Sunflower downy mildew is a disease of high global economic impact as well as a causal agent that is extremely difficult to eradicate. During the past decades, several approaches for the determination of Plasmopara halstedii (Ph) races have been used worldwide and are discussed in this review. 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. ‘Soil drench’ inoculation allows more precise control of the 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 determination, 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 determination, single zoosporangia or single zoospore isolates are advisable. However, due to low success of isolation, approximately 1–2%, this method cannot be applied in routine Ph race screening. Methods surveyed in this review have a broad spectrum of applications, including taxonomic studies.

Resistant Lactuca spp. genotypes can efficiently modulate levels of S-nitrosothiols as reactive nitrogen species derived from nitric oxide in their defence mechanism against invading biotrophic pathogens including lettuce downy mildew. S-Nitrosylation belongs to principal signalling pathways of nitric oxide in plant development and stress responses. Protein S-nitrosylation is regulated by S-nitrosoglutathione reductase (GSNOR) as a key catabolic enzyme of S-nitrosoglutathione (GSNO), the major intracellular S-nitrosothiol. GSNOR expression, level and activity were studied in leaves of selected genotypes of lettuce (Lactuca sativa) and wild Lactuca spp. during interactions with biotrophic mildews, Bremia lactucae (lettuce downy mildew), Golovinomyces cichoracearum (lettuce powdery mildew) and non-pathogen Pseudoidium neolycopersici (tomato powdery mildew) during 168 h post inoculation (hpi). GSNOR expression was increased in all genotypes both in the early phase at 6 hpi and later phase at 72 hpi, with a high increase observed in L. sativa UCDM2 responses to all three pathogens. GSNOR protein also showed two-phase increase, with highest changes in L. virosa-B. lactucae and L. sativa cv. UCDM2-G. cichoracearum pathosystems, whereas P. neolycopersici induced GSNOR protein at 72 hpi in all genotypes. Similarly, a general pattern of modulated GSNOR activities in response to biotrophic mildews involves a two-phase increase at 6 and 72 hpi. Lettuce downy mildew infection caused GSNOR activity slightly increased only in resistant L. saligna and L. virosa genotypes; however, all genotypes showed increased GSNOR activity both at 6 and 72 hpi by lettuce powdery mildew. We observed GSNOR-mediated decrease of S-nitrosothiols as a general feature of Lactuca spp. response to mildew infection, which was also confirmed by immunohistochemical detection of GSNOR and GSNO in infected plant tissues. Our results demonstrate that GSNOR is differentially modulated in interactions of susceptible and resistant Lactuca spp. genotypes with fungal mildews and uncover the role of S-nitrosylation in molecular mechanisms of plant responses to biotrophic pathogens.

Plasmopara halstedii was isolated from diseased sunflowers collected from eight locations in the Czech Republic from 2007 to 2014. Races of the pathogen were determined based on 84 isolates collected during the study. In total, eight races of P. halstedii were detected using a set of nine sunflower differential lines. Races 700, 704, 705, 710, 714 and 715 were proven by soil drench inoculation, and two additional races (730 and 770) proposed by the previously applied leaf disc inoculation method. Race 700 was the most dominant in the Czech P. halstedii populations, with race 710 being the second most frequent. Races 704 and 714 were found over three seasons, while other races were recorded only in one growing season (race 730 in 2010, and the new races 705 and 715 in 2014). A comprehensive study was further conducted for isolates collected in 2013-14 using an extended differential set consisting of 15 sunflower lines. According to the latter methodology which marks races with five-digit virulence codes, races 70060, 70471, 70571, 71060, 71461 and 71571 were recorded. The growing complexity of P. halstedii pathogenicity exhibited by the ability to infect higher numbers of differential genotypes and resulting in determination of the new pathogen races (virulence profiles) 70571, 71461 and 71571 is alarming. Although the limited number of isolates studied cannot characterize the entire pathogen diversity in the Czech Republic, the trend towards more diverse virulence in P. halstedii populations is clearly demonstrated by the new records of races 704, 705, 714 and 715, all capable of overcoming the resistance gene Pl 6 .

Sedlářová M., Trojanová Z., Lebeda A. (2013): Distribution and harmfulness of Plasmopara halstedii on sunflower in the Czech Republic. Plant Protect. Sci., 49: 1-10.In 2007-2012 the first detailed study of P. halstedii distribution and severity was performed in the Czech Republic by monitoring altogether 128 localities. Incidence of the pathogen was recorded at seven localities in south-eastern (Central and South Moravia) and central parts (East and Central Bohemia) of the country; at only four localities the occurrence of sunflower downy mildew symptoms and pathogen sporulation were recorded repeatedly. In all cases the primary infection prevailed, and the severity of infections was rather low. The majority of records were for sunflower experimental fields (e.g. fields of Central Institute for Supervising and Testing in Agriculture. Brno-Chrlice, Czech Republic), one record came from a commercial field and one record from a hobby field. Results of surveys indicate that reservoirs of primary inoculum likely exist in the soil contaminated by P. halstedii oospores in these habitats, but additional transfer by infected seed may be possible. Historical and geographical consequences of recorded infections are discussed, as well as virulence variation in the Czech pathogen populations which is currently in the process of investigation.

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Sedlářová M., Pospíchalová R., Drábková Trojanová Z., Bartůšek T., Slobodianová L., Lebeda A.