used resistance screening protocol is a ‘whole seedling immersion’ inoculation. ʻSoil drenchʼ inoculation allows more precise control of a number of PH zoosporangia applied to a single sunflower seedling. A detached leaf assay has been described, but it has been used mainly for PH subcultivation and fungicide tests. For race determinations, a differential set consisting of nine sunflower genotypes has been used since 1988, coupled with a numerical triplet code for virulence phenotyping of PH. The increasing variability in global PH populations has demonstrated the inadequacy of the current set of differentials, and several researchers have proposed additional public lines as new differentials. Furthermore, bulk isolates may show different results in repeated tests, as PH may contain genetically distinct zoospores within a single zoosporangium. For precise race determinations, single zoosporangia or single zoospore isolates are advisable. However, due to low success of isolation, reaching 1-2%, these cannot be applied in routine PH race screening. Methods surveyed in this review have a broad spectrum of applications, including taxonomic studies. This article is protected by copyright. All rights reserved.
Article
Full-text available
The obligate biotrophic Oomycete, Plasmopara halstedii, causal agent of sunflower downy mildew, is capable of producing new pathogenic races over time. Although changes in the P. halstedii race composition were reviewed for the first time in 2007, since then the pathogen has continued to change its virulence character dramatically. There was a need, therefore, to update information on pathogenic diversity of P. halstedii by making accounts of the temporal and spacial changes in the pathogen populations in North and South America and Europe. This paper, based on current publications and personal communications, attempts to present an accurate overview of races in Europe and Americas for the last 7 years.
Article
Full-text available
Sunflower downy mildew, caused by Plasmopara halstedii, can be an economic problem in North Dakota, where approximately half of the United States sunflower crop is grown. Prevalence and incidence of downy mildew in North Dakota was assessed in mid-season and late-season surveys annually from 2001 to 2011. In aggregate, 2,772 fields were evaluated. Downy mildew levels fluctuated annually, and no clear connection between statewide rainfall or management tools available to growers could be made. Surveys at mid season enabled a two-fold higher detection in prevalence and incidence of downy mildew than in late-season surveys due to the disappearance of early infected plants by end of season. Both surveys identified years with relatively higher levels of downy mildew but were inconsistent when lower levels of the pathogen were recorded. The results of this study suggest that mid-season assessment of downy mildew more accurately estimates disease pressure than late-season assessment. Accepted for publication 3 February 2013. Published 22 May 2013.
Article
Unlabelled: Downy mildew of sunflower is caused by Plasmopara halstedii (Farlow) Berlese & de Toni. Plasmopara halstedii is an obligate biotrophic oomycete pathogen that attacks annual Helianthus species and cultivated sunflower, Helianthus annuus. Depending on the sunflower developmental stage at which infection occurs, the characteristic symptoms range from young seedling death, plant dwarfing, leaf bleaching and sporulation to the production of infertile flowers. Downy mildew attacks can have a great economic impact on sunflower crops, and several Pl resistance genes are present in cultivars to protect them against the disease. Nevertheless, some of these resistances have been overcome by the occurrence of novel isolates of the pathogen showing increased virulence. A better characterization of P. halstedii infection and dissemination mechanisms, and the identification of the molecular basis of the interaction with sunflower, is a prerequisite to efficiently fight this pathogen. This review summarizes what is currently known about P. halstedii, provides new insights into its infection cycle on resistant and susceptible sunflower lines using scanning electron and light microscopy imaging, and sheds light on the pathogenicity factors of P. halstedii obtained from recent molecular data. Taxonomy: Kingdom Stramenopila; Phylum Oomycota; Class Oomycetes; Order Peronosporales; Family Peronosporaceae; Genus Plasmopara; Species Plasmopara halstedii. Disease symptoms: Sunflower seedling damping off, dwarfing of the plant, bleaching of leaves, starting from veins, and visible white sporulation, initially on the lower side of cotyledons and leaves. Plasmopara halstedii infection may severely impact sunflower seed yield. Infection process: In spring, germination of overwintered sexual oospores leads to sunflower root infection. Intercellular hyphae are responsible for systemic plant colonization and the induction of disease symptoms. Under humid and fresh conditions, dissemination structures are produced by the pathogen on all plant organs to release asexual zoosporangia. These zoosporangia play an important role in pathogen dissemination, as they release motile zoospores that are responsible for leaf infections on neighbouring plants. Disease control: Disease control is obtained by both chemical seed treatment (mefenoxam) and the deployment of dominant major resistance genes, denoted Pl. However, the pathogen has developed fungicide resistance and has overcome some plant resistance genes. Research for more sustainable strategies based on the identification of the molecular basis of the interaction are in progress. Useful websites: http://www.heliagene.org/HP, http://lipm-helianthus.toulouse.inra.fr/dokuwiki/doku.php?id=start, https://www.heliagene.org/PlasmoparaSpecies (soon available).
Article
Plants infected with downy mildew were collected from 1994 to 2000 in sunflower fields in Spain. The race of 102 bulk isolates of Plasmopara halstedii obtained from them was determined by inoculation of sunflower lines traditionally used as differentials for characterization of the pathogen. Nine different races of the fungus were determined. Although race I was most common and was the only one found in central Spain, races 4, 6, and 7 were widespread in southern Spain. The results allowed the identification of a new race of the pathogen, race 10, and of a race not previously reported in Europe, race 8. According to the proposal of a new system for characterization of the isolates of P halstedii in the late 1990s, Coded Virulence Formulae (CVF) were assigned to bulk isolates and single-sporangium (ss) isolates obtained from them. The CVF of the bulk isolates (CVFi) did not always fit with the previous designation. Similarly, ss isolates from the same bulk isolate exhibited different CVF, not only among themselves, but also compared with the CVF of the source isolate. Although a revision of the differential lines used to perform the racial characterization of fungal isolates seems to be needed, the occurrence of a diversity of genotypes in field populations of R halstedii and a high frequency of recombination and/or mutation of the fungal genome is also suggested.
Article
Sunflowers bud-inoculated with Plasmopara halstedii became systemically infected with downy mildew and produced infected seeds at 15 to 30 °C. Oospores were observed in seeds of inoculated and also naturally infected plants from the field. Infected seeds gave rise to symptomless plants. Infected seeds proved effective as inoculum, inducing infection in 14 to 89% of the plants inoculated at 20 °C. Most of the infections (80%) were symptomless (latent).Systemic infection occurred, but latent infection was more frequent, in plants grown in soil containing debris of mildewed plants. Latent infection also occurred in uninoculated plants through belowground contact with systemically infected plants. Seed produced by plants with latent infection may carry the pathogen, and may give rise to more plants with latent infection, accounting for widespread dissemination of the disease before plants with typical symptoms are observed.
Article
Studies on the effects of solvents on germinability of seeds of various crops established the optimum conditions for immersing seeds in several solvents. Reduction in germinability, in vitro and in soil, of seeds of cotton, bean, pea, soya bean, and sugar beet after immersion in acetone, dichloromethane, or ethanol for various periods up to 24 h depended upon the length of immersion and the seed type and solvent used. Soya-bean and sugar-beet seeds were the least sensitive to the solvents whereas bean and cotton seeds were the most sensitive. Various cultivars of the same crop behaved similarly. The amount of seed germination in vitro was similar to that in soil. Solvent combinations consisting of acetone with dimethylsulphoxide, dichloroethane. polyethylene glycol, or triethanolamine did not affect cotton-seed germination in soil whereas soya-bean seeds were adversely affected by some materials. The amount of solvent taken up by the seeds and the amount of oxidizable matter extracted by the solvent depended upon the solvent and the crop. Maximum absorption of solvent occurred within 1 h. The amount of oxidizable material diffusing from seeds immersed in solvents was not correlated with the amount of solvent uptake or extent of seed germination.(Received October 02 1978)
Article
A technique was developed to select vital zoospores of Plasmopara haistedii after zoosporogenesis for the infection of sunflower leaf disks. Successful infections were performed with samples containing several zoospores originated from a single sporangium as well as with samples containing only one single zoospore. Infections were indicated by spontaneous sporulation at the surface of inoculated sunflower leaf discs and were secured by transfer to whole seedlings. Using this method it was possible to generate unithallic infections and genetically homogenous strains of sunflower downy mildew. Es wurde ein Verfahren zur selektiven Gewinnung einzelner, frisch geschltüpfter Zoosporen von Plasmopara halstedii für die Infektion von Blattscheiben der Sonnenblume entwickelt. Erfolgreiche Infektionen wurden einerseits mit Proben erhalten, die mehrere, gemeinsam entstandene Zoosporen aus einem einzelnen Sporangium enthielten, als auch mit solchen, in denen sich nur eine einzige Zoospore befand. Die Infektionen zeigten sich durch spontane Sporulation an der Oberfläche inokulierter Blattscheiben und wurden danach auf Sonnenblumenkeimlinge übertragen. Die Methode erlaubt die Erzeugung unithallischer Infektionen und die Herstellung genetisch einheitlicher Stämme und von Plasmopara halstedii.
Article
Twelve expressed sequence tags-derived markers were isolated from Plasmopara halstedii (Oomycetes), the causal agent of sunflower downy mildew. A total of 25 single nucleotide polymorphisms and five indels were detected by single-strand conformation polymorphism analysis and developed for high-throughput genotyping of 32 isolates. There was a high level of genetic diversity (HE = 0.484). Observed heterozygosity ranged from 0 to 0.143 indicating that P. halstedii is probably a selfing species. These markers were also useful in detecting significant genetic variations among French populations (FST = 0.193) and between French and Russian populations (FST = 0.23). Cross-amplification tests on three closely related species indicated that no loci amplified in other Oomycete species.
Proposed internationally standardized methods for race identification of Plasmopara halstedii
  • T J Gulya
  • J F Miller
  • F Viranyi
  • W E Sackston
Gulya, T.J., Miller, J.F., Viranyi, F., Sackston, W.E., 1991. Proposed internationally standardized methods for race identification of Plasmopara halstedii. Helia 14(15): 11-20.
CMI descriptions of pathogenic fungi and bacteria, Set 98, Nos. 971-980
Hall, G., 1989. CMI descriptions of pathogenic fungi and bacteria, Set 98, Nos. 971-980. Mycopathologia 106(3): 183-211.
Laboratory protocols: CIMMYT applied molecular genetics laboratory
  • D Hoisington
  • D Gonzalez De Leon
  • M Khairallah
Hoisington, D., Gonzalez de Leon, D., Khairallah, M., 1992. Laboratory protocols: CIMMYT applied molecular genetics laboratory (No. 14756).
Prevalence and incidence of sunflower downy mildew in North Dakota between
  • T Gulya
  • H Kandel
  • M Mcmullen
  • J Knodel
  • D Berglund
  • F Mathew
  • S Markell
Gulya, T., Kandel, H., McMullen, M., Knodel, J., Berglund, D., Mathew, F., … Markell, S., 2013. Prevalence and incidence of sunflower downy mildew in North Dakota between 2001 and 2011. Plant Health Progress 14(1): 20. DOI:10.1094/PHP-2013-0522-01-RS.
Evaluation of wild annual Helianthus species for resistance to downy mildew and Sclerotinia stalk rot
  • T J Gulya
Gulya, T.J., 2005. Evaluation of wild annual Helianthus species for resistance to downy mildew and Sclerotinia stalk rot. In: Proc 27th Sunflower Res forum, Fargo, N.D., pp. 12-13.