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Sweet basil (Ocimum basilicum) is susceptible to downy mildew caused by the oomycete foliar pathogen Peronospora belbahrii. No resistant varieties of sweet basil are commercially available. Here, we report on the transfer of resistance gene Pb1 from the highly resistant tetraploid wild basil O. americanum var. americanum (PI 500945, 2n = 4x = 48) to the tetraploid susceptible O. basilicum ‘Sweet basil’ (2n = 4x = 48). F1 progeny plants derived from the interspecific hybridization PI 500945 × Sweet basil were resistant, indicating that the gene controlling resistance (Pb1) is dominant, but sterile due to the genetic distance between the parents. Despite their sterility, F1 plants were pollinated with the susceptible parent and 115 first backcross generation to the susceptible parent (BCs1) embryos were rescued in vitro. The emerging BCs1 plants segregated, upon inoculation, 5:1 resistant/susceptible, suggesting that resistance in F1 was controlled by a pair of dominant genes (Pb1A and Pb1A’). Thirty-one partially fertile BCs1 plants were self-pollinated to obtain BCs1-F2 or were backcrossed to Sweet basil to obtain the second backcross generation to the susceptible parent (BCs2). In total, 1 BCs1-F2 and 22 BCs2 progenies were obtained. The BCs1-F2 progeny segregated 35:1 resistant/susceptible, as expected from a tetraploid parent with two dominant resistant genes. The 22 BCs2 progenies segregated 1:1 resistant/susceptible (for a BCs1 parent that carried one dominant gene for resistance) or 5:1 (for a BCs1 parent that carried two dominant genes for resistance) at a ratio of 4:1. The data suggest that a pair of dominant genes (Pb1A and Pb1A’) residing on a two homeologous chromosomes is responsible for resistance of PI 500945 against P. belbahrii.

During the past two decades, a resurgence of cucurbit downy mildew has occurred around the world, resulting in severe disease epidemics. In the United States, resurgence of the disease occurred in 2004 and several hypotheses, including introduction of a new genetic recombinant or pathotype of the pathogen, have been suggested as potential causes for this resurgence. Occurrence and distribution of mating types of Pseudoperonospora cubensis in the United States were investigated using 40 isolates collected from cucurbits across 11 states from 2005 to 2013. Pairing of unknown isolates with known mating-type tester strains on detached leaves of cantaloupe or cucumber resulted in oospore formation 8 to 10 days after inoculation. Isolates differed in their ability to form oospores across all coinoculation pairings, with oospore numbers ranging from 280 to 1,000 oospores/cm2 of leaf tissue. Oospores were hyaline to golden-yellow, spherical, and approximately 36 μm in diameter. Of the 40 isolates tested, 24 were found to be of the A1 mating type, while 16 were of the A2 mating type. Mating type was significantly (P < 0.0001) associated with host type, whereby all isolates collected from cucumber were of the A1 mating type, while isolates from squash and watermelon were of the A2 mating type. Similarly, mating type was significantly (P = 0.0287) associated with geographical region, where isolates from northern-tier states of Michigan, New Jersey, New York, and Ohio were all A1, while isolates belonging to either A1 or A2 mating type were present in equal proportions in southern-tier states of Alabama, Florida, Georgia, North Carolina, South Carolina, and Texas. Viability assays showed that oospores were viable and, on average, approximately 40% of the oospores produced were viable as determined by the plasmolysis method. This study showed that A1 and A2 mating types of P. cubensis are present and the pathogen could potentially reproduce sexually in cucurbits within the United States. In addition, the production of viable oospores reported in this study suggests that oospores could have an important role in the biology of P. cubensis and could potentially influence the epidemiology of cucurbit downy mildew in the United States.

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.

The oomycete Plasmopara halstedii emerged at the onset of the 21st century as a destructive new pathogen causing downy mildew disease of ornamental Rudbeckia fulgida (rudbeckia) in the United States. The pathogen is also a significant global problem of sunflower (Helianthus annuus) and is widely regarded as the cause of downy mildew affecting 35 Asteraceae genera. To determine whether rudbeckia and sunflower downy mildew are caused by the same genotypes, population genetic and phylogenetic analyses were performed. A draft genome assembly of a P. halstedii isolate from sunflower was generated and used to design 15 polymorphic simple sequence repeat (SSR) markers. SSRs and two sequenced phylogenetic markers measured differentiation between 232 P. halstedii samples collected from 1883 to 2014. Samples clustered into two main groups, corresponding to host origin. Sunflower-derived samples separated into eight admixed subclusters, and rudbeckia-derived samples further separated into three subclusters. Pre-epidemic rudbeckia samples clustered separately from modern strains. Despite the observed genetic distinction based on host origin, P. halstedii from rudbeckia could infect sunflower, and exhibited the virulence phenotype of race 734. These data indicate that the newly emergent pathogen populations infecting commercial rudbeckia are a different species from sunflower-infecting strains, notwithstanding cross-infectivity, and genetically distinct from pre-epidemic populations infecting native rudbeckia hosts.

Cucurbit downy mildew (CDM), caused by the oomycete pathogen Pseudoperonospora cubensis, is a devastating disease that affects cucurbit species worldwide. This obligate, wind-dispersed pathogen does not overwinter in Michigan or other northern regions and new isolates can enter the state throughout the growing season. To evaluate the regional and temporal population structure of P. cubensis, sporangia from CDM lesions were collected from cucurbit foliage grown in Michigan and Ontario field locations in 2011. Population structure and genetic diversity were assessed in 257 isolates using nine simple sequence repeat markers. Genetic diversity was high for isolates from Michigan and Canada (0.6627 and 0.6131, respectively). Five genetic clusters were detected and changes in population structure varied by site and sampling date within a growing season. The Michigan and Canada populations were significantly differentiated, and a unique genetic cluster was detected in Michigan.

The deposition of callose is a common plant defense response to intruding pathogens and part of the plant’s innate immunity. In this study, eight grapevine callose synthase (CalS) genes were identified and characterized. To investigate biological function of CalS in grapevine against the infection of Plasmopara viticola, expression patterns of grapevine CalS family genes were analyzed among resistant/susceptible cultivars. After P. viticola infection, expression of CalS1, 3, 7, 8, 9, 10, and 11 were significantly modified among the grapevine cultivars. For example, the expression of CalS1 and CalS10 were greatly increased in downy mildew (DM)-immune Muscadinia rotundifolia ‘Carlos’ and ‘Noble’. Transient expression assay with promoters of the CalS1 and CalS10 genes confirmed that they were regulated by the oomycete pathogen P. viticola. CalS1 promoter activity was also significantly up-regulated by ABA in DM-immune M. rotundifolia ‘Noble’, but down-regulated in DM-susceptible Vitis vinifera ‘Chardonnay’. The CalS1 promoter, however, was also down-regulated by GA in ‘Chardonnay’, but not affected in ‘Noble’. The promoter activity of CalS10 was significantly up-regulated by GA in ‘Chardonnay’, but not regulated by ABA at all. It is proposed that CalS1 and CalS10 were involved in grapevine defense against DM disease.

Many cultivars of lettuce (Lactuca sativa L.) are susceptible to downy mildew, a nearly globally ubiquitous disease caused by Bremia lactucae. We previously determined that Batavia type cultivar ‘La Brillante’ has a high level of field resistance to the disease in California. Testing of a mapping population developed from a cross between ‘Salinas 88’ and La Brillante in multiple field and laboratory experiments revealed that at least five loci conferred resistance in La Brillante. The presence of a new dominant resistance gene (designated Dm50) that confers complete resistance to specific isolates was detected in laboratory tests of seedlings inoculated with multiple diverse isolates. Dm50 is located in the major resistance cluster on linkage group 2 that contains at least eight major, dominant Dm genes conferring resistance to downy mildew. However, this Dm gene is ineffective against the isolates of B. lactucae prevalent in the field in California and the Netherlands. A quantitative trait locus (QTL) located at the Dm50 chromosomal region (qDM2.2) was detected, though, when the amount of disease was evaluated a month before plants reached harvest maturity. Four additional QTL for resistance to B. lactucae were identified on linkage groups 4 (qDM4.1 and qDM4.2), 7 (qDM7.1), and 9 (qDM9.2). The largest effect was associated with qDM7.1 (up to 32.9% of the total phenotypic variance) that determined resistance in multiple field experiments. Markers identified in the present study will facilitate introduction of these resistance loci into commercial cultivars of lettuce.

Downy mildew, caused by the oomycete Peronospora belbahrii, is a devastating disease of sweet basil. In this study, 113 accessions of Ocimum species (83 Plant Introduction entries and 30 commercial entries) were tested for resistance against downy mildew at the seedling stage in growth chambers, and during three seasons, in the field. Most entries belonging to O. basilicum were highly susceptible whereas most entries belonging to O. americanum, O. kilimanadascharicum, O. gratissimum, O. campechianum, or O. tenuiflorum were highly resistant at both the seedling stage and the field. Twenty-seven highly resistant individual plants were each crossed with the susceptible sweet basil ‘Peri’, and the F1 progeny plants were examined for disease resistance. The F1 plants of two crosses were highly resistant, F1 plants of 24 crosses were moderately resistant, and F1 plants of one cross were susceptible, suggesting full, partial, or no dominance of the resistance gene(s), respectively. These data confirm the feasibility of producing downy mildew-resistant cultivars of sweet basil by crossing with wild Ocimum species.

Experiments to identify the factors affecting survival of Bremia lactucae sporangia after deposition on lettuce leaves were conducted in growth chambers and outdoors under ambient conditions. Lettuce seedlings at the four-leaf stage were inoculated with B. lactucae sporangia under dry conditions. Sporangia deposited on lettuce seedlings were incubated at different temperature and relative humidity (RH) combinations, exposed to 100, 50, 25, and 0␜unlight in the second experiment, and exposed to different artificial lights in wavelength ranges of UVA (315 to 400 nm), UVB (280 to 315 nm), or fluorescent light in the third experiment. After exposure for 0 to 48 h in the first experiment and 0 to 12 h in the second and third experiments, seedlings in two pots were sampled for each treatment, and sporangia were washed from 15 leaves excised from the sampled seedlings. Germination of sporangia was determined in water after incubation in the dark at 15°C for 24 h. The sampled seedlings with remaining leaves were first transferred to optimal conditions for infection (24 h), for the development of downy mildew, and then assessed for disease after 9 days. Sporangia survived much longer at 23°C (>12 h) than at 31°C (2 to 5 h), regardless of RH (33 to 76Ž Germination percentage was significantly reduced after exposure to 50 and 100␜unlight. UVB significantly reduced sporangium viability, while fluorescent light and UVA had no effect relative to incubation in the dark. Infection of seedlings followed a pattern similar to germination of sporangia. Solar radiation is the dominant factor determining survival of B. lactucae sporangia, while temperature and RH have small, insignificant effects in coastal areas of California. This suggests that infections by sporangia that survived a day are probable only on cloudy days or on leaves that are highly shaded. Additional keywords: lettuce downy mildew, ultraviolet radiation.

Enhancement of plant defense through the application of resistance inducers seems a promising alternative to chemical fungicides for controlling crop diseases but the efficacy can be affected by abiotic factors in the field. Plants respond to abiotic stresses with hormonal signals that may interfere with the mechanisms of induced systemic resistance (ISR) to pathogens. In this study, we exposed grapevines to heat, drought, or both to investigate the effects of abiotic stresses on grapevine resistance induced by Trichoderma harzianum T39 (T39) to downy mildew. Whereas the efficacy of T39-induced resistance was not affected by exposure to heat or drought, it was significantly reduced by combined abiotic stresses. Decrease of leaf water potential and upregulation of heat-stress markers confirmed that plants reacted to abiotic stresses. Basal expression of defense-related genes and their upregulation during T39-induced resistance were attenuated by abiotic stresses, in agreement with the reduced efficacy of T39. The evidence reported here suggests that exposure of crops to abiotic stress should be carefully considered to optimize the use of resistance inducers, especially in view of future global climate changes. Expression analysis of ISR marker genes could be helpful to identify when plants are responding to abiotic stresses, in order to optimize treatments with resistance inducers in field.

International audience; Plant diseases caused by fungi and oomycetes result in significant economic losses every year. Although phylogenetically distant, these organisms share many common features during infection. We identified genes in the oomycete Plasmopara viticola that are potentially involved in pathogenesis in grapevine by using fungal databases and degenerate primers. Fragments of P. viticola genes encoding NADH-ubiquinone oxidoreductase (PvNuo), laccase (PvLac), and invertase (PvInv) were obtained. PvNuo was overexpressed at 2 days postinoculation (dpi), during the development of the first hyphal structures and haustoria. PvLac was overexpressed at 5 dpi when genes related to pterostilbene biosynthesis were induced in grapevine. Transcript level for PvInv increased between 1 and 4 dpi before reaching a plateau. These results might suggest a finely tuned strategy of infection depending on nutrition and plant response. Phylogenetic analyses of PvNuo showed that P. viticola clustered with other oomycetes and was associated with brown algae and diatoms, forming a typical Straminipila clade. Based on the comparison of available sequences for laccases and invertases, the group formed by P. viticola and other oomycetes tended to be more closely related to Opisthokonta than to Straminipila. Convergent evolution or horizontal gene transfer could explain the presence of fungus-like genes in P. viticola.

Cucurbit downy mildew caused by Pseudoperonospora cubensis is economically the most important disease of cucurbits globally, and the pathogen is disseminated aerially over a large spatial scale. Spatio-temporal spread of the disease was characterized during phase I (low and sporadic disease outbreaks) and II (rapid increase in disease outbreaks) of the epidemic using records collected from sentinel plots from 2008 to 2009 in 23 states in the eastern United States as part of the United States Department of Agriculture Cucurbit Downy Mildew ipmPIPE network. A substantive goal of this study was to explain the pattern of time to disease outbreak using important covariates while accounting for spatially correlated differences in risk of disease outbreak among the states. Survival analyses that accounts for spatial dependence were performed on time to disease outbreak, and posterior median frailties (or random effects) were mapped to identify states with high or low risk for disease outbreak. From February to October, disease occurred in 195 and 172 out of 413 and 556 cases monitored in 2008 and 2009, respectively. Disease outbreaks were spatially aggregated, with a spatial dependence of up to ≈1,025 km where clustering of outbreaks in phase I and II of the epidemic were similar. However, unlike in phase I of the epidemic, space–time point pattern analysis was significant (P < 0.0001) for outbreaks in phase II, during which the highest risk window as estimated by the space–time function was within 1.5 months and 500 km of the initial outbreak. The risk of disease outbreak peaked around July and decreased thereafter until the end of the study period. Spatially correlated analysis of time to disease outbreak indicated the need to incorporate spatial frailties in standard survival analysis models. Evaluation of alternative formulations of the spatial models demonstrated that a Bayesian hierarchical spatially structured frailty model best described time to disease outbreak. This frailty model showed clustering of outbreaks at the state level and indicated that states in the mid-Atlantic region have high spatial frailties and a high risk of downy mildew outbreak.

Mitchell, M. N., Ocamb, C. M., Grunwald, N. J., Mancino, L. E., and Gent, D. H. 2011. Genetic and pathogenic relatedness of Pseudoperonospora cubensis and P. humuli . Phytopathology 101:805-818. The most economically important plant pathogens in the genus Pseudoperonospora (family Peronosporaceae) are Pseudoperonospora cubensis and P. humuli , causal agents of downy mildew on cucurbits and hop, respectively. Recently, P. humuli was reduced to a taxonomic synonym of P. cubensis based on internal transcribed spacer (ITS) sequence data and morphological characteristics. Nomenclature has many practical implications for pathogen identification and regulatory considerations; therefore, further clarification of the genetic and pathogenic relatedness of these organisms is needed. Phylogenetic analyses were conducted considering two nuclear and three mitochondrial loci for 21 isolates of P. cubensis and 14 isolates of P. humuli, and all published ITS sequences of the pathogens in GenBank. There was a consistent separation of the majority of the P. humuli isolates and the P. cubensis isolates in nuclear, mitochondrial, and ITS phylogenetic analyses, with the exception of isolates of P. humuli from Humulus japonicus from Korea. The P. cubensis isolates appeared to contain the P. humuli cluster, which may indicate that P. humuli descended from P. cubensis. Host-specificity experiments were conducted with two reportedly universally susceptible hosts of P. cubensis and two hop cultivars highly susceptible to P. humuli. P. cubensis consistently infected the hop cultivars at very low rates, and sporangiophores invariably emerged from necrotic or chlorotic hypersensitive-like lesions. Only a single sporangiophore of P. humuli was observed on a cucurbit plant during the course of the studies. Together, molecular data and host specificity indicate that there are biologically relevant characteristics that differentiate P. cubensis and P. humuli that may be obfuscated if P. humuli were reduced to a taxonomic synonym of P. cubensis . Thus, we recommend retaining the two species names P. cubensis and P. humuli until the species boundaries can be resolved unambiguously.

The individual and joint effects of covering the soil with polyethylene mulch before planting and fungicides commonly used by organic growers on tomato late blight (caused by Phytophthora infestans) were studied in three experiments conducted from 2002 to 2005. Application of fungicides resulted in inconsistent and insufficient late blight suppression (control efficacy ± standard error of 34.5 ± 14.3%) but the polyethylene mulch resulted in consistent, effective, and highly significant suppression (control efficacy of 83.6 ± 5.5%) of the disease. The combined effect of the two measures was additive. In a second set of three experiments carried out between 2004 and 2006, it was found that the type of polyethylene mulch used (bicolor aluminized, clear, or black) did not affect the efficacy of late blight suppression (control efficacy of 60.1 to 95.8%) and the differences in the effects among the different polyethylene mulches used were insignificant. Next, the ability of the mulch to suppress cucumber downy mildew (caused by Pseudoperonospora cubensis) was studied in four experiments carried out between 2006 and 2008. The mulch effectively suppressed cucumber downy mildew but the effect was less substantial (control efficacy of 34.9 ± 4.8%) than that achieved for tomato late blight. The disease-suppressing effect of mulch appeared to come from a reduction in leaf wetness duration, because mulching led to reductions in both the frequency of nights when dew formed and the number of dew hours per night when it formed. Mulching also reduced relative humidity in the canopy, which may have reduced sporulation.

The epidemiology of lettuce downy mildew has been investigated extensively in coastal California. However, the spatial patterns of the disease and the distance that Bremia lactucae spores can be transported have not been determined. During 1995 to 1998, we conducted several field- and valley-scale surveys to determine spatial patterns of this disease in the Salinas valley. Geostatistical analyses of the survey data at both scales showed that the influence range of downy mildew incidence at one location on incidence at other locations was between 80 and 3,000 m. A linear relationship was detected between semivariance and lag distance at the field scale, although no single statistical model could fit the semi-variograms at the valley scale. Spatial interpolation by the inverse distance weighting method with a power of 2 resulted in plausible estimates of incidence throughout the valley. Cluster analysis in geographic information systems on the interpolated disease incidence from different dates demonstrated that the Salinas valley could be divided into two areas, north and south of Salinas City, with high and low disease pressure, respectively. Seasonal and spatial trends along the valley suggested that the distinction between the downy mildew conducive and nonconducive areas might be determined by environmental factors.

A cold-tolerant strain of the mycoparasite Fusarium proliferatum was isolated following UV mutagenesis of the G6 strain, which is a biocontrol agent of grape downy mildew. The isolated strain (designated 1505) exhibited radial growth two to threefold that of the parent strain when grown at 13 degrees C, which is generally suboptimal for growth of Fusarium spp., but desirable for its host, Plasmopara viticola. This rapid growth was correlated with improved biological control of P. viticola, determined by a detached-leaf assay. Even though radial growth of strain 1505 at higher temperatures was slower than that of G6 and the strain failed to conidiate, there was no reduction in biocontrol efficacy. Significantly higher levels of extracellular beta-glucosidase and endo-1,4-beta-glucanase activity were measured in the culture filtrate of strain 1505 relative to that of strain G6. A DNA-mediated transformation procedure that included the introduction of antibiotic resistance and a GUS reporter gene system was adapted for F. proliferatum. Using the GUS-engineered strains, we demonstrated that both G6 and 1505 exhibit the characteristic coiling and penetration of host structures.

Downy mildew is a destructive disease of spinach worldwide. There have been 10 races described since 1824, six of which have been identified in the past 10 years. Race identification is based on qualitative disease reactions on a set of diverse host differentials which include open-pollinated cultivars, contemporary hybrid cultivars, and older hybrid cultivars that are no longer produced. The development of a set of near-isogenic open-pollinated spinach lines (NILs), having different resistance loci in a susceptible and otherwise common genetic background, would facilitate identification of races of the downy mildew pathogen, provide a tool to better understand the genetics of resistance, and expedite the development of molecular markers linked to these disease resistance loci. To achieve this objective, the spinach cv. Viroflay, susceptible to race 6 of Peronospora farinosa f. sp. spinaciae, was used as the recurrent susceptible parent in crosses with the hybrid spinach cv. Lion, resistant to race 6. Resistant F1 progeny were subsequently backcrossed to Viroflay four times with selection for race 6 resistance each time. Analysis of the segregation data showed that resistance was controlled by a single dominant gene, and the resistance locus was designated Pfs-1. By bulk segregant analysis, an amplified fragment length polymorphism (AFLP) marker (E-ACT/M-CTG) linked to Pfs-1 was identified and used to develop a co-dominant Sequence characterized amplified region (SCAR) marker. This SCAR marker, designated Dm-1, was closely linked (≈1.7 cM) to the Pfs-1 locus and could discriminate among spinach genotypes that were homozygous resistant (Pfs-1Pfs-1), heterozygous resistant (Pfs-1pfs-1), or homozygous susceptible (pfs-1pfs-1) to race 6 within the original mapping population. Evaluation of a wide range of commercial spinach lines outside of the mapping population indicated that Dm-1 could effectively identify Pfs-1 resistant genotypes; the Dm-1 marker correctly predicted the disease resistance phenotype in 120 out of 123 lines tested. In addition, the NIL containing the Pfs-1 locus (Pfs-1Pfs-1) was resistant to multiple races of the downy mildew pathogen indicating Pfs-1 locus may contain a cluster of resistance genes.

The effect of temperature on infection of lettuce by Bremia lactucae was investigated in controlled environment studies and in the field. In controlled conditions, lettuce seedlings inoculated with B. lactucae were incubated at 15, 20, 25, or 30°C during a 4-h wet period immediately after inoculation or at the same temperatures during an 8-h dry period after the 4-h postinoculation wet period at 15°C. High temperatures during wet and dry periods reduced subsequent disease incidence. Historical data from field studies in 1991 and 1992, in which days with or without infection had been identified, were analyzed by comparing average air temperatures during 0600 to 1000 and 1000 to 1400 Pacific standard time (PST) between the two groups of days. Days without infection had significantly higher temperatures (mean 21.4°C) than days with infection (20.3°C) during 1000 to 1400 PST (P < 0.01) but not during 0600 to 1000 PST. Therefore, temperature thresholds of 20 and 22°C for the 3-h wet period after sunrise and the subsequent 4-h postpenetration period, respectively, were added to a previously developed disease warning system that predicts infection when morning leaf wetness lasts ≥4 h from 0600 PST. No infection was assumed to occur if average temperature during these periods exceeded the thresholds. Based on nonlinear regression and receiver operating characteristic curve analysis, the leaf wetness threshold of the previous warning system was also modified to ≥3-h leaf wetness (≥0900 PST). Furthermore, by comparing solar radiation on days with infection and without infection, we determined that high solar radiation during 0500 to 0600 PST in conjunction with leaf wetness ending between 0900 and 1000 PST was associated with downy mildew infection. Therefore, instead of starting at 0600 PST, the calculation of the 3-h morning leaf wetness period was modified to start after sunrise, defined as the hour when measured solar radiation exceeded 8 W m-2 (or 41 μmol m-2 s-1 for photon flux density). The modified warning system was compared with the previously developed system using historical weather and downy mildew data collected in coastal California. The modified system was more conservative when disease potential was high and recommended fewer fungicide applications when conditions were not conducive to downy mildew development.

The asexual phase of the life cycle of Plasmopara halstedii, the causal agent of downy mildew of sunflower, plays a key role in the propagation of the disease. We investigated the morphological and ultrastructural changes that occur during the asexual development of the pathogen. Direct examination of infected cotyledons confirmed the presence of sporangiophores. In contact with water, important ultrastructural changes occurred, affecting the surface of zoosporangia, which became smoother, and their cytoplasm, which differentiated into flagellate zoospores. The subsequent encystment of zoospores was characterized by the synthesis of a cell wall and the loss of the flagella. In addition, two monoclonal antibodies (MAbs) specific for P. halstedii were used to analyze the immunochemical changes associated with these modifications. MAb 16A6, which bound to a 48-kDa glycoprotein, mainly labeled the surface of mobile or encysted zoospores and of mother cells of germ tubes. Conversely, MAb 2F9, which recognized highly glycosylated antigens, labeled the surface of zoosporangia and of flagellate zoospores, but not the encysted zoospores. These results provide new insights into the morphological and ultrastructural changes associated with the release and the encystment of zoospores which may be interesting targets for the development of new antimicrobial products.

International audience; Following inoculation of host and nonhost plants with Plasmopara viticola, the grapevine downy mildew, a histological survey was undertaken to identify the stage where its development is contained in nonhosts and in resistant host plants. Three herbaceous nonhost species, Beta vulgaris, Lactuca sativa, and Capsicum annuum, and three grapevine species displaying different level of resistance (Vitis vinifera [susceptible], Vitis riparia [partially resistant] and Muscadinia rotundifolia [totally resistant]) where inoculated by P. viticola using a controlled leaf disk inoculation bioassay. During the early steps of infection, defined as encystment of zoospores on stomata, penetration of the germ tube, and production of the vesicle with the primary hypha, there was no evidence of a clear-cut preference to grapevine tissues that could attest to host specificity. The main difference between host grapevine species and nonhosts was observed during the haustorium formation stage. In nonhost tissues, the infection was stopped by cell wall-associated defense responses before any mature haustorium could appear. Defense responses in resistant grapevines were triggered when haustoria were fully visible and corresponded to hypersensitive responses. These observations illustrate that, for P. viticola, haustorium formation is not only a key stage for the establishment of biotrophy but also for the host specificity and the recognition by grapevine resistance factors.

Peronospora tabacina is an obligate plant pathogen that causes downy mildew disease on several species of Nicotiana, including N. tabacum (tobacco). The primary objective of this study was to use gnotobiotic associations to describe interactions between the pathogen and roots of either N. tabacum (cv. KY14) or N. repanda. We found that the pathogen was capable of moving systemically from shoots to roots of both host species and emerged from the root tissues as hyphae. We also demonstrated that root-associated hyphae were infectious on roots of nearby plants and resulted in new systemic infections. Following overnight darkness, sporulation of the pathogen was observed on infected roots exposed to air on both host species. We also found that within 2 months in culture, structures resembling resting stages of Peronospora tabacina were produced on hyphae emerging from roots of N. repanda but not N. tabacum. These findings appear relevant to both the epidemiology of the disease and to future studies of this and other downy mildew pathosystems.

The oily paste extracts of Inula viscosa leaves made with organic solvents were effective in controlling downy mildew caused by Plasmopara viticola in detached leaf tissues of grapes in growth chambers. Thin-layer chromatography of such extracts revealed the presence of 11 Rf regions of which four contained highly effective compounds against the disease. Two major inhibitory compounds, each comprising 10.6% of the total paste weight, were identified as tomentosin and costic acid. An emulsified concentrate formulation of the oily paste extracts provided excellent activity against the disease in the field. The effective dose (concentration) required for 90% control of the disease in treated shoots in the field was below 0.125% (paste in water). No seasonal fluctuations were observed in the control efficacy of six extracts made from I. viscosa leaves harvested during the period of May to July. In whole vines, treated and artificially inoculated, the paste concentration required for 90% control of the disease ranged between 0.30 to 0.37%, whereas in naturally infected vines it was 0.58%. It appears that I. viscosa is a valuable source for fungicidal preparations against downy mildew of grapes. This is the first report on the control of a disease with I. viscosa extracts under field conditions.

Several aspects of grapevine downy mildew epidemiology that are fundamental to model predictions were investigated. Simple rainfall-, temperature-, and phenology-based thresholds (rain > 2.5 mm; temperature > 11°C; and phenology > Eichorn and Lorenz [E&L] growth stage 12) were evaluated to forecast primary (oosporic) infection by Plasmopara viticola. The threshold was consistent across 15 years of historical data on the highly susceptible cv. Chancellor at one site, and successfully predicted the initial outbreak of downy mildew for 2 of 3 years at three additional sites. Field inoculations demonstrated that shoot tissue was susceptible to infection as early as E&L stage 5, suggesting that initial germination of oospores, rather than acquisition of host susceptibility, was probably the limiting factor in the initiation of disease outbreaks. We also found that oospores may continue to germinate and cause infections throughout the growing season, in contrast to the widely-held assumption that the supply of oospores is depleted shortly after bloom. Lesion productivity (sporangia/lesion) did not decline with age of a lesion in the absence of suitable weather to induce sporulation. However, the productivity of all lesions declined rapidly through repeated cycles of sporulation. Extremely high temperatures (i.e., one day reaching 42.8°C) had an eradicative effect under vineyard conditions, and permanently reduced sporulation from existing (but not incubating) lesions to trace levels, despite a later return to weather conducive to sporulation. In fair weather, most sporangia died sometime during the daylight period immediately following their production. However, over 50% of sporangia still released zoospores after 12 to 24 h of exposure to overcast conditions.

Ishii, H., Fraaije, B. A., Sugiyama, T., Noguchi, K., Nishimura, K., Takeda, T., Amano, T., and Hollomon, D. W. 2001. Occurrence and molecular characterization of strobilurin resistance in cucumber powdery mildew and downy mildew. Phytopathology 91:1166-1171. Between 1998 and 1999, control failure of powdery mildew (Podosphaera fusca) and downy mildew (Pseudoperonospora cubensis) by the strobilurin fungicides azoxystrobin and kresoxim-methyl was observed in cucumber-growing areas of Japan. Results from inoculation tests carried out on intact cucumber plants and leaf disks clearly showed the distribution of pathogen isolates highly resistant to azoxystrobin and kresoximmethyl. Fragments of the fungicide-targeted mitochondrial cytochrome b gene were polymerase chain reaction amplified from total pathogen DNA and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochrome b gene, resulting in substitution of glycine by alanine at position 143, was found in resistant isolates of downy mildew. This substitution in cytochrome b seemed to result in high resistance to strobilurins in this pathogen. The same mutation was found in some but not all resistant isolates of powdery mildew. This study suggests that a mutation at position 143 in the target-encoding gene, resulting in an amino acid substitution, was probably a major cause of the rapid development of high strobilurin resistance in these two pathogens.

Severe downy mildew diseases of opium poppy (Papaver somniferum) can be caused by Peronospora arborescens and P. cristata, but differentiating between the two pathogens is difficult because they share morphological features and a similar host range. In Spain, where severe epidemics of downy mildew of opium poppy have occurred recently, the pathogen was identified as P. arborescens on the basis of morphological traits. In this current study, sequence homology and phylogenetic analyses of the internal transcribed spacer regions (ITS) of the ribosomal DNA (rDNA) were carried out with DNA from P. arborescens and P. cristata from diverse geographic origins, which suggested that only P. arborescens occurs in cultivated Papaver somniferum in Spain. Moreover, analyses of the rDNA ITS region from 27 samples of downy-mildew-affected tissues from all opium-poppy-growing regions in Spain showed that genetic diversity exists within P. arborescens populations in Spain and that these are phylogenetically distinct from P. cristata. P. cristata instead shares a more recent, common ancestor with a range of Peronospora species that includes those found on host plants that are not members of the Papaveraceae. Species-specific primers and a PCR assay protocol were developed that differentiated P. arborescens and P. cristata and proved useful for the detection of P. arborescens in symptomatic and asymptomatic opium poppy plant parts. Use of these primers demonstrated that P. arborescens can be transmitted in seeds and that commercial seed stocks collected from crops with high incidence of the disease were frequently infected. Field experiments conducted in microplots free from P. arborescens using seed stocks harvested from infected capsules further demonstrated that transmission from seedborne P. arborescens to opium poppy plants can occur. Therefore, the specific-PCR detection protocol developed in this study can be of use for epidemiological studies and diagnosing the pathogen in commercial seed stocks; thus facilitating the sanitary control of the disease and avoidance of the pathogen distribution in seeds.

Unger, S., Buche, C., Boso, S. and Kassemeyer, H.-H. 2007. The course of the colonization of two different Vitis genotypes by Plasmopara viticola indicates compatible and incompatible host–pathogen interactions. Phytopathology 97:780-786. The course of colonization of leaf mesophyll by the causal agent of grapevine downy mildew, Plasmopara viticola, in a susceptible and a resistant grapevine genotype was examined in order to characterize the development of the pathogen in compatible and incompatible host–pathogen interactions. Within a few hours after inoculation, the pathogen was established in the susceptible Vitis vinifera cv. Muller-Thurgau and formed primary hyphae with a first haustorium. No further development occurred in the following 10 to 18 h. The next step, in which the hyphae grew and branched to colonize the intercellular space of the host tissue, was observed 1.5 days after inoculation. After 3 days, the intercostal fields were entirely filled with mycelium and sporulation was abundant under favorable environmental conditions. The first infection steps were essentially the same in the resistant V. rupestris. However, the invasive growth of P. viticola was delayed, and further development ceased before the intercostal fields were filled with mycelium. Additional keyword: resistance.

Postinfection applications of microconidial suspensions of Fusarium proliferatum G6 reduced sporangial production of Plasmopara viticola on grape leaf disks by 97% and prevented resporulation. Microscopic examination of the hyphal interaction in vitro showed hyphae of F. proliferatum G6 coiled around and inside sporangiophores of P. viticola. In the field from 1992 to 1995, where substantial natural grape downy mildew developed, weekly applications of F. proliferatum G6 microconidia reduced disease development on leaves and fruit clusters of Vitis interspecific hybrid cultivars Chancellor and Lakemont. Severity of downy mildew on Chancellor clusters was reduced by 77% in 1992, 80% in 1993, and 53% in 1994 and was reduced on leaves by 71% in 1992. On Lakemont, disease severity on clusters was reduced by 99% in 1993, 94% in 1994, and 81% in 1995 and was reduced on leaves by 79% in 1992, 67% in 1994, and 60% in 1995. Fumonisin mycotoxins, which are produced by F. proliferatum, including F. proliferatum G6, were not found in grape berries or juice made from berries sprayed with microconidia in the field. Agar tests with fungicides showed sulfur, copper, and metalaxyl to be minimally detrimental to F. proliferatum G6. Biological control with F. proliferatum might be practical in conjunction with a management program in which sulfur and copper are used or in an antiresistance management program with metalaxyl, on moderately resistant cultivars, or in areas where downy mildew is not severe.

In a previous field study in California, infection of lettuce by Bremia lactucae, the downy mildew pathogen, occurred mainly on days on which leaves dried late in the morning. This observation led to the hypothesis that spore release and infection take place concurrently during mornings with prolonged leaf wetness. To test this hypothesis, spore dispersal experiments were carried out during 13 nights and mornings for which prolonged leaf wetness was expected. At sunset, diseased spore-source plants (infected with a metalaxyl-insensitive isolate of B. latucae) and healthy spore-trap plants (grown in metalaxyl-amended nutrient solution) were placed next to each other outdoors. Lesions on the source plants sporulated at night, and spore release (measured with a volumetric spore sampler) began at sunrise. After leaf wetness had ended later in the day, the trap plants were moved to a growth chamber and incubated in conditions favorable for colonization by B. lactucae, but not for infection. They were inspected for disease 10 to 14 days after exposure. Trap plants developed signs and symptoms of downy mildew after experiments from five nights, all of which had leaf wetness persisting for at least 3 h after sunrise. Infection on these plants must have taken place during the morning of the exposure period, concurrently with spore release from source plants. Since a metalaxyl-insensitive isolate of B. lactucae was used in all experiments and trap plants were grown in metalaxyl-amended nutrient solution, the possibility of infection by inoculum occurring naturally at the experimental site could be excluded

Dimethomorph (DMM) was effective in controlling late blight in potato and tomato caused by either metalaxyl-sensitive (MS) or metalaxyl-resistant (MR) field isolates of Phytophthora infestans and downy mildew in cucumbers and melons caused by MS or MR isolates of Pseudoperonospora cubensis. The fungicide did not affect zoospore discharge from sporangia of P. infestans but strongly inhibited zoospore encystment, cystospore germination, and mycelial growth in vitro. DMM showed translaminar activity and local systemic activity in intact plants but failed to translocate from one leaf to another in either acropetal or basipetal direction. DMM applied as a soil drench strongly protected tomatoes against late blight but failed to protect cucumbers against downy mildew. When applied in lanolin paste to the stem surface, but not when applied to the hypocotyl, it provided excellent control of both diseases in the foliage. DMM applied 1 day postinoculation partially protected potato against late blight and cucumber against downy mildew, and in both systems reduced sporulation of the pathogen. DMM diminished the sporulation of P. infestans and P. cubensis when applied to normally developed infected tissue. In P cubensis it induced enhanced callose-encasement of haustoria. It showed a remarkable persistence on foliage subsequent to excessive washing with water as well as a high residual activity lasting for 9 days. DMM seems to be a good candidate for the control of oomycete diseases in the field especially in growing areas where phenylamide-resistant fungal populations prevail.

Relationships between weather and downy mildew epidemics of hop in the Yakima Valley of Washington State over a 38-yr period were examined with linear discriminant and logistic regression analyses. A linear discriminant function with minimum temperature in April X the total number of rainfall days in April and May, an indicator variable for the presence of an epidemic the preceding year, and the minimum temperature in May X the number of occurrences of three or more consecutive rainfall days in May correctly classified the epidemic status for 92% of the years. The percentage of years with mild epidemics correctly classified (specificity) and years with severe epidemics correctly classified (sensitivity) for a 10-yr test data set were 86 and 100%, respectively [...]

The short-lived effectiveness of major gene resistance in lettuce to downy mildew (Bremia lactucae) means that plant breeders are in constant need of new resistance genes. In this study, the genetics of resistance to B. lactucae in nine accessions of Lactuca sativa or L. serriola was analyzed using segregating F 2 populations. This analysis was aimed at establishing the number of resistance factors in these accessions, their phenotypic effects, and the mapping of these factors in linkage groups. The cultivar Mariska (R18) and the line CS-RL both carry a widely effective resistance factor, which is located in linkage group 1. In seven other accessions, eight novel resistance factors, dissimilar to recognized major genes, were detected and designated R23 to R30 [...]

Spatial and temporal data on disease incidence in an experimental field of red cabbage in which a downy mildew epidemic developed from a point source was analyzed by a geostatistical model. A two-step prediction procedure was defined to formulate a linear predictor for space and time variables based on a spatial semivariogram that changes in time. The model accommodated any pattern of spatial variables, including patterns of healthy and diseased plants. The spatial transposition of the epidemic focus was determined with high precision. Time of initiation of the epidemic was determined with lower precision due to extrapolation in time. The results were compared with data from three other experimental plots: one red cabbage plot from the previous year and two from the same year

A 232-bp DNA sequence was obtained from random amplified polymorphic DNA of Peronospora tabacina, the blue mold pathogen of tobacco. This sequence had homology to P. tabacina DNA but not to DNA of other downy mildew or oomycetous fungi tested. The fragment was determined to be part of a repetitive DNA sequence that was ubiquitous in a worldwide collection of P. tabacina. Oligonucleotides were designed for amplification of this sequence by polymerase chain reaction. The fragment was reliably amplified with amounts of DNA (1-10 fg) that are less than that contained in a single P. tabacina sporangiospore. Use of this technique enabled the detection of P. tabacina DNA directly in local lesions, systemic vascular infections, and other infected parts of tobacco plants [...]

Weather conditions and downy mildew (Bremia lactucae) development were monitored in 13 commercial lettuce fields in the central coast production areas of California during 1991 and 1992. Days on which infection likely had occurred were identified based on observations of the appearance of new lesions combined with quantitative information about the length of the latent period of the disease. Univariate (Kolmogorov-Smirnov tests) and multivariate (stepwise discriminant analysis) procedures were applied to statistically differentiate infection days from days on which infection had not occurred and to characterize infection days in terms RACT of weather [...]

Resistance of spinach to white rust (Albugo occidentalis) and races 3 and 4 of downy mildew (Peronospora farinosa f. sp. spinaciae) was quantified on several cultivars and breeding lines in separate field inoculation experiments. Several cultivars and breeding lines from the Arkansas breeding program had undergone repeated field selections for white rust resistance (Fall Green, Ozarka II, FA88-310, FA88-354, and FA86-70) while others had not (Grandstand, St. Helens, and Hybrid 424). Resistance to both diseases was quantified by measuring disease incidence and severity at regular intervals 7-32 days after inoculation. The latent periods and the area under the disease progress curve (AUDPC) also were compared [...]

Plasmopara lactucae-radicis recently has been described as the causal agent of downy mildew on roots of cultivated lettuce. This fungus is the only known causal agent of downy mildew that is restricted to the roots of its host. Thirty-seven cultivars of Lactuca sativa were screened for resistance to P. lactucae-radicis. Two-week-old lettuce plants grown under hydroponic conditions were challenged with this fungus and evaluated 2 and 3 wk after inoculation for resistance. Root necrosis and production of sporangia on roots was considered a susceptible reaction (...)

The effects of 10 different foliar diseases on the photosynthetic and transpirational activities of five annual and three perennial crops were examined under field conditions. The following pathosystems were investigated: leaf rust, powdery mildew, and Septoria tritici blotch of wheat; rust of corn; downy mildew of cucumber; Alternaria leaf spot of cotton; powdery mildew of pepper; Pestalotia of mango; powdery mildew of peach; and powdery mildew of grapes. Visual estimates of infection do not always indicate adequately the effect of a pathogen on these physiological processes (...)

The causal organism of an incidence of maize downy mildew in Southern China proved difficult to classify by standard techniques. The pathogen, subsequently identified as Peronosclerospora sacchari, was detected by DNA hybridization in endosperm, pericarp, and pedicel tissues, but not in embryos of infected maize seeds. Plasmid pCLY83, which had been selected from a P. maydis DNA library, served as the probe (...)

Ninety-seven California isolates of Bremia lactucae were characterized for virulence phenotype, metalaxyl insensitivity, and sexual compability type from 1987 to 1989. Seventy-eight lettuce cultivars and 85 lettuce accessions were screened for resistance to lettuce downy mildew caused by the three major California pathotypes of B. lactucae. Of the 97 B. lactucae isolates, two were California pathotype IA, four were pathotype II, 30 were pathotype III, and 61 seemed to be pathotype IV. Several pathotype IV isolates were separated easily into derivatives having virulence phenotypes identical to either pathotype II or pathotype III (...)

Hop plants in hills in three or four hop yards were assessed visually during severe and mild epidemics of downy mildew in the Yakima Valley of Washington. Several methods of analysis were used to investigate the spatial pattern of hop plants systemically infected with Pseudoperonospora humuli during the two types of epidemics. When disease incidences were low during the mild epidemics and early in the development of the severe epidemics, distributions associated with aggregation of disease provided a good representation of the frequency distribution of systemically infected shoots (...)

Maize stripe virus (MStpV) was identified in maize leaves by either diagnostic symptoms or needle-shaped crystals. The symptoms consisted of chlorotic patterns of overlapping circles with distinct margins when magnified 3-10×. The crystals were found in abundance by phase-contrast light microscopy in sap from symptomatic leaf areas. The crystals were similar to crystals of MStpV noncapsid protein in immunofluorescence microscopy. Diagnostic symptoms and the crystals could be found readily in naturally and experimentally MStpV-infected maize plants with a wide range of gross symptoms types and throughout disease development (...)

Exposure of field plants to sporangial inoculum provided by earlier planted "infector rows" resulted in considerably more downy mildew on all Pennisetum americanum cvs. tested than was obtained by exposure of the cvs. to oospores of Sclerospora graminicola. In large, isolated field plots of susceptible hybrids, downy mildew incidence and severity were considerably greater in plots with a central source of sporangia (potted plants with sporulating infected leaves) present from crop emergence than in plots with no such initial sporangial source. Sporangia remained infective for up to 340 m downwind from a sporangial source during the rainy season, but during the dry, post-rainy season no infection occurred at >80 m from a sporangial source. The significance of these results in epidemiology and their application for large scale field screening for resistance are discussed