A potential perennial host for <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis> in temperate regions

Pseudoperonospora cubensis causes great losses in cucurbitaceous crops worldwide. In cool temperate climates of northern Europe or North America overwintering as active mycelium is not possible, because all hosts so far reported there are summer annuals. Oospores have not yet been found in these regions under field conditions. The only perennial member of the Cucurbitaceae found naturally in central and northern Europe is Bryonia dioica. To date this plant has not been recorded as a host for downy mildews, but our infection trials demonstrate that P. cubensis is able to infest this plant. Amplification and sequencing of the ITS rDNA confirmed the observed downy mildew disease on B. dioica as P. cubensis. From these findings, the possibility that P. cubensis may be able to overwinter on this perennial host cannot be excluded. Whether or not B. dioica plays a part in the epidemology of P. cubensis in Europe requires evaluation by further studies.     

Phylogenetic investigations in the genus <Emphasis Type="Italic">Pseudoperonospora</Emphasis> reveal overlooked species and cryptic diversity in the <Emphasis Type="Italic">P. cubensis</Emphasis> species cluster

Pseudoperonospora cubensis is one of the most devastating diseases of cucurbitaceous crops. The pathogen has a worldwide distribution and occurs in all major cucurbit growing areas. It had been noticed for the first time at the end of the 19th century, but it became a globally severe disease as recently as 1984 in Europe and 2004 in North America. Despite its economic importance, species concepts in Pseudoperonospora are debated. Here, we report that the genus Pseudoperonospora contains cryptic species distinct from the currently accepted ones. Pseudoperonospora on Celtis is split into two phylogenetic lineages and Pseudoperonospora humuli is confirmed as a species distinct from the Cucurbitaceae-infecting lineages. A cryptic species occupying a basal position within the Pseudoperonospora cubensis complex is revealed to be present on Humulus japonicus, thus providing evidence that the host jump that gave rise to Pseudoperonospora cubensis likely occurred from hops. Notably, Cucurbitaceae infecting pathogens are present in two cryptic sister species or subspecies. Clade 1 contains primarily specimens from North America and likely represents Pseudoperonospora cubensis s.str.. Pre-epidemic isolates in clade 2 originate from Japan and Korea, suggesting this cryptic species or subspecies is indigenous to East Asia. Recent samples of this lineage from epidemics in Europe and the United States cluster together with clade 2. It thus seems possible that this lineage is associated with the recent severe epidemics of cucurbit downy mildew and is now naturalised in North America and Europe.     

Coincidence of virulence shifts and population genetic changes of Pseudoperonospora cubensis in the Czech Republic

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.     

Phylogenetic investigations in the downy mildew genus <Emphasis Type="Italic">Bremia</Emphasis> reveal several distinct lineages and a species with a presumably exceptional wide host range

Bremia lactucae is one of the most devastating and widespread pathogens in lettuce production worldwide. Despite its economical importance, uncertainty prevails about the species delimitation in the genus Bremia. Commonly, Bremia is considered to be monotypic, containing only Bremia lactucae, while taxonomists have described additional species, and molecular phylogenetic studies have shown significant sequence divergence between accessions from different hosts. Here, we report that several previously described species are genetically highly distinct from Bremia lactucae parasitic to Lactuca sativa. These include Bremia lapsanae, Bremia sonchicola, and Bremia taraxaci. In addition to these host-specific species, a plurivorous species is revealed, which infects hosts from three different tribes in the Asteraceae subfamilies Asteroideae and Carduoideae. The broad host range of clade 1 is exceptional for downy mildews and only paralleled by Pseudoperonospora cubensis, which infects a broad range of Cucurbitaceae. The taxonomic status of Bremia cirsii and of Bremia centaureae remains unresolved, as the accessions from Cirsium and Centaurea, respectively, did not form a monophylum but were partly contained in the plurivorous clade 1. Bremia lactucae was found to be restricted to Lactuca sativa and Lactuca serriola. Thus, it can be assumed that Bremia infections on weeds apart from Lactuca species do not pose a significant risk for lettuce production. However, it is unlikely that breeding resistance genes from Lactuca serriola into Lactuca sativa will result in durable resistance of lettuce to downy mildew disease, because the current study provides additional evidence that Bremia accessions from both hosts form a population continuum.     

Cucurbit downy mildew (<Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>)—biology,ecology, epidemiology,host-pathogen interaction and control

Cucurbit downy mildew, caused by the oomycete Pseudoperonospora cubensis, is a devastating, worldwide-distributed disease of cucurbit crops in the open field and under cover. This review provides recent data on the taxonomy, biology, ecology, host range, geographic distribution and epidemiology of P. cubensis. Special attention is given to host-pathogen interactions between P. cubensis and its economically-important cucurbit hosts (Cucumis sativus, C. melo, Cucurbita pepo, C. maxima, and Citrullus lanatus); pathogenic variability in P. cubensis at the species, genus, and population levels; and, differentiation of pathotypes and races. Genetics and variability of host resistance and cellular and molecular aspects of such resistance are considered. A focus is given to methods of crop protection, including prevention and agrotechnical aspects, breeding for resistance—classical and transgenic approaches, chemical control and fungicide resistance. Novel technologies in biological and integrated control are also discussed. This review also summarizes the most important topics for future research and international collaboration.     

Screening of wildcucumis species against downy mildew (Pseudoperonospora cubensis) isolates from cucumbers

Under controlled inoculation, a set of 56 accessions belonging to 19 wild species of the genusCucumis was studied for resistance to seven isolates of cucurbit downy mildew (Pseudoperonospora cubensis (Berk. et Curt.) Rostow.) from cucumber. No resistance toP. cubensis was detected in theseCucumis accessions. In three host accession/pathogen isolate combinations, limited sporulation was observed. Nine newCucumis species are described as hosts forP. cubensis: C. africanus, C. ficifolius, C. figarei, C. meeusii, C. metuliferus, C. myriocarpus, C. leptodermis, C. sagittatus andC. zeyheri. Results are discussed in relation to the origin and evolution ofCucumis species.     

Molecular phylogenetic analysis of <Emphasis Type="Italic">Peronosclerospora</Emphasis> (Oomycetes) reveals cryptic species and genetically distinct species parasitic to maize

Downy mildews are amongst the most widespread and economically important pathogens of cultivated grasses in the tropics and subtropics. Despite their importance, molecular methods, particularly DNA sequence analysis, have rarely been applied to either species identification or to the determination of phylogenetic relationships between species. Here we report the presence of several cryptic species in the genus Peronosclerospora. Further we confirm that maize can be parasitised by several species of Peronosclerospora, including P. eriochloae, which has not been reported previously as a pathogen of maize. The presence of 14 distinct phylogenetic lineages, including three that are parasitic to maize, highlights the current fragmentary knowledge on the diversity and classification of species within Peronosclerospora. Species identification in Peronosclerospora has been traditionally based on the host genus and a set of variable morphological characteristics, which has meant that the identification of species is often unreliable. This situation is primed for the application of molecular techniques for the identification of species. One of the lineages parasitic to maize in Australia has not yet been formally described and its distribution is not known. Future investigation including a broad sampling of downy mildews from maize and other cultivated and native grasses on a world-wide basis is a prerequisite to a re-evaluation of quarantine regulations aimed at restricting or limiting their spread.     

Resistance mechanism to carboxylic acid amide fungicides in the cucurbit downy mildew pathogen Pseudoperonospora cubensis

BACKGROUND: Pseudoperonospora cubensis, the causal oomycete agent of cucurbit downy mildew, is responsible for enormous crop losses in many species of Cucurbitaceae, particularly in cucumber and melon. Disease control is mainly achieved by combinations of host resistance and fungicide applications. However, since 2004, resistance to downy mildew in cucumber has been overcome by the pathogen, thus driving farmers to rely only on fungicide spray applications, including carboxylic acid amide (CAA) fungicides. Recently, CAA‐resistant isolates of P. cubensis were recovered, but the underlying mechanism of resistance was not revealed. The purpose of the present study was to identify the molecular mechanism controlling resistance to CAAs in P. cubensis. RESULTS: The four CesA (cellulose synthase) genes responsible for cellulose biosynthesis in P. cubensis were characterised. Resistant strains showed a mutation in the CesA3 gene, at position 1105, leading to an amino acid exchange from glycine to valine or tryptophan. Cross‐resistance tests with different CAAs indicated that these mutations lead to resistance against all tested CAAs. CONCLUSION: Point mutations in the CesA3 gene of P. cubensis lead to CAA resistance. Accurate monitoring of these mutations among P. cubensis populations may improve/facilitate adequate recommendation/deployment of fungicides in the field. Copyright © 2011 Society of Chemical Industry     

Tracking host infection and reproduction of Peronospora salviae-officinalis using an improved method for confocal laser scanning microscopy

Peronospora salviae-officinalis, the causal agent of downy mildew on common sage, is an obligate biotrophic pathogen. It grows in the intercellular spaces of the leaf tissue of sage and forms intracellular haustoria to interface with host cells. Although P. salviae-officinalis was described as a species of its own 10 years ago, the infection process remains obscure. To address this, a histological study of various infection events, from the adhesion of conidia on the leaf surface to de novo sporulation is presented here. As histological studies of oomycetes are challenging due to the lack of chitin in their cell wall, we also present an improved method for staining downy mildews for confocal laser scanning microscopy as well as evaluating the potential of autofluorescence of fixed nonstained samples. For staining, a 1:1 mixture of aniline blue and trypan blue was found most suitable and was used for staining of oomycete and plant structures, allowing discrimination between them as well as the visualization of plant immune responses. The method was also used to examine samples of Peronospora lamii on Lamium purpureum and Peronospora belbahrii on Ocimum basilicum, demonstrating the potential of the presented histological method for studying the infection processes of downy mildews in general.     

Resistance to acylalanines in Pseudoperonospora cubensis1

Resistance to acylalanine fungicides in Pseudoperonospora cubensis, the downy mildew of cucurbits, is reviewed. Insensitive strains of the fungus predominated during the second year of metalaxyl application in cucumber glasshouses in Greece and Israel. Resistance was detected either on detached cucumber leaves floated on metalaxyl solutions or on potted plants treated (sprayed or drenched) with this fungicide. Metalaxyl-resistant strains of P. cubensis showed good pathogenicity and fitness, and competed favorably with the sensitive strains in the absence of the fungicide. A synergism between these two biotypes resulted in an increased virulence of the resistant forms on metalaxyl-treated plants. Metalaxyl-resistant strains of the pathogen exhibited cross resistance to other acylalanine fungicides. Strains resistant to acylalanines and to the chemically unrelated oomycete-fungicides fosetyl-Al and propamocarb were isolated in Israel, as well. In Greece, however, protective sprays with fosetyl-Al, cymoxanil and mancozeb gave good control of cucumber downy mildew when the pathogen was resistant to metalaxyl. P. cubensis is the first case of fungus which developed resistance to acylalanines in the field.     

Downy mildew of Cucurbits (Pseudoperonospora Cubensis): the Fungus and its hosts,distribution, epidemiology and control

Taxonomy of the genusPseudoperonospora, morphology ofPseudoperonospora cubensis (Berk, et Curt.) Rostow. and occurrence of its oospores, are described briefly. A list is presented of over 40 cucurbitaceous host species, representing about 20 genera, on whichP. cubensis has been recorded. Two or more races exist in Japan and the United States, but not in Europe or the Middle East. The distribution ofP. cubensis is widest on all continents on cucumbers (70 countries) and muskmelon (50 countries); onCucurbita and watermelons it extends to about 40 and 25 countries, respectively. P. cubensis may overwinter as oospores, though this seems rare, and on wild hosts or crops grown in the open or under cover. Airborne sporangia may also reach cooler countries from regions with mild winters. Apart from the leaf wetness essential for infection, the factors determining disease progress are: rate of foliage growth and physiological age of the host; amount of primary inoculum available, light, and the rate at which lesions necrotize. The interaction of these factors is described for early, mid-season, and late crops. Losses caused byP. cubensis depend on the growth stage at which the crop is attacked, and on the rate of foliage and pathogen development. Breeding has produced downy mildew resistant lines of cucumbers, used chiefly in the United States, and some resistant lines of melons and watermelons. The most important agricultural practices used to restrict downy mildew development are proper irrigation management and avoidance of sowing in proximity to infected crops. Success of control by protectant chemicals depends largely on proper timing of applications. Proximity of inoculum sources, hours of leaf wetness, age of crop, and irrigation practices are the principal factors that determine when to begin treatments. These factors and rate of leaf formation determine the frequency of applications. Application of systemic fungicides is much easier to time correctly.     

Host range expansion in a powdery mildew fungus (<Emphasis Type="Italic">Golovinomyces</Emphasis> sp.) infecting <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: <Emphasis Type="Italic">Torenia fournieri</Emphasis> as a new host

Since 2003, Torenia fournieri plants grown for experimental purposes were repeatedly infected by powdery mildew in a laboratory in Hungary. Based on morphological characteristics, the pathogen belonged to the mitosporic genus Oidium subgen. Reticuloidium, the anamorph stage of Golovinomyces. The rDNA ITS sequence was identical to that of two other powdery mildew fungi, infecting Arabidopsis and Veronica, respectively, in different parts of the world. According to a previous phylogenetic analysis of ITS and 28S rDNA sequences, those two powdery mildews belong to a recently evolved group of Golovinomyces characterized by multiple host range expansions during their evolution. Both the ITS sequence and the morphological data indicate that the powdery mildew anamorph infecting Torenia also belongs to this group. It is likely that the powdery mildew infections of the experimental T. fournieri plants, native to south-east Asia, were the result of a very recent host range expansion of a polyphagous Golovinomyces because (i) T. fournieri is absent from our region, except as an experimental plant grown in the laboratory, (ii) the powdery mildew fungus infecting this exotic plant belongs to a group of Golovinomyces where host range expansion is a frequent evolutionary scenario, (iii) cross-inoculation tests showed that this pathogen is also able to infect other plant species, notably A. thaliana and tobacco, and (iv) no Golovinomyces species are known to infect T. fournieri anywhere in the world. Although host range expansion has often been proposed as a common evolutionary process in the Erysiphales, and also in other biotrophic plant pathogens, this has not been clearly demonstrated in any case studies so far. To our knowledge, this is the first convincing case of a host range expansion event in the Erysiphales.     

Structure and temporal shifts in virulence of Pseudoperonospora cubensis populations in the Czech Republic

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, which 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.     

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.     

Identification of <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis> using real-time PCR and high resolution melting (HRM) analysis

To develop a molecular method to identify Pseudoperonospora cubensis in cucumber leaves with signs of downy mildew, we compared the nucleotide sequences of ribosomal DNA-internal transcribed spacer, cytochrome oxidase II, and β-tubulin genes of P. cubensis and P. humuli isolates. Seven single nucleotide polymorphisms (SNPs) distinguished P. cubensis and P. humuli based on variations in β-tubulin sequences, and one specific primer set was designed for further analysis. Real-time PCR and high resolution melting analysis showed that the primer set can be used to specifically identify P. cubensis in cucumber leaves with downy mildew.     

Department of Crop Science,Herbicide Section,Report October 1967 - August 1968

Abstract

A serious outbreak of muskmelon (Cucumis melo L.) downy mildew caused by Pseudoperonospora cubensis (Berk. &; Curt) Rostow, occurred in the Punjab (India) due to unusual rain showers accompanied by high humidity. Nine different fungicides were evaluated and of these zineb 75% (Dithane Z-78) and copper oxychloride (Blitox 50%) gave reasonable control of the disease.     

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.     

Synergistic interaction between BABA and mancozeb in controlling<Emphasis Type="Italic">Phytophthora infestans</Emphasis> in potato and tomato and<Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis> in cucumber

Spray mixtures consisting of the plant activator BABA (DL-3-aminobutyric acid) and the protectant fungicide mancozeb were significantly more effective than BABA or mancozeb alone in controlling late blight (Phytophthora infestans) in potato and tomato and downy mildew (Pseudoperonospora cubensis) in cucumber. A mixture composed of 5 parts BABA and 1 part mancozeb (w/w, a.i.) exhibited a higher synergy factor than the 1+1 or the 1+5 (BABA + mancozeb) mixtures. No synergistic interaction was detected between BABA plus mancozeb in controlling sporangial or cystospore germination, nor mycelial growth ofP. infestans in vitro. The results showed enhanced effect of mancozeb in BABA-induced plants, suggesting, therefore, that lower dosages of this fungicide may be sufficient to control late blight or downy mildew under field conditions. http://www.phytoparasitica.org posting July 15, 2003.     

Increased susceptibility toPseudoperonospora cubensis in virus-infectedCucurbita pepo genotypes

Pseudoperonospora cubensis developed more and larger lesions, infected greater leaf areas and produced more sporangia on virus-infected (Vi) than on virus-free (Vf) leaves ofCucurbita pepo genotypes.Pseudoperonospora cubensis from Vi and Vf leaves did not vary in sporangial dimensions (length x width) or pathogenicity to different cucurbits. The preferential development and sporulation ofP. cubensis in Vi than in Vf leaves was due to increased host susceptibility.     

A new pathotype of<Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis> causing downy mildew in cucurbits in Israel

Downy mildew, incited by the comycetePseudoperonospora cubensis (Berk.et Curt.) Rost., was recorded in Israel during the years 1979–2001 on cucumber (Cucumis sativus) and melon (Cucumis melo) and classified as pathotype 3. In July 2002 severe outbreaks of downy mildew occurred on pumpkin (Cucurbita moschata) and summer squash (Cucurbita pepo subsp.pepo). Host range pathogenicity studies revealed high compatibility of the new population with cucumber, melon, pumpkin and summer squash but low compatibility with watermelon. This new population was therefore designated as pathotype 6. The possible origin of this new pathotype in Israel is discussed. http://www.phytoparasitica.org posting Oct. 9, 2003.