The hop downy mildew pathogen Pseudoperonospora humuli

Pseudoperonospora humuli is an obligate biotrophic oomycete that causes downy mildew, one of the most devastating diseases of cultivated hop, Humulus lupulus. Downy mildew occurs in all production areas of the crop in the Northern Hemisphere and Argentina. The pathogen overwinters in hop crowns and roots, and causes considerable crop loss. Downy mildew is managed by sanitation practices, planting of resistant cultivars, and fungicide applications. However, the scarcity of sources of host resistance and fungicide resistance in pathogen populations complicates disease management. This review summarizes the current knowledge on the symptoms of the disease, life cycle, virulence factors, and management of hop downy mildew, including various forecasting systems available in the world. Additionally, recent developments in genomics and effector discovery, and the future prospects of using such resources in successful disease management are also discussed.TaxonomyClass: Oomycota; Order: Peronosporales; Family: Peronosporaceae; Genus: Pseudoperonospora; Species: Pseudoperonospora humuli.Disease symptomsThe disease is characterized by systemically infected chlorotic shoots called “spikes". Leaf symptoms and signs include angular chlorotic lesions and profuse sporulation on the abaxial side of the leaf. Under severe disease pressure, dark brown discolouration or lesions are observed on cones. Infected crowns have brown to black streaks when cut open. Cultivars highly susceptible to crown rot may die at this phase of the disease cycle without producing shoots. However, foliar symptoms may not be present on plants with systemically infected root systems.Infection processPathogen mycelium overwinters in buds and crowns, and emerges on infected shoots in spring. Profuse sporulation occurs on infected tissues and sporangia are released and dispersed by air currents. Under favourable conditions, sporangia germinate and produce biflagellate zoospores that infect healthy tissue, thus perpetuating the infection cycle. Though oospores are produced in infected tissues, their role in the infection cycle is not defined.ControlDowny mildew on hop is managed by a combination of sanitation practices and timely fungicide applications. Forecasting systems are used to time fungicide applications for successful management of the disease.Useful Websites https://content.ces.ncsu.edu/hop‐downy‐mildew (North Carolina State University disease factsheet), https://www.canr.msu.edu/resources/michigan‐hop‐management‐guide (Michigan Hop Management Guide), http://uspest.org/risk/models (Oregon State University Integrated Plant Protection Center degree‐day model for hop downy mildew), https://www.usahops.org/cabinet/data/Field‐Guide.pdf (Field Guide for Integrated Pest Management in Hops).     

The cucurbit downy mildew pathogen Pseudoperonospora cubensis

Pseudoperonospora cubensis[(Berkeley & M. A. Curtis) Rostovzev], the causal agent of cucurbit downy mildew, is responsible for devastating losses worldwide of cucumber, cantaloupe, pumpkin, watermelon and squash. Although downy mildew has been a major issue in Europe since the mid-1980s, in the USA, downy mildew on cucumber has been successfully controlled for many years through host resistance. However, since the 2004 growing season, host resistance has been effective no longer and, as a result, the control of downy mildew on cucurbits now requires an intensive fungicide programme. Chemical control is not always feasible because of the high costs associated with fungicides and their application. Moreover, the presence of pathogen populations resistant to commonly used fungicides limits the long-term viability of chemical control. This review summarizes the current knowledge of taxonomy, disease development, virulence, pathogenicity and control of Ps. cubensis. In addition, topics for future research that aim to develop both short- and long-term control measures of cucurbit downy mildew are discussed. TAXONOMY: Kingdom Straminipila; Phylum Oomycota; Class Oomycetes; Order Peronosporales; Family Peronosporaceae; Genus Pseudoperonospora; Species Pseudoperonospora cubensis. DISEASE SYMPTOMS: Angular chlorotic lesions bound by leaf veins on the foliage of cucumber. Symptoms vary on different cucurbit species and varieties, specifically in terms of lesion development, shape and size. Infection of cucurbits by Ps. cubensis impacts fruit yield and overall plant health. INFECTION PROCESS: Sporulation on the underside of leaves results in the production of sporangia that are dispersed by wind. On arrival on a susceptible host, sporangia germinate in free water on the leaf surface, producing biflagellate zoospores that swim to and encyst on stomata, where they form germ tubes. An appressorium is produced and forms a penetration hypha, which enters the leaf tissue through the stomata. Hyphae grow through the mesophyll and establish haustoria, specialized structures for the transfer of nutrients and signals between host and pathogen. CONTROL: Management of downy mildew in Europe requires the use of tolerant cucurbit cultivars in conjunction with fungicide applications. In the USA, an aggressive fungicide programme, with sprays every 5-7 days for cucumber and every 7-10 days for other cucurbits, has been necessary to control outbreaks and to prevent crop loss. USEFUL WEBSITES: http://www.daylab.plp.msu.edu/pseudoperonospora-cubensis/ (Day Laboratory website with research advances in downy mildew); http://veggies.msu.edu/ (Hausbeck Laboratory website with downy mildew news for growers); http://cdm.ipmpipe.org/ (Cucurbit downy mildew forecasting homepage); http://ipm.msu.edu/downymildew.htm (Downy mildew information for Michigan's vegetable growers).     

Infection periods in relation to the natural development of hop downy mildew (Pseudoperonospora humuli)

The relationships between temperature and surface wetness and subsequent infection of hop tissues by P. humuli were examined on potted plants and detached leaves kept in temperature-controlled growth rooms. Periods of wetness which would just allow leaf infection ranged from 1 1/2 h at 30d? to 24 h at 5d?. The corresponding ranges for shoots were: light infection, 3 h at 19–23d? to 6 h at 8–10d?; severe infection, 4 h at 19–23d? to 8 h at 12–13d?. These data were used to relate the development of downy mildew in an unsprayed hop garden during 1967 and 1968 to periods with temperature/surface wetness suitable for minimum (minor infection periods) and severe infection (major infection periods). In 1967 a sudden outbreak of infected basal shoots (spikes) was related to an isolated major infection period. By contrast, early in 1968, major shoot infection periods did not arise and spikes appeared gradually in response to a succession of minor infection periods. More spikes were formed in 196 than in 1967; this was not related to the incidence of infection periods but probably reflected the relatively higher concentrations of airborne sporangia early in 1968. In both years outbreaks of leaf and lateral shoot infection could be traced to major infection periods caused by rain; sudden disease increases again originated from isolated infection periods. There was a close similarity between the incubation period for each principal disease outbreak and that expected from growth-room experiments. Major infection periods occurred more frequently at the end of June 1968, resulting in a higher final concentration of diseased tissue than in 1967. Predicted major infection periods failed to induce large disease increases when dew alone provided wetness or when no airborne sporangia could be detected.     

Small RNA inhibits infection by downy mildew pathogen Hyaloperonospora arabidopsidis

Gene silencing exists in eukaryotic organisms as a conserved regulation of the gene expression mechanism. In general, small RNAs (sRNAs) are produced within the eukaryotic cells and incorporated into an RNA-induced silencing complex (RISC) within cells. However, exogenous sRNAs, once delivered into cells, can also silence target genes via the same RISC. Here, we explored this concept by targeting the Cellulose synthase A3 (CesA3) gene of Hyaloperonospora arabidopsidis (Hpa), the downy mildew pathogen of Arabidopsis thaliana. Hpa spore suspensions were mixed with sense or antisense sRNAs and inoculated onto susceptible Arabidopsis seedlings. While sense sRNAs had no obvious effect on Hpa pathogenicity, antisense sRNAs inhibited spore germination and hence infection. Such inhibition of infection was not race-specific, but dependent on the length and capping of sRNAs. Inhibition of infection by double stranded sRNA was more efficient than that observed with antisense sRNA. Thus, exogenous sRNA targeting conserved CesA3 could suppress Hpa infection in Arabidopsis, indicating the potential of this simple and efficient sRNA-based approach for deciphering gene functions in obligate biotrophic pathogens as well as for R-gene independent control of diseases in plants.     

Zoosporogenesis in Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew

During sporulation of Pseudoperonospora cubensis on cucumber leaves ( Cucumis saliva ) zoosporangia are formed on the dichotomously branched sporangiophore. The mature zoosporangium has a preformed discharge papilla and the cytoplasm is uncleaved. The zoosporangium wall is decorated and the outer layer of the wall is electron opaque in ultrathin sections. As the zoosporangium is able to survive freezing (- 18°C) for prolonged periods of time (3–4 months) the zoosporangium may serve as the "resting" structure which survives overwintering in Northern latitudes in the absence of oospore formation.
Zoospore cleavage can be synchronized by placing freshly harvested zoosporangia in distilled water. Cleavage of the zoosporangial cytoplasm is by means of the fusion of small vesicles apparently derived from dictyosomes which become highly active after zoosporogenesis is induced.
Vesicles with an osmiophilic electron opaque content are the dominant type of vesicle found in the zoosporangia. The content of these vesicles undergoes dynamic changes during zoosporogenesis and during the late stages of sporogenesis the content becomes finely striated as is typical of these vesicles when observed in the zoospore. On the basis of the results presented here it is suggested that zoosporangium formation and zoosporogenesis in P. cubensis could serve as a model system for assays with obligate oomycetous plant pathogens, also in relation to fungicide mode of action studies.     

The zoospore of Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew

The zoospore of Pseudosporonospora cubensis is typical of the secondary zoospore of the Peronosporales. The reniform zoospore contains a central nucleus with a prominent beak-like extension to the kinetosomes on the lateral side of the spore in the groove region. "Fuzzy" vesicles derived from dictyosomes surround and fuse with the contractile vacuole. Mitochondria and microbodies are located in the peripheral cytoplasm of the zoospore but the latter are confined to the groove region of the spore. The microbodies usually contain a laminate inclusion and the microbodies are not in a fixed position in relation to the peripheral cisternae. Neither a microbody-lipid body complex nor a "U-body" were observed.
The kinetosomes of the spore are almost perpendicular to each other at the distal end of the beak-like extension of the nucleus. A complex system of cytoplasmic microtu-bules flare out from the kinetosomes to surround the nucleus and bundles of cytoplasmic microtubules extend under the plasmalemma of the spore. The zoospore contain numerous vesicles with osmiophilic inclusions which are finely striated; these are the so-called finger-print vesicles.     

Internal mycelium of Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew

The internal mycelium of Pseudoperonospora cubensis has been observed in transmission and scanning electron microscopic preparations. The internal mycelium may be inter- or intracellular. Haustoria of short swollen bundles of hyphae have been observed. Actively growing hyphae contain numerous mitochondria, nuclei, active dictyosomes, low amounts of storage materials (lipid) and microbody-like structures with a laminate inclusion. Thick walled hyphae with a diameter which is smaller than the actively growing hyphae have been observed. These thick wallcd hyphae contain large amounts of reserve material (lipid) and it is suggested that they may function as resting propagules.     

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.     

Arabidopsis is susceptible to infection by a downy mildew fungus.

A population of Arabidopsis thaliana growing locally in a suburb of Zürich called Weiningen was observed to be infected with downy mildew. Plants were collected and the progress of infection was investigated in artificial inoculations in the laboratory. The plants proved to be highly susceptible, and pronounced intercellular mycelial growth, haustoria formation, conidiophore production, and sporulation of the causal organism Peronospora parasitica were all observed. The formation of oogonia, antheridia, and oospores also occurred. In contrast, Arabidopsis strain RLD was resistant to infection and none of the above structures was formed. The fungus was localized very soon after penetration of RLD leaf cells, which responded with a typical hypersensitive reaction. The differential interaction of an isolate of P. parasitica with two strains of Arabidopsis opens up the possibility of cloning resistance determinants from a host that is very amenable to genetic and molecular analysis.     

Resurgence of cucurbit downy mildew in the United States: Insights from comparative genomic analysis of Pseudoperonospora cubensis

Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew (CDM), is known to exhibit host specialization. The virulence of different isolates of the pathogen can be classified into pathotypes based on their compatibility with a differential set composed of specific cucurbit host types. However, the genetic basis of host specialization within P. cubensis is not yet known. Total genomic DNA extracted from nine isolates of P. cubensis collected from 2008 to 2013 from diverse cucurbit host types (Cucumis sativus, C. melo var. reticulatus, Cucurbita maxima, C. moschata, C. pepo, and Citrullus lanatus) in the United States were subjected to whole‐genome sequencing. Comparative analysis of these nine genomes confirmed the presence of two distinct evolutionary lineages (lineages I and II) of P. cubensis. Many fixed polymorphisms separated lineage I comprising isolates from Cucurbita pepo, C. moschata, and Citrullus lanatus from lineage II comprising isolates from Cucumis spp. and Cucurbita maxima. Phenotypic characterization showed that lineage II isolates were of the A1 mating type and belonged to pathotypes 1 and 3 that were not known to be present in the United States prior to the resurgence of CDM in 2004. The association of lineage II isolates with the new pathotypes and a lack of genetic diversity among these isolates suggest that lineage II of P. cubensis is associated with the resurgence of CDM on cucumber in the United States.     

Identification of arabidopsis loci required for susceptibility to the downy mildew pathogen Hyaloperonospora parasitica

Plants are susceptible to a limited number of pathogens. Most infections fail due to active defense or absence of compatibility. Many components of the plant's surveillance system and defense arsenal have been identified in the last decades. However, knowledge is limited on compatibility; in particular, the role of plant factors in the infection process. To gain insight into these processes, we have initiated an Arabidopsis thaliana mutant screen for reduced susceptibility to the downy mildew pathogen Hyaloperonospora parasitica. Ethyl methane sulfonate (EMS) mutants were generated in the highly susceptible Arabidopsis line Ler eds1-2. Eight downy mildew-resistant (dmr) mutants were analyzed in detail, corresponding to six different loci. Microscopic analysis showed that, in all mutants, H. parasitica growth was severely reduced. Resistance of dmr3, dmr4, and dmr5 was associated with constitutive expression of PR-1. Furthermore, dmr3 and dmr4, but not dmr5, also were resistant to Pseudomonas syringae and Golovinomyces orontii, respectively. However, enhanced activation of plant defense was not observed in dmr1, dmr2, and dmr6. We postulate that, in these susceptibility mutants, cellular processes are disrupted which are required for H. parasitica infection. This interesting new set of mutants provides a basis to elucidate the molecular processes underlying susceptibility to downy mildew in Arabidopsis.     

Emerging virulence arising from hybridisation facilitated by multiple introductions of the sunflower downy mildew pathogen Plasmopara halstedii

The sunflower downy mildew pathogen Plasmopara halstedii is an invasive plant pathogen in Europe of American origin. Despite efforts to produce resistant host varieties, nationwide monitoring in France has revealed the rapid emergence of new virulent races increasing the number from one founder identified in 1966 to as many as 14 today. We have genotyped 146 samples (including all 14 races) using 13 nuclear and one mtDNA marker. Samples of the same race were found to share alleles/mtDNA haplotype and the two most common races had individuals with multiple matching genotypes. Cluster analyses confirmed that the samples form three groups to which races strongly adhere. Clusters were highly differentiated (F(ST) 0.65) and characterised by high inbreeding coefficients. Despite this, samples of recently emergent races, including six that are unique to France had mixed ancestry between the groups suggesting they have arisen in situ due to hybridisation. Five such samples also had conflicting mtDNA and nuclear DNA profiles. This demonstrates that multiple introductions have aided the establishment of this pathogen in France, and suggests recombination facilitated by these introductions is driving the emergence of new and endemic races in response to host resistance.     

Host‐induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce

Host‐induced gene silencing (HIGS) is an RNA interference‐based approach in which small interfering RNAs (siRNAs) are produced in the host plant and subsequently move into the pathogen to silence pathogen genes. As a proof‐of‐concept, we generated stable transgenic lettuce plants expressing siRNAs targeting potentially vital genes of Bremia lactucae, a biotrophic oomycete that causes downy mildew, the most important disease of lettuce worldwide. Transgenic plants, expressing inverted repeats of fragments of either the Highly Abundant Message #34 (HAM34) or Cellulose Synthase (CES1) genes of B. lactucae, specifically suppressed expression of these genes, resulting in greatly reduced growth and inhibition of sporulation of B. lactucae. This demonstrates that HIGS can provide effective control of B. lactucae in lettuce; such control does not rely on ephemeral resistance conferred by major resistance genes and therefore offers new opportunities for durable control of diverse diseases in numerous crops.     

A genetic map of the lettuce downy mildew pathogen,Bremia lactucae,constructed from molecular markers and avirulence genes

The genetic map of Bremia lactucae was expanded utilizing 97 F(1) progeny derived from a cross between Finnish and Californian isolates (SF5xC82P24). Genetic maps were constructed for each parent utilizing 7 avirulence genes, 83 RFLP markers, and 347 AFLP markers, and a consensus map was constructed from the complete data set. The framework map for SF5 contained 24 linkage groups distributed over 835cM; the map for C82P24 contained 21 linkage groups distributed over 606cM. The consensus map contained 12 linkage groups with markers from both parents and 24 parent-specific groups. Six avirulence genes mapped to different linkage groups; four were located at the ends of linkage groups. The closest linkages between molecular markers and avirulence genes were 3cM to Avr4 and 1cM to Avr7. Mating type seemed to be determined by a single locus, where the heterozygote determined the B(2) type and the homozygous recessive genotype determined the B(1) type.     

Antioxidant metabolism in pearl millet genotypes during compatible and incompatible interaction with downy mildew pathogen

In this study, the ascorbic acid content, lipid peroxidation product, reactive oxygen generation and scavenging enzyme activities were determined in pearl millet [Pennisetum glaucum (L.) R.Br.] leaves. These parameters were analysed at two stages: (i) pre-infection [45 days after sowing (DAS)] and (ii) post-infection [7 days after infection (DAI), i.e. 57 DAS]. Lipid peroxidation product (malondialdehyde content) was recorded higher in compatible interaction at pre-infection stage while it was increased in incompatible interaction at post-infection stage. Resistant genotypes had higher ascorbic acid content at both the stages of analysis. Superoxide dismutase (SOD) activity was higher in susceptible genotypes at pre-infection but after infection it was found to be higher in resistant genotypes. Ascorbate peroxidase, catalase (CAT) and lipoxygenase activities were higher in resistant genotypes at both the stages of analysis. Native PAGE isozyme banding pattern of SOD, CAT, APX and esterase showed some inducible band(s) due to disease infection.