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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. Race 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 2013-2014 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 ability to infect higher number of differential genotypes and resulting in determination of the new pathogen races (virulence profiles) 70571, 71461 and 71571 is alarming. Though 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 Pl6. This article is protected by copyright. All rights reserved.

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.

Pseudoperonospora cubensis is an oomycete pathogen causing downy mildew disease on a variety of Cucurbitaceae, and has recently re-emerged as a destructive disease on crops in this family, mainly on cucumber and squash. Multilocus sequence analysis (MLSA) of four mitochondrial and two nuclear DNA regions was used to detect changes in the genetic structure of P. cubensis populations occurring in the Czech Republic that might be associated with recently reported shifts in virulence. The analysed sample set contains 67 P. cubensis isolates collected from 1995 to 2012 in the Czech Republic and some other European countries. Sequence analyses revealed differences and changes in the genetic backgrounds of P. cubensis isolates. While all isolates sampled before 2009 exhibited the genotype of the subspecies of Clade II and were collected from cucumber, all samples collected from other hosts belonged to Clade I (P. cubensis sensu stricto) or were sampled from 2009 onwards. In addition, 67·16% of all post-2009 isolates from Clade II had two heterozygous positions in their nrITS sequence, which suggests sexual reproduction and/or a mutational origin. Thus, the results indicate that, apart from the rise in prevalence of Clade I, the change in the genetic structure of P. cubensis populations may be linked with a hybridization or, less likely, a mutation event that rendered strains able to infect a broader spectrum of host species.

The structure and temporal dynamics of the virulence of Pseudoperonospora cubensis (causal agent of cucurbit downy mildew) were studied in pathogen populations in the Czech Republic from 2001 to 2010. A total of 398 P. cubensis isolates collected from Cucumis (Cm.) sativus, Cm. melo, Cucurbita (Cr.) maxima, Cr. pepo, Cr. moschata and Citrullus lanatus were analysed for variation in virulence (pathotypes). Virulence was evaluated on a differential set of 12 genotypes of cucurbitaceous plants. All isolates of P. cubensis were characterized by their level of virulence (classified according the number of virulence factors, VF; low VF = 1‐4, medium VF = 5‐8, high VF = 9‐12): high (75%), medium (24%) and low (1%). The structure and dynamics of virulence in the pathogen populations were expressed by pathotypes using tetrad numerical codes and a total of 67 different pathotypes of P. cubensis were determined. The most susceptible group of differentials was Cucumis spp., while the lowest frequency of virulence was recorded on Cr. pepo ssp. pepo, Ci. lanatus and Luffa cylindrica. A high proportion (c. 90%) of isolates was able to infect cucurbit species Benincasa hispida and Lagenaria siceraria ,w hich are not commonly cultivated in the Czech Republic or elsewhere in central Europe. In the recent pathogen populations (2008‐2010) there was prevailing frequency (70‐100%) of isolates with high numbers (9‐12) of virulence factors. ‘Super pathotype’ 15.15.15 was often observed in the study within the pathogen populations and was one of the four most frequently recorded pathotypes. Pseudoperonospora cubensis populations shifted to a higher virulence over time. From 2009 the pathogen population changed dramatically and new pathotypes appeared able to establish natural and serious infection of Cucurbita spp. and Ci. lanatus, which was not observed in 2001‐2008. Generally, virulence structure and dynamics of P. cubensis populations are extremely variable in the Czech Republic.

This paper reviews the taxonomy, biology, importance, host–pathogen interactions and control of lettuce powdery mildew. The main causal agent of this disease, Golovinomyces cichoracearum s.s., is an important powdery mildew pathogen of many members of the family Asteraceae. The pathogen is distributed worldwide and occurs on Lactuca sativa as well as wild Lactuca spp. and related taxa (e.g. Cichorium spp.). Powdery mildew of lettuce can be a major problem in production areas with favourable environmental conditions for disease development (dry, hot weather). The fungus grows ectophytically and appears as white, powdery growth on both the upper and lower sides of leaves. There is rather limited information on the geographic distribution of powdery mildew on wild Lactuca spp. Most L. sativa cultivars have been found to be susceptible. Large variability in virulence was confirmed and existence of different races is supposed. Resistance in L. sativa and some related wild Lactuca spp. is characterized by race-specificity, but the genetic background of resistance is poorly understood. Sources of resistance are known in L. saligna and L. virosa. Lettuce powdery mildew can be effectively controlled by common fungicides (e.g. sulphur, myclobutanil, quinoline, strobilurins, etc.) and protective compounds (e.g. extract of neem oil, Reynoutria sachaliensis extracts). However, fungicide resistance may arise. Non-fungicidal activators of plant systemic acquired resistance (SAR) had no direct effect on the causal agent. Future issues regarding lettuce powdery mildew research are summarized.

Molecular genetic polymorphisms within Pseudoperonospora cubensis isolates of different geographic origins were investigated to establish their phylogenetic relationships and to assess genetic variability between two distant pathogen populations. Thirty isolates originating from Greece (Crete; 15), the Czech Republic (13), the Netherlands (one) and France (one) were analysed by AFLP fingerprinting and ITS 5·8S rDNA sequence analysis. All isolates were obtained from cucumber (Cucumis sativus) plants showing typical downy mildew symptoms. Four AFLP primer combinations produced a total of 288 high-quality bands of which 45% were polymorphic, allowing isolates to be grouped into two separate clusters: one including the Central European (Czech Republic) and Western European (the Netherlands and France) and the other the Cretan isolates. Within each AFLP cluster there was some variation, which could be accounted for by geographic origin or pathogenicity. The two populations (Cretan vs. Central and Western European) exhibited a high degree of genetic isolation. There was no clear AFLP grouping of isolates on the basis of pathotypes. No variability was detected in the ITS1 region; however, ITS2 sequences grouped P. cubensis isolates in two subclusters: one with all investigated European and the other with Asian isolates. The two subclusters formed a larger P. cubensis cluster which was differentiated from the cluster of the neighbouring species Pseudoperonospora humuli. Within P. cubensis, AFLP fingerprints could resolve genetically isolated populations, even on small or medium geographic scales, while ITS2 sequence showed differences on a global scale, being only suitable for phylogenetic analyses.

During 1997–2000 the virulence variation and distribution of virulence phenotypes of Bremia lactucae (lettuce downy mildew) in natural populations of Lactuca serriola (prickly lettuce) were studied. Altogether 139 isolates of B. lactucae originating from the Czech Republic, France and Germany were examined for the presence of 27 virulence factors (v-factors) and their combinations. In the Czech population, 37 different v-phenotypes (P1–P37) of B. lactucae were found to occur on L. serriola. Most v-phenotypes were characterized by v-factors that match resistance (Dm genes/R-factors) carried by L. serriola. A wide diversity of v-phenotypes was recorded every year, but most were rare and did not reappear in other populations of B. lactucae. The three v-phenotypes P21, P28 and P29 were most frequent and widely distributed. There was variation both between spatially isolated populations and within populations of the pathogen. Geographic differences in virulence were found for the southern parts of Moravia, where the phenotypic composition of pathogen populations was completely different from the remaining part of the area investigated. However, some populations had v-phenotypes similar to those of spatially distant populations in Moravia.

Changes in the genetic structure of the lettuce crop (Lactuca sativa) and populations of the lettuce downy mildew pathogen (Bremia lactucae) in Germany from 1974 to 1997 were studied and the genetic and population genetic results of race surveys of B. lactucae were reinterpreted. Data on 176 B. lactucae isolates collected from L. sativa cultivars at 48 sites in 11 German federal states were analysed. The host population contained 12 well-defined race-specific resistance genes (Dm1, Dm2, Dm3, Dm4, Dm5/8, Dm6, Dm7, Dm11, R12, Dm13, Dm16 and R18). However, only some of these genes (e.g. Dm3, Dm7, Dm11, Dm16 and R18) played an important role in management of resistance in the host crop. Substantial changes in the frequencies of these R-genes were recorded over the period as a reaction to the occurrence of new virulent phenotypes in the pathogen population. Generally, the durability of resistance was very short. However, distinct regional patterns were observed for the frequencies of different virulence (v) factors. The data on changes in the virulence of B. lactucae population exemplified well the process of host–pathogen coevolution. During the period studied, there was a considerable increase in the frequency of some v-factors (v2, v3, v5/8, v6, v7, v11 and v16) and in the complexity of v-phenotypes, while the diversity in the pathogen population in general decreased. In 1996/97, 12 of the 14 v-factors evaluated reached frequencies higher than 0·90 in the pathogen population. Relatively low frequencies were observed only for factors v14 (0·17) and v18 (0·24). The presence of 23 v-factors was studied in six German isolates virulent on lettuce cultivar Titan, possessing the resistance gene R36 derived from Lactuca saligna and introduced in German lettuce crops during the mid-1990s. Isolate DEG2 showed the highest complexity of v-phenotype; it overcame the resistance in cv. Mariska (R18) and line CS-RL (R18+?), but some sporulation occurred also on cv. Titan (Dm6 + R36). Conversely, the isolates SR2 and SAW1 overcame resistance based on R36, but were avirulent to R18. Future utilization of R-genes in commercial lettuce cultivars is proposed; R-genes R18 and R36 could be considered most efficient for this purpose. However, they may be effective only in certain regions.

The early stages of development of Bremia lactucae (lettuce downy mildew) were examined on lettuce cultivars possessing high (Iceberg and Regina di Maggio) and low (Great Lakes and Plenos) levels of field resistance. Germ tubes, appressoria, penetration, primary and secondary vesicles, intercellular hyphae and haustoria were observed 3. 6 and 24 h after inoculation of cotyledons and of leaf discs from adult plants. Differences were observed between cv. Iceberg and susceptible genotypes in the percentage of spores germinating and the incidence and speed of development of infection structures. Secondary vesicles were first observed 24 h and 6 h after inoculation in Iceberg and susceptible genotypes, respectively. The lowest incidence of secondary vesicle formation 24 h after inoculation (48 and/or 43%) was recorded in Iceberg and Regina di Maggio, and the highest incidence (68%) occurred in Plenos. The formation of intercellular hyphae and haustoria was not observed in cv. Iceberg some 24 h after inoculation. There were significant differences in the lengths of germ tubes formed on different cultivars. Those on cv. Iceberg were longer than those formed on susceptible cultivars. The results indicate that the field resistance of B. lactucae may result from mechanisms which are effective in the early stages of infection.

Verbena (Verbena officinalis) is a perennial herb native to Europe, which is widely grown for traditional medicine and tea or for ornamental purposes. In August 2009, plants showing typical symptoms of downy mildew disease were collected in Brno, Czech Republic. In September 2009, plants with symptoms were found also in Olomouc. Infected leaves turned yellow or pale green, with a greyish to brownish fungal-like downy growth developing on the abaxial leaf surface. Detailed microscopic examination of a representative sample (KUS-F24423) was made to identify the pathogen. Conidiophores were subhyaline, tree-like, straight, 250–400 · 7–12 lm, monopodially branched in 5–7 orders, and emergent from stomata. Ultimate branchlets were substraight to slightly curved, 5–12 lm long, 2–3 lm wide, and had obtuse or subacute tips. Conidia were greyish brown to violet, ellipsoidal, with size in the range 25–38 · 17–20 lm, and a length ⁄ width ratio of 1AE3–2AE0. These data are in full agreement with those given forPeronosporaverbenae (Braunetal., 2009). The amplification and sequencing of the ITS rDNA were performed with primers DC6 and ITS4 (Cookeet al., 2000), with the sequence of the region deposited in GenBank (Accession No. GQ919014). Comparison with the sequences available in the GenBank database revealed that the ITS sequence shares a 94% similarity with Peronospora flava. Since this was the first sequence submitted for P. verbenae, no comparable data were available. Downy mildew disease on verbena was first recorded under Plasmopara halstedii from New Mexico, USA (Anonymous 1960, cited in Farr & Rossman, 2009). Plasmopara halstedii, however, is reported only on Helianthus or some related Asteraceae (Leppik, 1966).