Characteristics of a <Emphasis Type="Italic">Plasmopara angustiterminalis</Emphasis> isolate from <Emphasis Type="Italic">Xanthium strumarium</Emphasis>

Leaves of Xanthium strumarium infected with downy mildew were collected in the vicinity of a sunflower field in southern Hungary in 2003. Based on phenotypic characteristics of sporangiophores, sporangia and oospores as well as host preference the pathogen was classified as Plasmopara angustiterminalis. Additional phenotypic characters were investigated such as the size of sporangia, the number of zoospores per sporangium and the time-course of their release. Infection studies revealed infectivity of the P. angustiterminalis isolate to both X. strumarium and Helianthus annuus. Inoculation of the sunflower inbred line, HA-335 with resistance to all known P. halstedii pathotypes, resulted in profuse sporulation on cotyledons and formation of oospores in the bases of hypocotyls. Infections of sunflower differential lines often led to damping-off. Molecular genetic analysis using simple sequence repeat primers and nuclear rDNA sequences revealed clear differences to Plasmopara halstedii, the downy mildew pathogen of sunflower.     

Homothallism in Pseudoperonospora humuli

The downy mildew pathogen, Pseudoperonospora humuli, forms oospores abundantly in diseased hop tissue. Diverse monosporangial isolates of P. humuli derived from samples collected in Japan, Germany and the USA readily formed oospores within hop leaves when inoculated singly, suggesting homothallism. Single zoospore isolates also readily formed oospores within hop leaves, further supporting the homothallic nature of this oomycete. The majority of oospores were deemed viable based on cytoplasm characteristics and plasmolysis assays. However, disease symptoms failed to develop when hop leaves were inoculated with newly formed oospores, even when oospore conditioning was attempted with treatment with potassium permanganate or β‐glucuronidase/arylsulphatase, brief exposure to freezing temperature, or passage through an earthworm. Oospores derived from a monosporangial isolate of P. humuli that overwintered outdoors in infested leaves buried in soil also failed to cause downy mildew. Pseudoperonospora humuli is homothallic and oospores of the organism appear to require as yet unknown conditions to stimulate their germination and/or infection.     

Exceptional length of ITS in <Emphasis Type="Italic">Plasmopara halstedii</Emphasis> is due to multiple repetitions in the ITS-2 region

Plasmopara halstedii, cause of downy mildew of sunflower, is a pathogen of worldwide economic importance. Efforts to amplify the ITS-region from this organism revealed an unexpected fragment length of about 2600 bp, in contrast to about 900 bp, reported for other members of the Peronosporaceae. First attempts to obtain the complete sequence of the P. halstedii fragment were unsuccessful, due to repeated elements in ITS, which were uncovered later on. The presence of a single EcoRI-site allowed us to apply a restriction-ligation procedure to amplify parts of the ITS fragment separately. Sequencing of these fragments revealed the presence of four copies of a tandemly arranged repetitive element in the ITS-2 region. The complete sequence was obtained by using a sequencing primer which annealed shortly before the repetitions so covering the gap in the sequence around the restriction site. The ITS sequence in P. halstedii (AY773346) consisted of 2587 bp in total, with ITS-2 accounting for 2212 bp alone. This is the longest ITS-2 sequence reported so far for any examined species.     

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.     

Pathogenic variability of Plasmopara halstedii infecting sunflower in the Czech Republic

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 Pl₆.     

Heterothallism in Plasmopara viticola

Sexuality in the oomycete Plasmopara viticola , the causal agent of grapevine downy mildew, was studied using isolates from five populations from North America and Europe. Leaf discs of Vitis vinifera cv. 'Chardonnay' were inoculated with either individual single-sporangiophore isolates, or in all possible pairwise combinations of 25 isolates from New York State, USA. The occurrence of oospores in leaf discs indicated that the pathogen was heterothallic with two mating types, P1 and P2 in a ratio of 11 : 14 for this population. Heterothallism was confirmed when three representative isolates of each mating type from New York were coinoculated with each of 40 isolates from populations of P. viticola from Michigan, Missouri (USA), Germany and Italy. For each isolate tested, oospores formed with either test isolates of P1 or test isolates of P2 mating types, indicating that the isolates were exclusively P1 or P2 only. For these same isolates, no oospores formed as a result of self-crosses. The ratio of P1 : P2 mating types for all isolates in the study was 27 : 38, statistically equivalent to a 1 : 1 ratio according to χ² analysis ( P  = 0·68).     

Advances in sunflower downy mildew research

This review summarises the progress in research on sunflower downy mildew as reported in publications of the past 10 to 15 years, the period since the last comprehensive review on Plasmopara halstedii. Particular attention is paid to subjects that showed much progress and may be of particular interest to sunflower pathologists, mycologists or molecular biologists. Accordingly, single sections are devoted to the problems of taxonomic and phylogenetic aspects, host specificity, the host—pathogen interaction including resistance phenomena, as well as epidemiology and disease management. Reflecting the progress achieved in some fields and illuminating the deficits in others should stimulate the reader's interest in this very significant pathosystem.     

Reaction of maize,sorghum and Johnson grass to Peronosclerospora sorghi

Development of sorghum downy mildew, incited by Peronoscleospora sorghi (Weston and Uppal) C.G. Shaw, on maize, sorghum and Johnson grass was investigated at two locations in Uganda during three seasons (1994 and 1995). More sorghum downy mildew developed on the Johnson grass and sorghum than on the maize at all locations and in all seasons. No significant differences were observed in sporulation of P. soghi on the three hosts. Leaf shredding occurred on the three hosts but was the least on maize. Cross-inoculation with both conidia and oospores was achieved on the three hosts. Since the fungal population formed oospores and sporulated readily on the three hosts, which is typical of the sorghum strain, the disease in Uganda is attributed to the sorghum strain.     

Methodology of virulence screening and race characterization of Plasmopara halstedii,and resistance evaluation in sunflower – a review

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.     

Development of a PCR test to detect the downy mildew causal agent Plasmopara halstedii in sunflower seeds

Plasmopara halstedii , the causal agent of downy mildew of sunflower, is an obligate parasite but viable sporangia and oospores of the pathogen may be found in a quiescent state in seeds of sunflower and therefore may be transported with sunflower seeds in international commercial exchanges. In order to prevent the spread of this pathogen, especially the introduction of potentially new races, an efficient method to analyse sunflower seed samples is required. In this study, a P. halstedii -specific polymerase chain reaction (PCR) test was developed based on the ribosomal large sub unit (LSU) DNA. The forward (PHAL-F) and reverse (PHAL-R) PCR primers were designed from two polymorphic regions of LSU. After screening 22 isolates of P. halstedii corresponding to different races and countries and 32 other oomycete, deuteromycete and ascomycete isolates, the PHAL-F/R primers amplified only a single PCR band of c. 310 bp from P. halstedii . The PHAL-F/R PCR test could detect as little as 3 pg of P. halstedii genomic DNA per 20  µ L reaction volume and enabled the direct detection of P. halstedii in 35 g sunflower seed samples without the need for any prior biological baiting step. An internal amplification control (IAC) was developed to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. The test was successfully used on samples of naturally contaminated seeds. These new molecular tools should be of great interest for quarantine seed testing purposes.     

Occurrence and molecular characterization of azoxystrobin resistance in cucumber downy mildew in Shandong province of China

Cucumber downy mildew caused byPseudoperonospora cubensis (Berk. and Curt.) Rostov. limits crop production in Shandong Province of China. Since management of downy mildew is strongly dependent on fungicides, a rational design of control programs requires a good understanding of the fungicide resistance phenomenon in field populations of the pathogen. A total of 106 and 97 isolates ofP. cubensis were obtained in 2006 and 2007, respectively. The EC₅₀ values for the growth of all the 106 isolates collected in 2006 were 0.0063–0.0688μg ml⁻¹ (average: 0.0196±0.0048μg ml⁻¹) azoxystrobin and these were therefore considered sensitive isolates. However, 57 field isolates ofP. cubensis of the 97 collected in 2007 with EC₅₀ values that ranged from 0.609 to >51.2μg ml⁻¹ were considered resistant to azoxystrobin. Fragments of the fungicide-targeted mitochondrial cytochromeb gene from total pathogen DNA were amplified using polymerase chain reaction and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochromeb gene, resulting in substitution of glycine by alanine at position 143, was found in the three selected azoxystrobin-resistant isolates of downy mildew. This substitution in cytochromeb exhibited different resistance levels, with the resistance factor from 21.15 to greater than 2618.9. In addition, the different resistance levels seemed to appear within 1 year (between 2006 and 2007). Therefore, growers of Shandong Province in China now are faced with a challenge in managing the azoxystrobin resistance in cucumber downy mildew. http://www.phytoparasitica.org posting March 10, 2008.     

Mating type and sexual reproduction of <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>, the downy mildew agent of cucurbits

The oomycete Pseudoperonospora cubensis is a leaf pathogen causing severe damage to members of the Cucurbitaceae, especially cucumber and melon. It propagates clonally by sporangia. Oospores of P. cubensis were previously observed in nature but their formation in the laboratory was never reported nor their germination or infection. Here we report on the sexual reproduction of P. cubensis under controlled conditions in the laboratory. When field isolates were inoculated singly onto detached leaves of cucurbits in growth chambers no oospores were produced. However, when pairs of selected isolates were mixed and inoculated onto detached leaves, oospores were formed in the mesophyll within 6–11 days, suggesting that P. cubensis is heterothallic, having two opposite mating types, A1 and A2. Isolates belonging to pathotype 3 were all A1 whereas isolates belonging to the new pathotype 6 were either A1 or A2. Oospores were spherical, ~40 μm in diameter, hyaline to red-brown in color. Oospores were produced regularly, in large numbers, in Cucumis sativum and Cucumis melo, very seldom and in very small numbers in Cucurbita pepo, Cucurbita maxima and Citrullus lanatus, and not in Cucurbita moschata. Oospores were formed at 12.5–21°C but not at 25°C. Under moisture-saturated atmosphere oospores were also produced in leaves of intact plants. Oospores inoculated onto detached leaves in growth chambers produced F1 downy mildew lesions at 6–21 days after inoculation, many in Cucumis sativum, Cucumis melo and Cucurbita moschata, very few in Cucurbita pepo or Citrullus lanatus, and none in Cucurbita maxima. This report shows that P. cubensis is heterothallic, having A1 and A2 mating types which can cross and enable sexual reproduction in cucurbits. A preliminary report on part of the results has been published earlier.     

Homothallism in Peronospora viciae f.sp. pisi and the effect of temperature on oospore production

Monoconidial cultures derived from seven P. viciae f.sp. pisi isolates, obtained from different countries, were able lo produce oospores, Apparently, these isolates were homothallic. Oospore production of one isolate was studied at 5, 10, 15 and 20°C in systematically colonized shoots, and in local lesions on leaflets, stem parts and pods of the pisum sativum cv. Kelvedon Wonder. The number of oospores produced per gram systemically colonized tissue increased with temperature. In lesions of leaflets and of stem parts, including tendrils, petioles and main stem, most oospores were produced at 20°C. At 10°C, a few oospores were found in stem parts but none in leaflet lesions. At 5° C, no oospores were formed at all. In pods, moe oospores were produced at 15 and 20°C than at 10°C, but the numbers of oospores was smaller than in the other plant parts. Oospores formed at lower temperatures were larger than those formed at higher temperatures. At 20°C, similar oospore densities were found in leaflet lesions of three cultivars widely differing in resistance to downy mildew.     

First report of spinach downy mildew caused by race Pfs:8 of <Emphasis Type="Italic">Peronospora farinosa</Emphasis> f. sp. <Emphasis Type="Italic">spinaciae</Emphasis> in Japan

The race of field isolates of Peronospora farinosa f. sp. spinaciae (Pfs), causal agent of spinach downy mildew, were identified using race-differential cultivars. One isolate was similar to race Pfs:6. Three isolates were identified as race Pfs:8, the first time the race has been reported in Japan as far as we know. The differential reaction caused by the other two isolates did not match any known to be caused by races Pfs:1 through Pfs:11; thus, this strain appears to a new pathogenic strain in Japan.     

Temporal disease dynamics and relative importance of sexual and asexual reproduction of grape downy mildew (Plasmopara viticola) in an isolated vineyard in the North Georgia Mountains,USA

The North Georgia Mountains are the southernmost region along the United States East Coast where European wine grapes (Vitis vinifera) are grown commercially. Epidemics of downy mildew, caused by Plasmopara viticola, are frequent and severe, but little is known about the epidemiology and population biology of the pathogen in this region. Disease monitoring in an experimental vineyard from 2015 to 2017 indicated that times of disease onset and progress rates were highly variable across years and cultivars. Oospores were observed microscopically, and simulation with a process-based model indicated presence of conditions favourable for oospore germination in the spring and early summer each year. A total of 409 P. viticola isolates collected over three  years were genotyped with seven microsatellite markers, revealing very high genotypic diversity, which when combined with the observation of oospores is indicative of a sexually reproducing population. Among the 409 isolates, 225 multilocus genotypes (MLGs) were identified, of which 164 were detected only once and 61 were repeated (clonal). Eight MLGs (represented by 28 isolates) were detected across years, suggesting the possibility of asexual overwintering of P. viticola in this region. Across sampling dates, the percentage of isolates belonging to nonrepeated (unique) MLGs ranged from 27.3% to 63.2%. Even towards the end of the annual epidemic, the percentage of isolates in nonrepeated MLGs was still relatively high, around 30%. These MLGs may have originated from oospores germinating late during the growing season, although incomplete sampling at earlier dates and contribution by immigration cannot be fully excluded.     

First report of downy mildew of carnation caused by Peronospora dianthicola in Japan

Yellowish lesions with downy, gray growth developed on the leaves of carnations (Dianthus caryophyllus) in a greenhouse in Hokkaido Prefecture, Japan, in March 2011. We identified the fungus on diseased plants as Peronospora dianthicola on the basis of the morphologies of the conidia, conidiophores, and oospores. To confirm pathogenicity of the fungus, we inoculated healthy plants with a conidial suspension of the fungus, and the plants developed the same signs and symptoms as the naturally infected plants. We then reidentified P. dianthicola from the diseased lesions. This is the first report of downy mildew of carnation in Japan.     

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.