Agent Of Sunflower Downy Mildew Research Articles

Инкубационный период возбудителя ложной мучнистой росы подсолнечника разной расовой принадлежности в зависимости от температуры

Инкубационный период возбудителя ложной мучнистой росы подсолнечника разной расовой принадлежности в зависимости от температуры

Molecular mapping of the downy mildew and rust resistance genes in a sunflower germplasm line TX16R

The sunflower germplasm line TX16R is resistant to Plasmopara halstedii (causal agent of sunflower downy mildew) and Puccinia helianthi (causal agent of sunflower rust), which are two destructive foliar diseases in sunflower production worldwide. This study reports the mapping of the downy mildew and rust resistance genes Pl33 and R16 from TX16R, respectively. Progeny testing of test crosses for downy mildew resistance suggested that Pl33 localizes to linkage group (LG) 4 of the sunflower genome. Molecular mapping of Pl33 using simple sequence repeat (SSR) and single-nucleotide polymorphism (SNP) markers identified Pl33 cosegregating with ORS644, ORS963, SFW04901, and SFW04052, and linking to two SNPs, NSA_006089 and NSA_008496, at a genetic distance of 0.2 cM on the proximal side. Bulked segregant analysis using SSR and EST-SSR markers from LGs previously reported for rust genes identified polymorphic SSR markers associated with rust resistance on LG13. R16 was mapped between SFW08875 and SFW04317 on LG13, with a genetic distance of 1.8 and 1.1 cM, respectively. The 15 linked markers span a genetic distance of 27.4 cM in LG13. The cosegregating or closely linked markers to the two resistance genes will facilitate marker-assisted selection (MAS) and gene pyramiding, and will further assist in identifying genes responsible for DM and rust resistance.

Occurrence and Distribution of Races 713, 733 and 734 of Sunflower Downy Mildew Pathogen in the Russian Federation

AbstractThe samples of Plasmopara halstedii (the causal agent of sunflower downy mildew) were collected on sunflower fields in south of the Russian Federation: in the Krasnodar, Rostov and Stavropol regions, and in the Republic of Adygea in 2016 and 2017. Virulence code of 545 isolates was identified; i. e. 280 isolates originating from 24 fields in 2016, and 265 isolates from 17 fields in 2017. Races 330, 334, 710 and 730, widespread within the region in previous years, were found in all mentioned areas. In addition, races 713, 733 and 734 have been detected in several districts of the Krasnodar region since 2016. This is the first report of these races in Russia and above that the first record throughout Europe and Asia for the races 733 and 734. The identification of new P. halstedii races was confirmed by the method of cross inoculations from individual differential lines. The phenotype corresponding to virulence code 734 was in some cases disclosed as a mixture of race 334 with 710 or 730. The presence of several P. halstedii races on an individual plant was also confirmed for the first time in the territory of the Russian Federation.

Recent Changes in the Pathogenic Variability of Plasmopara Halstedii (Sunflower Downy Mildew) Populations from Different Continents

AbstractThe 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.

Pathogenic, morphological and genetic diversity in Plasmopara halstedii, the causal agent of sunflower downy mildew

Pathogenic, morphological and genetic variation was studied in 35 Plasmopara halstedii (sunflower downy mildew) isolates of different races. Virulence spectrum was analyzed in sunflower hybrids carrying effective Plgenes. Aggressiveness was analyzed in one sunflower inbred line showing a high level of quantitative resistance. There were differences in virulence spectrum for pathogen isolates. Index of aggressiveness was calculated for each isolate and two groups were revealed as more aggressive isolates of 100 and 3xx races, and less aggressive isolates of 7xx races. Significant morphological differences were found in zoosporangia and sporangiophores morphology. Genetic relationships were detected between the pathogen isolates using 12 EST-derived markers. Five multilocus genotypes (MLG) were identified among 35 P. halstedii isolates. Our results did not show a correlation between pathogen variation and both morphological and genetic characteristics.

Single nucleotide polymorphisms reveal multiple introductions into France of Plasmopara halstedii, the plant pathogen causing sunflower downy mildew

Plasmopara halstedii, the causal agent of sunflower downy mildew, displays a gene-for-gene interaction with its host plant, Helianthus annuus and other species of the genus. Monitoring of the evolution of virulent races in France over a 19-year period led to the identification of 14 different races (or pathotypes). Twelve expressed sequence tag (EST)-derived markers displaying SNPs and insertion–deletion variations have recently been identified in P. halstedii. We used these markers to study the genetic structure and the evolution of sunflower downy mildew races. Bayesian assignment analysis identified three genetically differentiated groups of isolates organized around the first three races described in France. Strong genetic substructuring according to geographic origin of races was observed, confirming that these three groups corresponded to three separate introductions into France of isolates with different genetic and phenotypic backgrounds. Our results suggest that multiple introductions of P. halstedii isolates may have provided the raw material for more complex processes in the evolution of races, such as recombination between races or clonal evolution through mitotic instability.

Isozyme analysis of Plasmopara halstedii using cellulose‐acetate gel electrophoresis

Isozyme analysis by cellulose‐acetate gel electrophoresis was used for the first time on Plasmopara halstedii, the causal agent of sunflower downy mildew. Forty‐five isolates originating from sunflower, cocklebur and Helianthus × laetiflorus were used, comprising 10 field isolates and 35 single‐spore lines of an additional 30 field isolates representing 10 different virulence phenotypes. Sixteen isozyme systems were analysed, of which three, isocitrate dehydrogenase, malate dehydrogenase and phosphoglucomutase, resulted in clear, reproducible banding patterns and revealed some polymorphism among the isolates. Phosphoglucomutase differentiated two groups among the isolates collected from cultivated sunflower, while the other enzymes were polymorphic between isolates from the different hosts. Polymorphisms were not related to virulence phenotype.

Twelve polymorphic expressed sequence tags‐derived markers for Plasmopara halstedii, the causal agent of sunflower downy mildew

AbstractTwelve 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.

Transient expression of gfp in the obligate biotrophic oomycete Plasmopara halstedii using electroporation and a mechanoperforation method

Studying plant biotrophic oomycetes is difficult, because they depend on living plant cells for nutrition, which necessitates investigations in planta. Internal optical labelling is a powerful tool for analysing intra‐ and interspecific interactions. Different approaches are described for establishing a gfp‐transformation system for Plasmopara halstedii, the causal agent of sunflower downy mildew. A vector containing gfp and the oomycete‐specific ham34 regulators from Bremia lactucae was constructed for transformation experiments. Particle bombardment of infected sunflower cotyledons during different developmental stages of the pathogen did not result in successful transformation. In contrast, transient gfp expression in sporangia was achieved using electroporation. However, gfp expression was lost during the subsequent round of infection. A novel transformation method for biotrophic organisms in planta employing mechanoperforation – so‐called Löchern–resulted in sporangiophores of P. halstedii transiently expressing gfp and emerging distant from the site of transformation. The new technique is advantageous compared with others as the transformed hyphae can recover during their vegetative growth, before reproductive structures occur. This first step lays an important foundation for further investigations of obligate biotrophic organisms.

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.

Microscopical studies of the effect of metalaxyl on the interaction between sunflower,Helianthus annuus L. and downy mildew,Plasmopara halstedii

The mode of action of the fungicide metalaxyl againstPlasmopara halstedii, the causal agent of sunflower downy mildew, was studied following its application before, during and after artificial contamination of seedlings. The development of the fungus within the treated seedlings was examined microscopically and compared to that occuring in untreated genetically susceptible or resistant genotypes. Hypersensitive-like reactions and necrotic zones leading to the inhibition of fungus growth within the hypocotyl were observed for the three modes of application. This suggests that sunflower defence mechanisms activated by genetical resistance are also involved in the control of downy mildew by metalaxyl.

Preservation of air-dried downy mildew sporangia in liquid nitrogen without cryoprotectants or controlled freezing

Zoosporangia of Plasmopara halstedii , causal agent of sunflower downy mildew, were infective after storage in liquid nitrogen for up to 4·75 yr, using a dry spore, fast-freeze (DSFF) technique. The DSFF technique consists of vacuuming air-dried zoosporangia with a cyclone collector, placing them in a polypropylene screw-cap tube without any cryoprotectant, and immediately placing them in a liquid nitrogen storage tank. Addition of a cryoprotectant or the use of a controlled freezing rate did not enhance infectivity of zoosporangia stored in liquid nitrogen over that of the basic DSFF method. The ability of air-dried zoosporangia to withstand liquid nitrogen storage without cryoprotectants appears to be due to their lower water content compared to that of spores collected by washing and filtering. Spores stored in liquid nitrogen by the DSFF technique were infective after five freeze/thaw cycles. The technique was also successful with Peronospora parasitica and Pseudoperonospora cubensis sporangia, but not with Peronosclerospora sorghi .

Isolation and characterization of a mycovirus from Plasmopara halstedii

A virus was isolated from Plasmopara halstedii, the causal agent of sunflower downy mildew. The virus contained two species of ssRNA with molecular weight (MW) of 1.05 × 106 and 0.53 × 106. One polypeptide with a MW of 37.5 × 103 was associated with whole virus. Purified preparations consisted of two components, the top component consisting primarily of empty shells. The A260/A280 UV absorbance ratio for the bottom component ranged from 1.25 to 1.35. The partially purified virions were isometric, approximately 32 nm in diameter, and had projections of approximately 4 nm on the virion surface. This virus may be related to the type A virus in Sclerophthora macrospora. Key words: mycovirus, downy mildew, Helianthus annuus.

Ultrastructure of virus-like particles in Plasmopara halstedii

Virus-like particles were observed in five North Dakota isolates of Plasmopara halstedii race 2, the causal agent of sunflower downy mildew. Virus-like particles were observed in fungal mycelium within sunflower seedlings showing typical symptoms of systemic infection as well as from seedlings displaying "twisted leaf syndrome." Virus-like particles were never observed in sunflower cells. The particles were isometric in shape and measured 23–26 nm in thin sections and 32 nm after phosphotungstic acid staining. Virus-like particles occurred dispersed throughout the cytoplasm and in membrane-bound organelles. Both crystalline and noncrystalline arrays were common. The particles were observed in haustoria, intercellular hypha, sporangiophores, zoosporangia, and oospores. Four species of double-stranded RNA were detected in sunflower tissue infected with P. halstedii; none were detected in healthy sunflower tissue. This is only the second report of an isometric, virus-like particle occurring in a member of the Peronosporales. Key words: mycovirus, downy mildew, Helianthus annuus.

New Races of the Sunflower Downy Mildew Pathogen (Plasmopara halstedii) in Europe and North and South America

AbstractCollections of the causal agent of sunflower downy mildew (Plasmopara balstedii) (PH) from several European and North and South American countries were used to inoculate a series of sunflower inbred lines to differentiate races. Race 3 was identified from Argentina; race 4 from France, Hungary, and Bulgaria; race 6 from Canada and France; and race 7 from Argentina. This is the first report of race 3 in South America and of race 4 in Europe, and is the first identification of races 6 and 7. Reactions of USDA lines RHA‐274, RHA‐325, and DM‐2 differentiate races 2, 6, and 7, with RHA‐274 resistant to all three. Thirty‐three commercial hybrids from 11 countries were tested; 70 % were resistant to race 2, only 27 % had resistance to race 6, and none were resistant to race 4. Surveys to determine the relative proportions of the various races within each country are needed to assess their potential impact. The USDA lines HA‐335, HA‐336, HA‐337, HA‐338, HA‐339, RHA‐340, HA‐R4, and HA‐R5 are resistant to all seven mildew races and could be utilized for the production of PH resistant hybrids. For the short term, it appears that seed treatment with metalaxyl may be the most effective control.

Ultrastructure of Plasmopara halstedii containing virus-like particles

Mycoviruses have been found in more than 100 species of fungi and it is estimated that they may occur in as many as 500 species (Buck, 1986; Hollings, 1978; and Lemke, 1981). Mycoviruses range in size from small, isometric particles (25 nm in diameter) to long, flexuous rods exceeding 2000 nm in length, with the majority being isometric particles ranging in size from 25-50 nm (Buck, 1986). Mycoviruses hae been reported in all groups of fungi, but have been least frequently observed in the Oomycetes.Sunflower (Helianthus annuus) seedlings were inoculated with a North Dakota isolate of race 2 Plasmopara halstedii, the causal agent of sunflower downy mildew. Systemic disease symptoms developed within 14 days and samples were prepared for transmission electron microscopy using standard techniques.