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).     

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.     

Relationship between virulence and aggressiveness in Plasmopara halstedii (sunflower downy mildew)

Relationship between virulence and aggressiveness was studied in seven Plasmopara halstedii (sunflower downy mildew) pathotypes including five progeny pathotypes of races 300, 304, 314, 704 and 714 arising from two parental pathotypes of races 100 and 710. Aggressiveness criteria including percentage infection, latent period, sporulation density and reduction of hypocotyl length were analysed in one sunflower inbred line showing a high level of quantitative resistance. There were significant differences between P. halstedii pathotypes for all aggressiveness criteria. Pathogenicity of progeny pathotypes as compared with parental ones (relationship between virulence and aggressiveness) seems to be positive, negative or uncorrelated. Hypothesis explaining these cases are discussed.     

Control of downy mildew (Pseudoperonospora cubensis) of greenhouse grown cucumbers with alternative biological agents

In organic cucumber production infection with downy mildew (Pseudoperonospora cubensis) is a major problem. Plant extracts from Glycyrrhiza glabra L. (licorice), a plant belonging to the family Fabaceae, and Salvia officinalis (sage) as well as cultures of the bacterium Aneurinibacillus migulanus were investigated for efficacy of disease control under commercial growing conditions. Contrary to bioassays, where sage extract and the microorganism showed highest activity, in the trials of 2008 G. glabra extract was more effective than sage extract or A. migulanus against P. cubensis. Parameters such as concentrations of the preparations or application intervals could have been the reason for this. In the following year's trial (2009) the concentration of these agents was therefore increased somewhat and plants were either treated in seven day application intervals or in ten day application intervals. In the semi-commercial trials of 2009 all alternative biological agents showed good efficacies up to around 80% against infection with downy mildew. The application interval seemed to have a marginal effect only. Again, the licorice extract tended to be the best agent.     

Relationship between aggressiveness and zoosporangia viability in Plasmopara halstedii (sunflower downy mildew)

Relationship between aggressiveness and zoosporangia viability was studied in seven Plasmopara halstedii (sunflower downy mildew) isolates of races 100, 300, 304, 314, 710, 704 and 714. Aggressiveness criteria including latent period and sporulation density were analysed on sunflower inbred line showing a high level of quantitative resistance. There were significant differences between pathogen isolates for the two aggressiveness criteria. Viability analyses were performed on oval and spheric zoosporangia. The number of zoospores released from oval zoosporangia was significantly higher than those released from spheric ones. The oval zoosporangia for more aggressive isolates of races 100 and 3xx produced more zoospores than the oval ones for less aggressive isolates of races 7xx. There was a significant correlation between aggressiveness criteria and the number of zoospores released from oval zoosporangia and vice versa for zoospores released from spheric ones. It is concluded that the relationship between aggressiveness and oval zoosporangia viability may be established in P. halstedii.     

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.     

The relation between cation-ratio and host-resistance to certain downy mildew and root-knot diseases

Summary The (Ca+Mg)/K ratio was found to have a relationship with the resistance of various crops to downy mildew and root-knot diseases. Predisposing factors such as host nutrition, prior infection by other pathogens and plant age changed (Ca+Mg)/K ratio. (Ca+Mg)/K ratio calculated from the published data of other workers also exhibited this relationship. It appears to play a role in several host-pathogen systems.     

Cytoskeletal responses during early development of the downy mildew of grapevine (Plasmopara viticola)

A host-free system was established to induce the early development of the obligate biotrophic pathogen Plasmopara viticola, the downy mildew of grapevine. This system was used to study cytoskeletal responses during encystation and germ tube formation. During these processes, both the actin and the tubulin cytoskeleton show a stage-specific pattern of distribution. Elimination of the cytoskeleton by the actin drug latrunculin B and the microtubule drug ethyl-N-phenyl-carbamate did not affect the release of mobile zoospores from the sporangia, nor the encystation process, but efficiently inhibited the formation of a germ tube. The data are discussed with respect to a role of both actin and microtubules for the establishment of the cell polarity guiding the emergence and the growth of the germ tube.     

Control of pearl millet downy mildew caused by Sclerospora graminicola with systemic fungicides in an artificially-contaminated plot

Among four fungicides, viz. metalaxyl (two formulations), fosetyl-Al, pro-pamocarb and cyomaxanil tested in vitro against sporangial germination inhibition of Sclerospora graminicola, cyomaxanil was found to be most inhibitory. In an artificially contaminated plot, when used as seed treatment or foliar spray for the control of downy mildew of pearl millet, only metalaxyl was effective. Metalaxyl 25 (Ridomil) and metalaxyl 35 (Apron) seed treatments protected the pearl millet plants from downy mildew up to 30 days. As a foliar spray, metalaxyl 25 used once at 20 days or twice after 20 and 38 days of plant growth gave less disease at harvest time. Seed treatment (metalaxyl 25 or 35) followed by one metalaxyl 25 spray was found to be effective in controlling the downy mildew. These treatments improved the growth of plants and yield significantly.     

Mapping of QTL for downy mildew resistance in maize

Quantitative trait loci (QTLs) of maize involved in mediating resistance to Peronosclerospora sorghi, the causative agent of sorghum downy mildew (SDM), were detected in a population of recombinant inbred lines (RILs) derived from the Zea mays L. cross between resistant (G62) and susceptible (G58) inbred lines. Field tests of 94 RILs were conducted over two growing seasons using artificial inoculation. Heritability of the disease reaction was high (around 70%). The mapping population of the RILs was also scored for restriction fragment length polymorphic (RFLP) markers. One hundred and six polymorphic RFLP markers were assigned to ten chromosomes covering 1648 cM. Three QTLs were detected that significantly affected resistance to SDM combined across seasons. Two of these mapped quite close together on chromosome 1, while the third one was on chromosome 9. The percentage of phenotypic variance explained by each QTL ranged from 12.4% to 23.8%. Collectively, the three QTLs identified in this study explained 53.6% of the phenotypic variation in susceptibility to the infection. The three resistant QTLs appeared to have additive effects. Increased susceptibility was contributed by the alleles of the susceptible parent. The detection of more than one QTL supports the hypothesis that several qualitative and quantitative genes control resistance to P. sorghi.     

Linkage between genes for resistance to downy mildew (Bremia lactucae) in lettuce

Data are presented for the joint segregation of the gene combinations: Dm-2/Dm-3, Dm-3/Dm-6, Dm-2/Dm-6 and Dm-6/Dm-8, following inoculation with five races of B. lactucae. It is postulated that Dm-2, 3 and 6 comprise a tight linkage group but that Dm-6 and Dm-8 are not linked as has been proposed previously. Information on the reaction of some resistant lettuce cultivars to certain B. lactucae races and some new postulated host genotypes is also presented.     

Bidirectional backcrosses between wild and cultivated lettuce identify loci involved in nonhost resistance to downy mildew

Key message

The nonhost resistance of wild lettuce to lettuce downy mildew seems explained by four components of a putative set of epistatic genes.

Abstract

The commonplace observation that plants are immune to most potential pathogens is known as nonhost resistance (NHR). The genetic basis of NHR is poorly understood. Inheritance studies of NHR require crosses of nonhost species with a host, but these crosses are usually unsuccessful. The plant-pathosystem of lettuce and downy mildew, Bremia lactucae, provides a rare opportunity to study the inheritance of NHR, because the nonhost wild lettuce species Lactuca saligna is sufficiently cross-compatible with the cultivated host Lactuca sativa. Our previous studies on NHR in one L. saligna accession led to the hypothesis that multi-locus epistatic interactions might explain NHR. Here, we studied NHR at the species level in nine accessions. Besides the commonly used approach of studying a target trait from a wild donor species in a cultivar genetic background, we also explored the opposite, complementary approach of cultivar introgression in a wild species background. This bidirectional approach encompassed (1) nonhost into host introgression: identification of L. saligna derived chromosome regions that were overrepresented in highly resistant BC1 plants (F1?×?L. sativa), (2) host into nonhost introgression: identification of L. sativa derived chromosome regions that were overrepresented in BC1 inbred lines (F1?×?L. saligna) with relatively high infection levels. We demonstrated that NHR is based on resistance factors from L. saligna and the genetic dose for NHR differs between accessions. NHR seemed explained by combinations of epistatic genes on three or four chromosome segments, of which one chromosome segment was validated by the host into nonhost approach.

     

Photosynthetic responses of lettuce to downy mildew infection and cytokinin treatment

Changes in primary metabolism of lettuce, Lactuca sativa L. (cv. Cobham Green), induced by compatible interaction with the biotrophic oomycete pathogen Bremia lactucae Regel (race BL 16), under two intensities of illumination in the presence and absence of exogenous cytokinins were studied by chlorophyll fluorescence imaging. Thirteen days post-inoculation leaf discs infected by B. lactucae exhibited impairments of photosynthesis associated with biotrophic infections, including: reductions in photosynthetic pigment contents and the maximum quantum yield of photosystem II photochemistry (F_V/F_M), inhibition of electron transport (Φ_PSII) and increased non-photochemical chlorophyll fluorescence quenching (NPQ). Detected changes in photosynthetic parameters correlated with the leaf area colonized by the pathogen’s intercellular hyphae. Applications of two cytokinins, benzylaminopurine and meta-topolin, previously shown to suppress B. lactucae sporulation if applied 24 h prior to inoculation at a concentration of 200 μM, retarded the pathogen’s asexual reproduction with no apparent negative effects on the host’s photosynthetic apparatus. However, long-lasting treatment of healthy tissues with this high concentration of exogenous cytokinin led to effects parallel to pathogenesis: reductions in photosynthetic pigment contents accompanied by inhibition of photosystem II photochemistry and electron transport. These effects of both prolonged exposure to cytokinins and the pathogenesis were weaker in discs exposed to the lower photosynthetic photon flux density. The role of cytokinins in plant-biotrophic pathogen interactions and their potential as disease control agents are discussed.     

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.     

Identification and validation of QTLs conferring resistance to sorghum downy mildew (Peronosclerospora sorghi) and Rajasthan downy mildew (P. heteropogoni) in maize

We have mapped the quantitative trait loci (QTLs) conferring resistance to sorghum downy mildew (Peronosclerospora sorghi; SDM) and Rajasthan downy mildew (P. heteropogoni; RDM), two species of DM prevalent throughout India. QTL mapping was carried out on a backcross population of 151 individuals derived from a cross between CM139 (susceptible parent) and NAI116 (highly resistant to both SDM and RDM). Heritability estimates were 0.74 for SDM and 0.67 for RDM. Composite interval mapping combined with a linkage map constructed with 80 simple sequence repeat (SSR) markers resulted in the identification of three QTLs (one each on chromosomes 2, 3 and 6) for SDM resistance and two QTLs (one each on chromosomes 3 and 6) for RDM resistance, all of which were contributed by NAI116. The significance of the major QTL on chromosome 6 (bin 6.05) that confers resistance to diverse DMs in tropical Asia, including SDM and RDM in India, was also verified. The results confirmed that some common QTLs contribute to both SDM and RDM resistance, while additional loci might specifically govern resistance to SDM. The QTL information generated in this study provide information that will aid in undertaking an integrated breeding strategy for the transfer of resistance to SDM and RDM in maize lines using marker-assisted selection.