Genetic variation for tolerance to the downy mildew pathogen Peronospora variabilis in genetic resources of quinoa (Chenopodium quinoa).

BACKGROUND: Quinoa (Chenopodium quinoa Willd.) is an ancient grain crop that is tolerant to abiotic stress and has favorable nutritional properties. Downy mildew is the main disease of quinoa and is caused by infections of the biotrophic oomycete Peronospora variabilis Gaüm. Since the disease causes major yield losses, identifying sources of downy mildew tolerance in genetic resources and understanding its genetic basis are important goals in quinoa breeding. RESULTS: We infected 132 South American genotypes, three Danish cultivars and the weedy relative C. album with a single isolate of P. variabilis under greenhouse conditions and observed a large variation in disease traits like severity of infection, which ranged from 5 to 83%. Linear mixed models revealed a significant effect of genotypes on disease traits with high heritabilities (0.72 to 0.81). Factors like altitude at site of origin or seed saponin content did not correlate with mildew tolerance, but stomatal width was weakly correlated with severity of infection. Despite the strong genotypic effects on mildew tolerance, genome-wide association mapping with 88 genotypes failed to identify significant marker-trait associations indicating a polygenic architecture of mildew tolerance. CONCLUSIONS: The strong genetic effects on mildew tolerance allow to identify genetic resources, which are valuable sources of resistance in future quinoa breeding.

Two-omics data revealed commonalities and differences between Rpv12- and Rpv3-mediated resistance in grapevine.

Plasmopara viticola is the causal agent of grapevine downy mildew (DM). DM resistant varieties deploy effector-triggered immunity (ETI) to inhibit pathogen growth, which is activated by major resistance loci, the most common of which are Rpv3 and Rpv12. We previously showed that a quick metabolome response lies behind the ETI conferred by Rpv3 TIR-NB-LRR genes. Here we used a grape variety operating Rpv12-mediated ETI, which is conferred by an independent locus containing CC-NB-LRR genes, to investigate the defence response using GC/MS, UPLC, UHPLC and RNA-Seq analyses. Eighty-eight metabolites showed significantly different concentration and 432 genes showed differential expression between inoculated resistant leaves and controls. Most metabolite changes in sugars, fatty acids and phenols were similar in timing and direction to those observed in Rpv3-mediated ETI but some of them were stronger or more persistent. Activators, elicitors and signal transducers for the formation of reactive oxygen species were early observed in samples undergoing Rpv12-mediated ETI and were paralleled and followed by the upregulation of genes belonging to ontology categories associated with salicylic acid signalling, signal transduction, WRKY transcription factors and synthesis of PR-1, PR-2, PR-5 pathogenesis-related proteins.

A secreted WY-domain-containing protein present in European isolates of the oomycete Plasmopara viticola induces cell death in grapevine and tobacco species.

Plasmopara viticola is a biotrophic oomycete pathogen causing grapevine downy mildew. We characterized the repertoire of P. viticola effector proteins which may be translocated into plants to support the disease. We found several secreted proteins that contain canonical dEER motifs and conserved WY-domains but lack the characteristic RXLR motif reported previously from oomycete effectors. We cloned four candidates and showed that one of them, Pv33, induces plant cell death in grapevine and Nicotiana species. This activity is dependent on the nuclear localization of the protein. Sequence similar effectors were present in seven European, but in none of the tested American isolates. Together our work contributes a new type of conserved P. viticola effector candidates.

Detection of Two Peronospora spp., Responsible for Downy Mildew, in Opium Poppy Seed.

Downy mildew is a serious threat to opium poppy production globally. In recent years, two pathogen species, Peronospora somniferi and Peronospora meconopsidis, which induce distinct symptoms, have been confirmed in Australia. In order to manage the spread of these pathogens, identifying the sources of inoculum is essential. In this study, we assessed pathogen presence associated with poppy seed. We developed PCR and qPCR assays targeting the coxI and coxII gene regions, for the detection, differentiation, and quantification of P. somniferi and P. meconopsidis in poppy seed. These results were complemented and compared with direct seed histological examination and a seed washing combined with viability staining for oospore detection. The majority of seed lots from all harvest years contained detectable P. meconopsidis, the earliest (1987) predating the first official record of the disease in Tasmania (1996). In contrast, only seed lots harvested in 2012 or later contained P. somniferi, evidence of its more recent introduction. P. meconopsidis contamination was estimated to be as high as 33.04 pg DNA/g of seed and P. somniferi as high as 35.17 pg DNA/g of seed. Incidence of pathogen contamination of seeds, estimated via a group testing protocol, ranged from 0 to 9% (P. meconopsidis) or 0 to 11% (P. somniferi). Mycelia were predominately found external to the seed coat. Seed washing and viability staining demonstrated that putatively viable oospores were present in the majority of seed lots. Transmission testing confirmed both pathogens can be successfully transmitted from infested seed to infected seedling. PCR and qPCR pathogen assays were found to be reliable and offer a routine test for determining pathogen inoculum in poppy seeds.

Detection of Latent Peronospora effusa Infections in Spinach.

Downy mildew disease of spinach, caused by Peronospora effusa, is managed in conventional fields by a combination of host resistance and scheduled fungicide applications. Fungicides are currently applied to prevent downy mildew epidemics regardless of the infection status of spinach crops. A more streamlined approach would be to develop methods to target either latent infections for fungicide application in conventional production systems or to hasten harvest in organic production. In this study, conventional polymerase chain reaction (PCR) was applied to detect P. effusa DNA in symptomless spinach leaves in three spatially and temporally separated field plots, each containing four 2-m beds, 35 m in length. Spinach leaves were sampled weekly at 3-m intervals at 48 locations throughout each plot. Initial samples were asymptomatic and yet PCR enabled detection of P. effusa DNA extracted from sampled spinach leaves. Detection of latent downy mildew infection in spinach leaves was confirmed by PCR as early as 7 days prior to symptom development. The limit of pathogen DNA detection in spinach leaves was calculated at 10 pg using the conventional PCR approach. Quantitative PCR with TaqMan methodology revealed the presence of inhibitors from spinach leaf DNA extracts and affected amplification efficiencies, but not when diluted, enabling detection of P. effusa DNA at a concentration of <0.1 pg. In conclusion, detection of latent infections may enable management decisions for earlier-than-normal harvest of infected, symptomless organic crops, and for timing fungicide applications on symptomless plants in conventional production.

New Races and Novel Strains of the Spinach Downy Mildew Pathogen Peronospora effusa.

Downy mildew disease, caused by Peronospora effusa (=P. farinosa f. sp. spinaciae [Pfs]), is the most economically important disease of spinach. Current high-density fresh-market spinach production provides conducive conditions for disease development, and downy mildew frequently forces growers to harvest early owing to disease development, to cull symptomatic leaves prior to harvest, or to abandon the field if the disease is too severe. The use of resistant cultivars to manage downy mildew, particularly on increasing acreages of organic spinach production, applies strong selection pressure on the pathogen, and many new races of Pfs have been identified in recent years in spinach production areas worldwide. To monitor the virulence diversity in the Pfs population, downy mildew samples were collected from spinach production areas and tested for race identification based on the disease reactions of a standard set of international spinach differentials. Two new races (designated races 15 and 16) and eight novel strains were identified between 2013 and 2017. The disease reaction of Pfs 15 was similar to race 4, except race 4 could not overcome the resistance imparted by the RPF9 locus. Several resistance loci (RPF1, 2, 4, and 6) were effective in preventing disease caused by Pfs 15. The race Pfs 16 could overcome several resistance loci (RPF2, 4, 5, 9, and 10) but not others (RPF1, 3, 6, and 7). One novel strain (UA1014) could overcome the resistance of spinach resistant loci RPF1 to RPF7 but only infected the cotyledons and not the true leaves of certain cultivars. A new set of near-isogenic lines has been developed and evaluated for disease reactions to the new races and novel strains as differentials. None of the 360 U.S. Department of Agriculture spinach germplasm accessions tested were resistant to Pfs 16 or UA1014. A survey of isolates over several years highlighted the dynamic nature of the virulence diversity of the Pfs population. Identification of virulence diversity and evaluation of the genetics of resistance to Pfs will continue to allow for a more effective disease management strategy through resistance gene deployment.

Multiple gene genealogy reveals high genetic diversity and evidence for multiple origins of Chinese Plasmopara viticola population.

Downy mildew caused by Plasmopara viticola is one of the most devastating diseases of grapevines worldwide. So far, the genetic diversity and origin of the Chinese P. viticola population are unclear. In the present study, 103 P. viticola isolates were sequenced at four gene regions: internal transcribed spacer one (ITS), large subunit of ribosomal RNA (LSU), actin gene (ACT) and beta-tubulin (TUB). The sequences were analyzed to obtain polymorphism and diversity information of the Chinese population as well as to infer the relationships between Chinese and American isolates. High genetic diversity was observed for the Chinese population, with evidence of sub-structuring based on climate. Phylogenetic analysis and haplotype networks showed evidence of close relationships between some American and Chinese isolates, consistent with recent introduction from America to China via planting materials. However, there is also evidence for endemic Chinese P. viticola isolates. Our results suggest that the current Chinese Plasmopara viticola population is an admixture of endemic and introduced isolates.

Computer Vision for High-Throughput Quantitative Phenotyping: A Case Study of Grapevine Downy Mildew Sporulation and Leaf Trichomes.

Quantitative phenotyping of downy mildew sporulation is frequently used in plant breeding and genetic studies, as well as in studies focused on pathogen biology such as chemical efficacy trials. In these scenarios, phenotyping a large number of genotypes or treatments can be advantageous but is often limited by time and cost. We present a novel computational pipeline dedicated to estimating the percent area of downy mildew sporulation from images of inoculated grapevine leaf discs in a manner that is time and cost efficient. The pipeline was tested on images from leaf disc assay experiments involving two F1 grapevine families, one that had glabrous leaves (Vitis rupestris B38 × 'Horizon' [RH]) and another that had leaf trichomes (Horizon × V. cinerea B9 [HC]). Correlations between computer vision and manual visual ratings reached 0.89 in the RH family and 0.43 in the HC family. Additionally, we were able to use the computer vision system prior to sporulation to measure the percent leaf trichome area. We estimate that an experienced rater scoring sporulation would spend at least 90% less time using the computer vision system compared with the manual visual method. This will allow more treatments to be phenotyped in order to better understand the genetic architecture of downy mildew resistance and of leaf trichome density. We anticipate that this computer vision system will find applications in other pathosystems or traits where responses can be imaged with sufficient contrast from the background.

Spatiotemporal Patterns in the Airborne Dispersal of Spinach Downy Mildew.

Downy mildew is the most devastating disease threatening sustainable spinach production, particularly in the organic sector. The disease is caused by the biotrophic oomycete pathogen Peronospora effusa, and the disease results in yellow lesions that render the crop unmarketable. In this study, the levels of DNA from airborne spores of P. effusa were assessed near a field of susceptible plants in Salinas, CA during the winter months of 2013-14 and 2014/15 using rotating-arm impaction spore-trap samplers that were assessed with a species-specific quantitative polymerase chain reaction (qPCR) assay. Low levels of P. effusa DNA were detectable from December through February in both winters but increased during January in both years, in correlation with observed disease incidence; sharp peaks in P. effusa DNA detection were associated with the onset of disease incidence. The incidence of downy mildew in the susceptible field displayed logistic-like dynamics but with considerable interseason variation. Analysis of the area under the disease progress curves suggested that the 2013-14 epidemic was significantly more severe than the 2014-15 epidemic. Spatial analyses indicated that disease incidence was dependent within an average range of 5.6 m, approximately equivalent to the width of three planted beds in a typical production field. The spatial distribution of spores captured during an active epidemic most closely fit a power-law distribution but could also be fit with an exponential distribution. These studies revealed two important results in the epidemiology of spinach downy mildew in California. First, they demonstrated the potential of impaction spore-trap samplers linked with a qPCR assay for indicating periods of high disease risk, as well as the detection of long-distance dispersal of P. effusa spores. Second, at the scale of individual crops, a high degree of spatial aggregation in disease incidence was revealed.

Season-Long Dynamics of Spinach Downy Mildew Determined by Spore Trapping and Disease Incidence.

Peronospora effusa is an obligate oomycete that causes downy mildew of spinach. Downy mildew threatens sustainable production of fresh market organic spinach in California, and routine fungicide sprays are often necessary for conventional production. In this study, airborne P. effusa spores were collected using rotating arm impaction spore trap samplers at four sites in the Salinas Valley between late January and early June in 2013 and 2014. Levels of P. effusa DNA were determined by a species-specific quantitative polymerase chain reaction assay. Peronospora effusa was detected prior to and during the growing season in both years. Nonlinear time series analyses on the data suggested that the within-season dynamics of P. effusa airborne inoculum are characterized by a mixture of chaotic, deterministic, and stochastic features, with successive data points somewhat predictable from the previous values in the series. Analyses of concentrations of airborne P. effusa suggest both an exponential increase in concentration over the course of the season and oscillations around the increasing average value that had season-specific periodicity around 30, 45, and 75 days, values that are close to whole multiples of the combined pathogen latent and infectious periods. Each unit increase in temperature was correlated with 1.7 to 6% increased odds of an increase in DNA copy numbers, while each unit decrease in wind speed was correlated with 4 to 12.7% increased odds of an increase in DNA copy numbers. Disease incidence was correlated with airborne P. effusa levels and weather variables, and a receiver operating characteristic curve analysis suggested that P. effusa DNA copy numbers determined from the spore traps nine days prior to disease rating could predict disease incidence.

Geographic distribution of cryptic species of Plasmopara viticola causing downy mildew on wild and cultivated grape in eastern North America.

The putative center of origin of Plasmopara viticola, the causal agent of grape downy mildew, is eastern North America, where it has been described on several members of the family Vitaceae (e.g., Vitis spp., Parthenocissus spp., and Ampelopsis spp.). We have completed the first large-scale sampling of P. viticola isolates across a range of wild and cultivated host species distributed throughout the above region. Sequencing results of four partial genes indicated the presence of a new P. viticola species on Vitis vulpina in Virginia, adding to the four cryptic species of P. viticola recently recorded. The phylogenetic analysis also indicated that the P. viticola species found on Parthenocissus quinquefolia in North America is identical to Plasmopara muralis in Europe. The geographic distribution and host range of five pathogen species was determined through analysis of the internal transcribed spacer polymorphism of 896 isolates of P. viticola. Among three P. viticola species found on cultivated grape, one was restricted to Vitis interspecific hybrids within the northern part of eastern North America. A second species was recovered from V. vinifera and V. labrusca, and was distributed across most of the sampled region. A third species, although less abundant, was distributed across a larger geographical range, including the southern part of eastern North America. P. viticola clade aestivalis predominated (83% of isolates) in vineyards of the European winegrape V. vinifera within the sampled area, indicating that a single pathogen species may represent the primary threat to the European host species within eastern North America.

Coupling Spore Traps and Quantitative PCR Assays for Detection of the Downy Mildew Pathogens of Spinach (Peronospora effusa) and Beet (P. schachtii).

Downy mildew of spinach (Spinacia oleracea), caused by Peronospora effusa, is a production constraint on production worldwide, including in California, where the majority of U.S. spinach is grown. The aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay for detection of airborne inoculum of P. effusa in California. Among oomycete ribosomal DNA (rDNA) sequences examined for assay development, the highest nucleotide sequence identity was observed between rDNA sequences of P. effusa and P. schachtii, the cause of downy mildew on sugar beet and Swiss chard in the leaf beet group (Beta vulgaris subsp. vulgaris). Single-nucleotide polymorphisms were detected between P. effusa and P. schachtii in the 18S rDNA regions for design of P. effusa- and P. schachtii-specific TaqMan probes and reverse primers. An allele-specific probe and primer amplification method was applied to determine the frequency of both P. effusa and P. schachtii rDNA target sequences in pooled DNA samples, enabling quantification of rDNA of P. effusa from impaction spore trap samples collected from spinach production fields. The rDNA copy numbers of P. effusa were, on average, ≈3,300-fold higher from trap samples collected near an infected field compared with those levels recorded at a site without a nearby spinach field. In combination with disease-conducive weather forecasting, application of the assays may be helpful to time fungicide applications for disease management.

Multiplex real-time PCR assays for detection of four seedborne spinach pathogens.

AIMS: To develop multiplex TaqMan real-time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach. METHODS AND RESULTS: Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae) and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nontarget, related fungi or oomycetes and numerous saprophytes commonly found on spinach seed. Multiplex real-time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real-time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed. CONCLUSIONS: The real-time PCR assays were species-specific and sensitive. Singular or multiplex real-time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed. SIGNIFICANCE AND IMPACT OF THE STUDY: The freeze-blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C. variabile, S. botryosum and V. dahliae) is quite laborious and takes several weeks to process. The real-time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry.

Molecular detection of Peronospora variabilis in quinoa seed and phylogeny of the quinoa downy mildew pathogen in South America and the United States.

Quinoa (Chenopodium quinoa) is an important export of the Andean region, and its key disease is quinoa downy mildew, caused by Peronospora variabilis. P. variabilis oospores can be seedborne and rapid methods to detect seedborne P. variabilis have not been developed. In this research, a polymerase chain reaction (PCR)-based detection method was developed to detect seedborne P. variabilis and a sequencing-based method was used to validate the PCR-based method. P. variabilis was detected in 31 of 33 quinoa seed lots using the PCR-based method and in 32 of 33 quinoa seed lots using the sequencing-based method. Thirty-one of the quinoa seed lots tested in this study were sold for human consumption, with seed originating from six different countries. Internal transcribed spacer (ITS) and cytochrome c oxidase subunit 2 (COX2) phylogenies were examined to determine whether geographical differences occurred in P. variabilis populations originating from Ecuador, Bolivia, and the United States. No geographical differences were observed in the ITS-derived phylogeny but the COX2 phylogeny indicated that geographical differences existed between U.S. and South American samples. Both ITS and COX2 phylogenies supported the existence of a Peronospora sp., distinct from P. variabilis, that causes systemic-like downy mildew symptoms on quinoa in Ecuador. The results of these studies allow for a better understanding of P. variabilis populations in South America and identified a new causal agent for quinoa downy mildew. The PCR-based seed detection method allows for the development of P. variabilis-free quinoa seed, which may prove important for management of quinoa downy mildew.

Metabolic study of grapevine leaves infected by downy mildew using negative ion electrospray--Fourier transform ion cyclotron resonance mass spectrometry.

Grapevine is of worldwide economic importance due to wine production. However, this culture is often affected by pathogens causing severe harvest losses. Understanding host-pathogen relationships may be a key to solve this problem. In this paper, we evaluate the direct flow injection by electrospray - Fourier transform ion cyclotron resonance mass spectrometry (MS) of leaf extracts as a rapid method for the study of grapevine response to downy mildew (Plasmopara viticola) attack. The comparison of MS profiles obtained from control and infected leaves of different levels of resistant grapevines highlights several classes of metabolites (mainly saccharides, acyl lipids, hydroxycinnamic acids derivatives and flavonoids) which are identified using high resolution MS and tandem MS (MS/MS). Statistical analyses of 19 markers show a clear segregation between inoculated and healthy samples. This study points out relative high levels of disaccharides, acyl lipids and glycerophosphoinositol in inoculated samples. Sulfoquinovosyl diacylglycerols also emerge as possible metabolites involved in plant defense.

Mitochondrial phylogeny reveals intraspecific variation in Peronospora effusa, the spinach downy mildew pathogen.

Since about two hundred years, downy mildew caused by Peronospora effusa is probably the most economically important disease of spinach (Spinacia oleracea). However, there is no information on the global phylogeographic structure of the pathogen and thus it is unclear whether a single genotype occurs worldwide or whether some local genetic variation exists. To investigate the genetic variability of this pathogen, a sequence analysis of two partial mitochondrial DNA genes, cox2 and nad1, was carried out. Thirty-three specimens of Peronospora effusa from four continents were analyzed, including samples from Australia, China, Japan, Korea, Mexico, Russia, Sweden, and the USA. Despite the potential anthropogenic admixture of genotypes, a phylogeographic pattern was observed, which corresponds to two major groups, an Asian/Oceanian clade and another group, which includes American/European specimens. Notably, two of six Japanese specimens investigated did not belong to the Asian/Oceanian clade, but were identical to three of the specimens from the USA, suggestive of a recent introduction from the USA to Japan. As similar introduction events may be occurring as a result of the globalised trade with plant and seed material, a better knowledge of the phylogeographic distribution of pathogens is highly warranted for food security purposes.

Characterization of single-nucleotide-polymorphism markers for Plasmopara viticola, the causal agent of grapevine downy mildew.

We report 34 new nuclear single-nucleotide-polymorphism (SNP) markers that have been developed from an expressed sequence tag library of Plasmopara viticola, the causal agent of grapevine downy mildew. This newly developed battery of markers will provide useful additional genetic tools for population genetic studies of this important agronomic species.

Reclassification of two peronospora species parasitic on draba in hyaloperonospora based on morphological and molecular phylogenetic data.

On the family Brassicaceae, the causal agent responsible for downy mildew disease was originally regarded as a single species, Peronospora parasitica (now under Hyaloperonospora), but it was recently reconsidered to consist of many distinct species. In this study, 11 specimens of Peronospora drabae and P. norvegica parasitic on the genus Draba were investigated morphologically and molecularly. Pronounced differences in conidial sizes (P. drabae: 14-20 × 12.5-15.5 µm; P. norvegica: 20-29 × 15.5-22 µm) and 7.8% sequence distance between their ITS1-5.8S-ITS2 rDNA sequences confirmed their status as distinct species. Based on ITS phylogeny and morphology (monopodially branching conidiophores, flexuous to sigmoid ultimate branchlets, hyaline conidia and lobate haustoria), the two species unequivocally belong to the genus Hyaloperonospora and not to Peronospora to which they were previously assigned. Therefore, two new combinations, Hyaloperonospora drabae and H. norvegica, are proposed. The two taxa are illustrated and compared using the type specimen for H. norvegica and authentic specimens for H. drabae, which is lectotypified.

Disease-specific expression of host genes during downy mildew infection of Arabidopsis.

Here, we report on the identification of Arabidopsis genes that are induced during compatible but not during incompatible interactions with the downy mildew pathogen Hyaloperonospora arabidopsidis. This set of so-called compatible specific (CS) genes contrasts with the large group of defense-associated genes that is differentially expressed during both compatible and incompatible interactions. From the 17 identified CS genes, 6 belong to the ethylene response factor (ERF) family of transcription factor genes, suggesting that these ERF have a role during compatibility. The majority of CS genes are differentially regulated in response to various forms of abiotic stress. In silico analysis of the CS genes revealed an over-representation of dehydration-responsive element/C-repeat binding factor (DREB1A/CBF3) binding sites and EveningElement motifs in their promoter regions. The CS-ERF are closely related to the CBF transcription factors and could potentially bind the DREB1A/CBF3 promoter elements in the CS genes. Transcript levels of CS genes peak at 2 to 3 days postinoculation, when pathogen growth is highest, and decline at later stages of infection. The induction of several CS genes was found to be isolate specific. This suggests that the identified CS genes could be the direct or indirect targets of downy mildew effector proteins that promote disease susceptibility.

A nested-polymerase chain reaction protocol for detection and population biology studies of Peronospora arborescens, the downy mildew pathogen of opium poppy, using herbarium specimens and asymptomatic, fresh plant tissues.

A sensitive nested-polymerase chain reaction (PCR) protocol was developed using either of two primer pairs that improves the in planta detection of Peronospora arborescens DNA. The new protocol represented an increase in sensitivity of 100- to 1,000-fold of detection of the oomycete in opium poppy tissue compared with the detection limit of single PCR using the same primer pairs. The new protocol allowed amplification of 5 to 0.5 fg of Peronospora arborescens DNA mixed with Papaver somniferum DNA. The protocol proved useful for amplifying Peronospora arborescens DNA from 96-year-old herbarium specimens of Papaver spp. and to demonstrate that asymptomatic, systemic infections by Peronospora arborescens can occur in wild Papaver spp. as well as in cultivated opium poppy. Also, the increase in sensitivity of the protocol made possible the detection of seedborne Peronospora arborescens in commercial opium poppy seed stocks in Spain with a high frequency, which poses a threat for pathogen spread. Direct sequencing of purified amplicons allowed alignment of a Peronospora arborescens internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequence up to 730-bp long when combining the sequences obtained with the two primer sets. Maximum parsimony analysis of amplified Peronospora arborescens ITS rDNA sequences from specimens of Papaver dubium, P. hybridum, P. rhoeas, and P. somniferum from different countries indicated for the first time that a degree of host specificity may exist within populations of Peronospora arborescens. The reported protocol will be useful for epidemiological and biogeographical studies of downy mildew diseases as well as to unravel misclassification of Peronospora arborescens and Peronospora cristata, the reported causal agents of the opium poppy downy mildew disease.