Downy Mildew Symptoms Research Articles

Molecular Detection of Peronospora variabilis in Quinoa Seed and Phylogeny of the Quinoa Downy Mildew Pathogen in South America and the United States

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

First Report of Downy Mildew Caused by Peronospora belbahrii on Sweet Basil (Ocimum basilicum) in Cyprus.

Sweet basil (Ocimum basilicum L.) is an economically important annual aromatic plant, grown mostly for culinary use for both fresh and dry consumption and as a source of essential oil. In Cyprus, approximately 4 ha are grown annually, either in greenhouses as a year-round crop or in open fields from April to November, and the majority of the production is exported to the European market. During May 2012, a sweet basil cv. Genovese Gigante greenhouse operation in the area of Limassol was severely affected by a foliar disease, causing almost 100% crop losses. Within a few days, a similar, heavy disease incidence was also reported from a nearby greenhouse facility on the Genovese-type cultivars Superbo, Aroma 2, and Bonazza, as well as on Thai basil (O. basilicum var. thyrsiflorum). Successively, destructive hits of similar symptomatology have been reported from other areas and since then the disease appears to have been well-established in the country, causing major economic damages. It is also noteworthy to mention that in greenhouse infections the disease remains active even during winter, considering the mild environmental conditions and the monoculture fashion followed. Symptoms appeared on the leaves initially as interveinal, zonal, chlorotic lesions, followed by the appearance of a fuzzy, purplish sporulation on the abaxial side. Progressively, infected leaves curled and sporadic necrotic spots were evident and finally abscised. Light microscopic examination of infected samples revealed the presence of straight, hyaline sporangiophores (n = 15) typical of downy mildew, 210 to 590 μm long (mean = 350.7 μm; SD ± 117.5 μm) × 12 to 15 μm wide (mean = 13.1 μm; SD ± 1.4 μm). Sporangiophores were monopodially branched three to five times, terminating with curved branchlets bearing single sporangia at their tips. The sporangia (n = 25) were purplish-grey, ovoid to subglobose, and measured 32 to 22 μm in length (mean = 27.2 μm; SD ± 2.8 μm) and 30 to 10 μm in breadth (mean = 21.7 μm; SD ± 4.8 μm). Based on these morphological characteristics, the causal agent was identified as Peronospora belbahrii Thines (1,4). Furthermore, genomic DNA was extracted from infected plant tissue from eight different samples according to Dellaporta et al. (2). The complete ITS rDNA region was amplified and sequenced using primers ITS5 and ITS4 (3). Two of the consensus sequences were deposited in GenBank (Accession Nos. KF419289 and KF419290) and a BLAST analysis in the NCBI database revealed 99% similarity to all of the P. belbahrii sequences and other Peronospora sp. previously reported on sweet basil (Accession Nos. AY831719, DQ479408, FJ394336, and FJ436024). In a pathogenicity trial, five 40-day-old potted sweet basil plants were spray-inoculated with a sporangial suspension (1 × 105 sporangia/ml) until runoff, bagged for 24 h, and placed in a growth chamber at 18°C. Subsequently, the plastic bags were removed and the plants were kept at 22°C with a 16-h photoperiod and 80% relative humidity. Additionally, five plants were water-sprayed and served as controls. Typical downy mildew symptoms appeared 6 to 8 days after inoculation, while the uninoculated plants remained disease-free. To our knowledge, this is first report of downy mildew on sweet basil in Cyprus.

First Report of Downy Mildew Caused by Plasmopara obducens on Impatiens in Taiwan.

In February 2013, single and double flowered impatiens (Impatiens walleriana Hook. f.) affected by downy mildew were observed in nurseries (cv. Accent) and in the wild in central Taiwan. More than 90% of the plants were infected in areas where the disease broke out. Symptomatic leaves showed yellowing, with white, fungal-like structure covering the lower leaf surfaces, causing the plants to become wilted and defoliated. Under microscopic observation, hyaline, thin-walled sporangiophores branched monopodially and had slightly swollen bases. Three apical branchlets were at right angles to the main axis, measuring 4.3-15.0 μm (average 8.5 μm). Sporangia were hyaline, ovoid, with an average length and width of 14.2 (10.0 to 18.0) × 12.1 (9.3 to 15.0) μm. For molecular categorization, PCR amplification of the 5' end of the large ribosomal subunit gene was performed with primers NL1 and NL4 (2). The amplicons were cloned, sequenced, and deposited in GenBank (Accession Nos. KC905620 and KC905621). The sequence similarities were 99% compared with that of Plasmopara obducens (J. Schröt.) J. Schröt from Florida (JX217746) (3). Based on morphological and molecular characters, the pathogen was identified as P. obducens. Three voucher specimens (TNM Nos. F0026644, F0026645, and F0026646) were deposited in the herbarium of the National Museum of Natural Science, Taichung, Taiwan. Pathogenicity was confirmed by inoculation of five young, potted impatiens plants with a suspension containing 1 × 105 sporangia/ml in 0.05% Tween 20 (approximately 8 ml/plant). An additional five plants sprayed with 0.05% Tween 20 served as negative controls. The plants were maintained in an outdoor ambient environment. After 2 weeks incubation at an average temperature of 20°C and approximately 80% relative humidity, the inoculated plants exhibited typical downy mildew symptoms, while the control plants remained healthy. The pathogenicity test was repeated in a dew chamber under 20°C with similar results. In the Asia-Pacific region, impatiens downy mildew was recently confirmed in Korea and Japan (1,4). To our knowledge, this is the first report of downy mildew on impatiens in Taiwan. Our further surveys indicated the disease has spread to other parts of the island and will become a potential problem requiring prevention.

First Report of Downy Mildew Caused by Plasmopara halstedii on Gerbera jamesonii in Brazil.

African daisy (Gerbera jamesonii Bolus ex Hook. f.) is an important species for both the cut flower and potted plant industries worldwide (4). Since the winter of 2009, plants showing severe downy mildew symptoms have been observed in a greenhouse located in an experimental area of the Universidade Federal de Viçosa (state of Minas Gerais, Brazil). The disease appeared as ill-delimited adaxial chlorosis of lamina; tissues became yellow and then brown with age with intense blighting of leaves of entire plants, leading to their death, when untreated. Dense, whitish sporulation was observed on the lower surfaces since early stages. A representative sample was dried in a plant press and deposited in the local herbarium under accession number VIC 32070. Slides were prepared with fungal structures mounted in lactofuchsin and observed under a light microscope (Olympus BX 51). Fungus morphology: Sporangiophores hypophyllous, emerging through stomata, cylindrical, up to 650 μm long and 5 to 10 μm wide, with slightly swollen base from 6.5 to 13 μm, hyaline, aseptate, straight, with up to 6 monopodial ramifications occurring mainly at right angles, the final branch ending in 3 or 4 ultimate branchlets; sporangia globose to ovoid, from 20 to 28 μm long and 13 to 18 μm wide, hyaline, smooth. Oospores were not observed. In order to further clarify the identity of the fungus on G. jamesonii, genomic DNA was extracted directly from the plant tissue and part of cytochrome c oxidase subunit 2 was amplified with the primers COX2 (3). The generated sequence was submitted to GenBank (Accession No. KC690148) and when compared with other entries revealed a high sequence similarity (99%) with Plasmopara halstedii (Farl.) Berl. & De Toni (EU743813) from Helianthus annuus L. This was also supported by the morphological data as compared with published descriptions (2) and it was then concluded that the chromistan fungus involved in downy mildew of African daisy was P. halstedii. Two different downy mildew genera, Bremia and Plasmopara, cause downy mildew disease on G. jamesoni. Bremia lactucae has been recorded in Argentina, Brazil, Germany, and Poland (4). There is only one record of a Plasmopara on this host in the United States (1), but this is an obscure report with no identification at the species level. Although P. halstedii has been commonly recorded on numerous hosts belonging to the Asteraceae worldwide, it has never been reported on G. jamesoni. To our knowledge, this is the first report of P. halstedii on G. jamesoni in Brazil. This disease has the potential to become important and cause significant losses because of a combination of the high severity to untreated plants and the increasing importance of African daisy in the flower market in Brazil.

First Report of Downy Mildew Caused by Peronospora belbahrii on Basil (Ocimum spp.) in Ontario.

Basil (Ocimum spp.) is one of the most commercially significant fresh culinary herb crops worldwide. In Ontario, basil is grown both in the field and in the greenhouse. In the summer of 2011, basil plants grown in a research field at the Simcoe Research Station in Norfolk County, Ontario, Canada (44°15'N, 77°35'W), were infected with downy mildew. Infected leaves exhibited interveinal chlorotic lesions on the upper surface and clear to black sporulation on the abaxial leaf surfaces. Leaf senescence and defoliation occurred at high disease severity, which reduced marketable yield. Basil downy mildew symptoms were severe on leaves of cultivars Genovese and Sweet Basil, with 40 to 100% disease incidence. Based on morphological characteristics, the basil downy mildew causal agent was identified as Peronospora belbahrii Thines (4). Infected leaves were collected and microscopic observations of the sporulating lesions were carried out and the structures measured. Sporangiophores (n = 20) were hyaline with relatively long, straight trunks and were monopodially branched, with a length of 150 to 360 μm (average 285 μm). Sporangiophores ended with two slightly curved branchlets, the longer one measuring 15 to 27 μm (average 19 μm) and the shorter one 5 to 15 μm (average 9 μm). Sporangia (n = 50) were round, or slightly ovoid, olive to brown in color, and measured 29 × 25 μm (25 to 35 × 20 to 30 μm). Genomic DNA was extracted from 10 isolates and the nuclear ribosomal internal transcribed spacer (ITS) region was amplified with ITS4 and ITS5 primers and sequenced. The sequences of the 10 isolates were nearly identical. A BLAST search of the NCBI database with the ITS sequences (GenBank Accession No. KC756923) revealed a 98 to 100% similarity to the sequences of P. belbahrii (HQ730979, FJ436024, and HQ702191) isolated from sweet basil in Florida (3), California (1), and Hungary (2), respectively. To confirm pathogenicity, 5-week-old 'Genovese' seedlings were sprayed with a suspension of 1 × 105 sporangia/ml. Plants were kept in a growth chamber maintained at 23/18°C, 60 to 85% relative humidity, and 12/12 h light/dark. Non-inoculated plants served as controls. Basil downy mildew symptoms developed after 8 days on the inoculated plants and the pathogen was identified in association with symptoms consistent with downy mildew. The non-inoculated controls remained healthy. In North America, the occurrence of basil downy mildew has been reported since 2007 (3) and the disease has spread into several U.S. states. To our knowledge, this is the first report of downy mildew on sweet basil in Canada.

First report of downy mildew (Plasmopara obducens) on impatiens walleriana in Italy.

During summer 2012, potted plants of impatiens (Impatiens walleriana) grown in gardens near Biella (northern Italy) showed symptoms of downy mildew. Infected leaves were paler green than normal and showed white, downy growth on the lower surface. Plants collapsed very rapidly, especially at high relative humidity (RH). Microscopical observations of infected leaves, maintained for 24 h at high RH, disclosed the presence of hyaline, tree-like, straight, 120-350×5.8-10 μm sporangiophores, with three sterigma. Sporangia were ovoid, hyaline and 10.7-15.4×11.7-16.6 (average 13.2×14.5) μm in size. Oospores were not observed in leaf tissue. The DNA region encoding for the large ribosomal subunit (LSU rDNA) was amplified using primers NL1 and NL4 (Maier et al., 2003) and sequenced (GenBank accession No. JX880252LSU). BLAST analysis of the 729 bp product obtained showed a similarity of 99% (E-value=0) with Plasmopara obducens from the USA (GenBank accession No. JX217746). To confirm pathogenicity, 60-day-old impatiens plants, grown singly in 15 litre pots in a growth chamber at 20±1°C, were inoculated by spraying leaves with a suspension of 1×105 sporangia/ml. Control plants were sprayed with distilled water. Plants were covered with plastic bags for 4 days. The first symptoms (chlorosis) developed 8 days post inoculation. Control plants remained healthy. This is the first report of P. obducens in Italy. The disease has been reported in several countries, including the USA (Wegulo et al., 2004), UK (Lane et al., 2005) and Serbia (Bulajic et al., 2011). Currently, this disease is present in several gardens in northern Italy, where its importance may increase rapidly due to the widespread cultivation of impatiens.

Characterization of resistance mechanisms activated by Trichoderma harzianum T39 and benzothiadiazole to downy mildew in different grapevine cultivars

Downy mildew, caused by Plasmopara viticola, is one of the most destructive diseases of grapevine and is controlled with intense application of chemical fungicides. Treatment with Trichoderma harzianum T39 (T39) or benzothiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) has been previously shown to activate grapevine resistance to downy mildew and reduce disease symptoms in the Pinot noir cultivar. However, enhancement of plant resistance can be affected by several factors, including plant genotype. In order to further extend the use of resistance inducers against downy mildew, the physiological and molecular properties of T39‐ and BTH‐activated resistance in different cultivars of table and wine grapes were characterized under greenhouse conditions. T39 treatment reduced downy mildew symptoms, but the degree of efficacy differed significantly among grapevine cultivars. However, efficacy of BTH‐activated resistance was consistently high in the different cultivars. Expression profiles of defence‐related genes differed among cultivars in response to resistance inducers and to pathogen inoculation. T39 treatment enhanced the expression of defence‐related genes in the responsive cultivars, before and after P. viticola inoculation. A positive correlation between the efficacy of T39 and the expression level of defence‐related genes was found in Primitivo and Pinot noir plants, while different genes or more complex processes were probably activated in Sugraone and Negroamaro. The data reported here suggest that the use of a responsive cultivar is particularly important to maximize the efficacy of resistance inducers and new natural inducers should be explored for the less responsive cultivars.

First Report of Downy Mildew Caused by Peronospora sordida on Butterflybush (Buddleja stachyoides) in Brazil

Butterflybush (common name in Brazil, verbasco), Buddleja stachyoides Cham. & Schltdl. (Buddlejaceae), is an erect herb or small shrub, native to Brazil, that is listed both as a folk medicinal plant and as a pasture weed (4). In March 2012, a group of B. stachyoides plants growing in a pasture in Viçosa (state of Minas Gerais, Brazil), were found bearing typical downy mildew symptoms. The only pathogen reported associated with this plant species is Podosphaera xanthii (1) and because there is no record of downy mildew on members of Buddleja in Brazil, an investigation was carried out to clarify the pathogen identity. Diseased plants had lesions on living leaves that were vein-delimited, chlorotic, coalescing, and becoming necrotic adaxially and bearing downy mildew-like colonies over diseased tissues abaxially. The samples were dried in a plant press and a representative specimen was deposited in the local herbarium at the Universidade Federal de Viçosa (Accession No. VIC 31836). Reproductive structures were scraped from leaves with a scalpel and mounted in lactophenol. Preliminary observations indicated the pathogen as belonging to Peronospora (Peronosporaceae). The pathogen had the following morphology: Sporangiophores are 288 to 641 μm long, dichotomously branching up to seven times, hyaline, smooth, 5 to 16 μm wide at the trunk, branches 63 to 202 μm long; tips subacute, in pairs or rarely single, 5 to 19 μm long; sporangia subglobose to ellipsoidal, 12 to 22 × 11 to 17 μm, pale yellowish brown, non-papillate. Only one species of Peronospora is known to infect members of Buddleja, namely Peronospora hariotii Gäum. (1). Nevertheless, the pathogen on B. stachyoides has smaller sporangia as compared to those of P. hariotii (20 to 26 × 16 to 21 μm) (2) and it was closer to P. sordida (3). DNA of the pathogen from B. stachyoides was extracted and the cytochrome c oxidase subunit II (COX2) and internal transcribed spacer (ITS) regions were sequenced. Sequences were deposited in GenBank (COX2 as JX982637; ITS as JX982638). A BLAST search yielded 99% and 100% of maximum identity with P. sordida for COX2 and ITS, respectively. A more detailed phylogenetic study is necessary to clarify the relationship between P. sordida, P. hariotii, and related species causing downy mildew on closely related hosts. To our knowledge, this is the first report of P. sordida occurring on a member of the genus Buddleja. This is also the first time that P. sordida is reported from South America.

First Report of Persian Buttercup Downy Mildew Caused by Peronospora sp. in Korea.

Persian buttercup (Ranunculus asiaticus L.) is an ornamental plant cultivated mainly in the countries surrounding the Mediterranean Sea, and has recently become popular in Korea. During March and April 2012, Persian buttercups 'Elegance' showing symptoms of downy mildew were found in plastic greenhouses in Hwaseong City of Korea. Infection resulted in chlorotic leaves with a dark greyish and dense fungal-like growth on the lower surfaces, and finally led to necrosis of the lesions. A sample was deposited in the Korea University herbarium (KUS-F26431). Conidiophores emerging from stomata were hyaline, 250 to 550 × 7 to 15 μm, straight, and dichotomously branched in 6 to 8 orders. Ultimate branchlets were mostly in pairs, slightly curved, 5 to 15 μm long, and had obtuse tips. Conidia were brown, broadly ellipsoidal to subglobose or ellipsoidal, often pedicellated, and measured 24 to 33 × 20 to 27 μm with a length/width ratio of 1.15 to 1.30. Fourteen species of Peronospora have previously been described on the genus Ranunculus (2), of which P. ficariae was mostly considered the causal agent of downy mildew on Persian buttercup (1,3). The present Korean accession is morphologically distinct from P. ficariae on R. ficaria (a synonym of Ficaria verna) by somewhat larger conidia with often pedicel-like ends. The nuclear ribosomal LSU and ITS regions were PCR-amplified and sequenced as described in Göker et al. (4), and the resulting sequences deposited in GenBank (Accession Nos. KC111207 and JX465737, respectively). A comparison with the GenBank sequences revealed that the present Korean pathogen differed from P. ficariae on R. ficaria at 10 of 688 characters (about 1.5%) in LSU (AF119600) and 11 of 802 characters (about 1.4%) in ITS sequences (unpublished sequence). In addition, the ITS sequence exhibits a dissimilarity of 1.5 to 2.0% from three species of Peronospora parasitic on Ranunculus; P. alpicola on R. aconitifolius (AY198271), P. illyrica on R. illyricus (AY198268), and P. ranunculi on R. acris (AY198267) and R. recurvatus (AY198269). Based on morphological and molecular distinction between P. ficariae and the Korean pathogen, we provisionally indicate this pathogen as an undetermined species of Peronospora. Pathogenicity was demonstrated by shaking diseased leaves onto the leaves of healthy Persian buttercup 'Elegance', incubating the plants in a dew chamber at 20°C for 24 h, and then maintaining them in a greenhouse (20 to 24°C and relative humidity 60 to 80%). After 3 to 4 days, inoculated plants developed downy mildew symptoms, from which an identical fungus was observed, thus fulfilling Koch's postulates. Control plants treated with sterile water did not develop any symptoms of downy mildew. To our knowledge, this is the first report of a downy mildew on Persian buttercup in Asia, although this disease has been found in other continental countries, such as Italy (1), New Zealand, South Africa, and the United States (3). The presence of a downy mildew on Persian buttercup in Asia can be considered as a potentially new and serious threat to commercial production of this ornamental plant.

Distribution and harmfulness of Plasmopara halstedii on sunflower in the Czech Republic

In 2007–2012 the first detailed study of P. halstedii distribution and severity was performed in the Czech Republic by monitoring altogether 128 localities. Incidence of the pathogen was recorded at seven localities in south-eastern (Central and South Moravia) and central parts (East and Central Bohemia) of the country; at only four localities the occurrence of sunflower downy mildew symptoms and pathogen sporulation were recorded repeatedly. In all cases the primary infection prevailed, and the severity of infections was rather low. The majority of records were for sunflower experimental fields (e.g. fields of Central Institute for Supervising and Testing in Agriculture. Brno-Chrlice, Czech Republic), one record came from a commercial field and one record from a hobby field. Results of surveys indicate that reservoirs of primary inoculum likely exist in the soil contaminated by P. halstedii oospores in these habitats, but additional transfer by infected seed may be possible. Historical and geographical consequences of recorded infections are discussed, as well as virulence variation in the Czech pathogen populations which is currently in the process of investigation.

Hyaloperonospora camelinae on Camelina sativa in Washington State: Detection, Seed Transmission, and Chemical Control.

Camelina (Camelina sativa) plants with symptoms of downy mildew were obtained from three different locations in Washington State. Based on polymerase chain reaction (PCR) and sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, the causal pathogen was identified as Hyaloperonospora camelinae. The PCR primers consistently amplified 699-bp bands from the infected plants but not from the asymptomatic plants. A comparison of the sequences with those in GenBank revealed 100% sequence similarity to H. camelinae. Growth and development of the H. camelinae was observed in different tissues using light microscopy and scanning electron microscopy (SEM). Light microscopic observation revealed the presence of oospores in the infected leaves and SEM revealed the presence of conidia and conidiophores on the seed surface. To determine whether H. camelinae is a seed-transmitted pathogen, seed collected from infected plants were planted in Sunshine professional growing mix maintained in a growth chamber. Disease symptoms were observed in 96% of the seedlings compared with 3% of the seedlings grown from seed from asymptomatic plants, which indicates that H. camelinae is a seed-transmitted pathogen. Seed treated with mefenoxam, a fungicide specific for Oomycetes, significantly reduced the incidence of the disease.

First Report of Impatiens Downy Mildew Caused by Plasmopara obducens in Ohio.

Single and double flowered impatiens (Impatiens walleriana Hook.f.) plants with symptoms of downy mildew were found in commercial greenhouses in Delaware, Wayne, and Holmes counties, Ohio, in April 2012. Plants were stunted and defoliated. Symptoms on remaining leaves included general chlorosis without discrete spots and downward curling of leaves. A downy white growth was observed on the lower surface of infected leaves. The disease was widespread in affected greenhouses and incidence in cvs. Shimmer Coral, Accent Mix, and Super Elfin was nearly 90%. The downy growth consisted of coenocytic mycelia, monopodial sporangiophores, and ovoid, hyaline sporangia typical of Plasmopara obducens (J. Schröt.) J. Schröt in Cohn (1,2,4). Sporangia were borne on branchlets measuring 5 to 15 μm long (average 10 μm) at right angles to the main axis of the sporangiophore. Sporangia were 9.4 to 17.5 × 12.8 to 16.3 μm. No oospores were observed. Total DNA was extracted directly from plant tissue with the Wizard SV Genomic DNA Purification System (Promega, Madison, WI) following the manufacturer's instructions. Large ribosomal subunit DNA was amplified by PCR using primers NL-1 and NL-4 (3). Amplicons of 690 bp and 834 bp were produced from each diseased sample, while only the 690-bp amplicon was produced from healthy tissue. DNA from each amplicon of sample IDM041712 was purified using the Wizard SV Gel and PCR Clean-Up System (Promega), sequenced, and the sequence of the diagnostic 834-bp amplicon was deposited in GenBank (JX142134). The sequence of the 834-bp amplicon was 99% similar to those of P. obducens isolates from Serbia (HQ246451) (1), the UK (AY587558), and Austria (EF196869). The sequence of the 690-bp amplicon (JX142135) was 99% similar to that of I. walleriana (HQ223336). Twelve young impatiens 'Shimmer Coral' plants were inoculated with sporangia washed from infected leaves (1 × 104 sporangia/ml). Plants were incubated at room temperature for 24 h in a moist chamber and then maintained in a greenhouse (21 to 23°C) until symptoms appeared. Control plants were sprayed with sterile water and maintained in the same environment. After 12 to 14 days, typical symptoms of downy mildew developed on the inoculated plants and microscopic examination revealed the same pathogen morphology as the original isolate. All non-inoculated control plants remained disease free. To our knowledge, this is the first report of downy mildew on impatiens in Ohio. This disease caused considerable economic losses in Ohio in 2012 and is likely to be a recurring problem requiring intensive preventative management.

First Report of Tatsoi Downy Mildew Caused by Hyaloperonospora brassicae in Korea.

Tatsoi (Brassica narinosa L.H. Bailey), also called spinach mustard or spoon mustard, is cultivated for edible greens in Asia. In Korea, this plant has recently become popular as a sprout vegetable that is grown to harvestable size in 5 to 6 days. During April 2012, tatsoi seedlings showing typical symptoms of downy mildew were found in plastic greenhouses in Pyeongtaek City of Korea. Infection resulted in chlorotic areas on the leaves with a white mildew developing on the abaxial surface, and finally led to necrosis of the lesions. Affected sprouts were unmarketable and abandoned without harvesting. A sample was deposited in the Korea University herbarium (Accession No. KUS-F26445). Microscopic examination of fresh material was performed under a light microscope. Conidiophores emerging from stomata were hyaline, 270 to 550 × 10 to 25 μm, straight, and monopodially branched in six to eight orders. Ultimate branchlets were mostly in pairs, flexuous, and 15 to 25 μm long. Conidia were hyaline, subglobose, and 20.5 to 26.5 × 19.5 to 24.5 μm with a length/width ratio of 1.05 to 1.20. These characteristics unequivocally indicate the genus Hyaloperonospora (1). Previously H. parasitica (formerly under Peronospora) has been considered a causal agent of downy mildew on tatsoi (2,4), but the present Korean accession is morphologically distinct from the former species by possessing subglobose conidia with a low length/width ratio. To confirm this morphological difference, amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA of the Korean specimen were performed using procedures outlined by Göker et al. (3). The resulting 874-bp sequence of the region was deposited in GenBank (Accession No. JX401551). A comparison with the ITS sequences available in the GenBank database revealed that it was identical to Hyaloperonospora brassicae found on Brassica oleracea var. italica (EU137726), and showed only one base pair substitution compared to pathogens from B. rapa ssp. pekinensis (JF975613) and B. napus spp. napus (EU049248), but is significantly different from H. parasitica on Capsella bursa-pastoris (AY210988) with a base-pair dissimilarity of about 13%. Therefore, the pathogen found in Korea was confirmed to be H. brassicae. Pathogenicity was demonstrated by shaking diseased leaves onto the leaves of healthy tatsoi seedlings, incubating the plants in a dew chamber at 20°C for 24 h, and then maintaining them in a greenhouse (22 to 26°C). After 3 days, inoculated plants developed downy mildew symptoms from which identical fungi were observed, thus fulfilling Koch's postulates. Control plants treated with sterile water did not develop any symptoms or signs of downy mildew. This is the first report of a downy mildew on tatsoi in Korea, although it has been found in China (2) and Japan (4). To our knowledge, there is no record of tatsoi downy mildew outside East Asia (2,4).

Plasmopara obducensoccurring onImpatiens wallerianahybrids and species in Hungary: Morphological and molecular characterization of the pathogen

In May 2011, young Impatiens walleriana Hook. f. plants showing downy mildew symptoms were collected from a greenhouse (Kecskemet, Hungary). The upper surface of affected leaves turned yellow and white fungal-like growth was observed on the underside. Diseased plants wilted and rapidly collapsed, resulting in losses of nearly 100%. Downy mildew of impatiens can be caused by two pathogens, Plasmopara obducens or Plasmopara constantinescui (syn: Bremiella sphaerosperma), which can be distinguished by the symptoms and by morphology of sporangiophores and sporangia. For molecular identification the 5′-end of the large subunit of ribosomal RNA gene was amplified by PCR using NL1 and NL4 primers and cloned and sequenced. The nucleotide sequence of the Hungarian isolate showed highest identity (99.72%) with two isolates of Plasmopara obducens from the United Kingdom and Serbia. On the basis of the symptoms, molecular and morphological characters, the pathogen was identified as P. obducens. The appearance of the pathogen in Hungary seriously endangers the production of I. walleriana, which is a popular ornamental in gardens and city parks. The chemical plant protection is not effective against the pathogen, by the occurrence of the first symptoms the diseased plants should be removed from the plantlet and must be destroyed.

Evaluation of “ground sensing” optical sensors for diagnosis of Plasmopara viticola on vines

The present work considers the possible use of two commercial optical devices, the GreenSeeker RT100 and the Crop Circle, in detecting different levels of grapevine downy mildew symptoms. The analysis was conducted on vine leaves that had been picked from plants of cv. Cabernet Franc infected by Plasmopara viticola. Leaves were divided into eight homogeneous infection classes and then analyzed (on the leaves’ adaxial surfaces) through the optical devices and a portable visible/near infrared (Vis/NIR) spectrophotometer used as tester. Data showed a linear relation between the percentage of symptomatic leaf area and the Normalized Difference Vegetation Index (NDVI) calculated through the optical sensors (R2 = 0.708 for GreenSeeker; R2 = 0.599 for Crop Circle; R2 = 0.950 for the spectrophotometer). The regression obtained for GreenSeeker is more significant than the one obtained for Crop Circle. This fact suggests a greater capability of GreenSeeker than Crop Circle in detecting different disease levels and its possible use in diagnosis application in the vineyard. Finally, the NDVI measurements carried out through the two commercial sensors, showed lower values on abaxial surfaces than on adaxial surfaces, and a reduced range of values. Moreover, the identification of different infection classes was more difficult on the abaxial surface. This is due to both the different structure of the leaf tissue and the different symptoms of P. viticola on the abaxial and adaxial surfaces. The present work will allow, in the future, applying these optical devices to diagnosis directly in vineyards.

First Report of Downy Mildew of Spider Flower Caused by a Hyaloperonospora sp. in Korea.

Spider flower, Tarenaya hassleriana (Chodat) H. H. Iltis (synonym Cleome hassleriana; C. spinosa), which is native to South America, is now cultivated as an ornamental plant worldwide. In Korea, this plant has recently become popular in gardens and parks because of its beautiful flowers. During July 2010, plants showing typical symptoms of downy mildew were observed in public gardens along the lakeside in Ganghwa, South Korea. Infection resulted in chlorotic areas on the leaves with a white mildew developing on the abaxial surface and finally leading to necrosis of the lesions. Representative samples of infected leaves were deposited at the herbarium of Korea University, Seoul, Korea (KUS-F25091 and F25462). Microscopic examination of fresh material was performed under a light microscope. Conidiophores emerging from stomata were hyaline, 250 to 650 × 10 to 15 μm, straight, and monopodially branched in five to eight orders. Ultimate branchlets were mostly in pairs, flexuous to sigmoid, 15 to 30 μm long, and had obtuse or subtruncate tips. Conidia were hyaline, subglobose, and measured 23 to 26.5 × 21 to 24 μm with a length/width ratio (L/W) of 1.05 to 1.15. Up to now, the downy mildew pathogen of the spider flower has been considered to be Hyaloperonospora parasitica, Peronospora capparidis or P. cleomes, but the latter two names were considered as synonyms of the former (1). In the current study, the spider flower pathogen was morphologically distinct from H. parasitica; in the Korean specimen, conidia were subglobose with a low L/W value, while in H. parasitica sensu stricto, originated from Capsella bursa-pastoris, conidia were broadly ellipsoidal and measured 22.5 to 26.5 × 18 to 21.5 μm with a L/W ratio of 1.17 to 1.31 (1). To confirm this morphological difference, the amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA of the Korean specimen were performed using procedures outlined by Göker et al. (3). The resulting 874-bp sequence of the region was deposited in GenBank (Accession No. JQ301468). A comparison with the ITS sequences available in the GenBank database revealed that the Korean accession exhibits a high dissimilarity of approximately 11% (99 of 882 characters are different) from that of H. parasitica from C. bursa-pastoris (AY210987). On the basis of morphological and molecular data, the spider flower pathogen found in Korea was clearly distinct from H. parasitica. Therefore, we provisionally indicate this pathogen as a Hyaloperonospora sp. To our knowledge, there is no previous record of a downy mildew on spider flower in Asia, although this disease has been previously recorded in Malawi, South Africa, Uganda, New Zealand, Poland, Romania, the United States, and Venezuela (2). The presence of a downy mildew on spider flower in Asia can be considered a potentially new and serious threat to this ornamental plant.

First Report of Plasmopara obducens Causing Downy Mildew on Impatiens walleriana in Hungary.

In May 2011, young impatiens plants (Impatiens walleriana Hook.f.) showing downy mildew symptoms were collected from a greenhouse in Kecskemét, Hungary. The disease was later reported from different parts of the country from a number of different cultivars. The upper surface of affected leaves turned yellow and white fungal-like growth was observed on the underside. Diseased plants wilted and rapidly collapsed, resulting in losses of nearly 100%. Appearance of the disease caused a loss of approximately 2,000 euros for the growers in Kecskemét. In Hungary, losses for growers and consumers could have reached half a million euros. Downy mildew of impatiens can be caused by two pathogens, Plasmopara obducens or Bremiella sphaerosperma (1). P. obducens differs from B. sphaerosperma by monopodial sporangiophores with straight branches and the lack of apical thickening on the branchlets. Sporangia of B. sphaerosperma are spherical, while those of P. obducens are subspherical. Sporangiophores of the fungus were colorless with straight trunks and monopodially branched four to seven times. Sporangiophores ended with three apical branchlets at right angles to the main axis, measuring 4.6 to 16.4 μm (average 9.6 μm). The ovoid and hyaline sporangia measured 11.04 (7.7 to 13.8) × 13.9 (9.9 to 17.4) μm. For molecular identification, total DNA was extracted from the pathogen scraped from the leaves using a cetyltrimethylammoniumbromide (CTAB) extraction method. The 5'-end of the large subunit of ribosomal RNA gene was amplified by PCR using NL1 and NL4 primers (3) and cloned and sequenced. The nucleotide sequence of IWPO-H1 Hungarian isolate (GenBank Accession No. HE577169) showed highest identity with Accession Nos. AY587558 and HQ246451 from the United Kingdom and Serbia (2), respectively, with 99.72% identity. On the basis of the symptoms and molecular and morphological characters, the pathogen was identified as P. obducens. Pathogenicity was confirmed by spraying young I. walleriana plants with a sporangial suspension (1 × 104 sporangiospores ml-1). Control plants were sprayed with sterile water. Plants were incubated at room temperature under glass vessels at approximately 90% relative humidity. Symptoms of downy mildew occurred 12 to 16 days after inoculation, while control plants remained healthy. Presence of P. obducens was reported from Austria, Australia, Bulgaria, China, the Czech Republic, Denmark, Germany, Finland, Korea, Lithuania, Norway, Pakistan, Poland, Romania, Russia, the United Kingdom, the United States, and recently from Serbia (2). To our knowledge, this is the first report of downy mildew of I. walleriana caused by P. obducens in Hungary. The appearance of P. obducens in Hungary seriously endangers the production of I. walleriana, which is an important and popular ornamental in gardens and city parks.

First report of Plasmopara obducens on impatiens (Impatiens walleriana) in Hungary

In late July 2010, diseased Impatiens walleriana hybrid plants were found with symptoms of downy mildew in a city garden of Budapest. Stunting of plants and poor flowering, curling of upper leaves and intensive development of a white …

First Report of Plasmopara obducens on Impatiens walleriana in Serbia.

In May 2010, Impatiens walleriana plants with single or double flowers that were showing symptoms resembling those of downy mildew were collected in a greenhouse in the vicinity of Mionica, Kolubara District, Serbia. Diseased plants were severely stunted, with mild inconspicuous mottling and yellowing on the upper surface of the leaves. The lower surface of the affected leaves was completely covered with distinctive thick, white fungal-like growth. Symptomatic leaves wilted very quickly and premature leaf fall was common, leaving plants with only a few of the youngest leaves and no or few and poorly developed flowers. Disease incidence was extremely high, approaching 100%, and wilting and collapse of affected plants was very rapid, resulting in losses of more than 90%. White downy growth developing on leaf undersurfaces consisted of hyaline, thin-walled sporangiophores with monopodial branching and numerous, ovoid and hyaline sporangia. Apical branchlets of sporangiophores were at right angles to the main axis, with no apical thickening. Downy mildew of impatiens can be caused by Plasmopara obducens or the less known Bremiella sphaerosperma. The two can be differentiated on the basis of symptomatology and morphology of sporangiophores (1). The absence of well-defined spots on the infected impatiens leaves and straight sporangiophores indicated that the pathogen was P. obducens, which was further supported by molecular identification. Total DNA was extracted directly from plant tissue with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions, and the 5'-end of the nuclear DNA coding for the large ribosomal subunit (LSU rDNA) was amplified by PCR using primers NL1 and NL4 (3). Each PCR amplification yielded two bands estimated at 800 and 650 bp, respectively. A representative isolate, 28-10, was sequenced and 727 bp of the larger band (GenBank Accession No. HQ246451) were found to be identical with P. obducens isolate (AY587558) from the United Kingdom. The sequence was almost identical with those of three P. obducens isolates deposited in NCBI GenBank: EF196869 and AY035522 differed from it by one base pair and FJ787308 by two base pairs. The sequence (HQ223336) of the smaller band was identical to that of three Impatiens accessions (AY727936, AF479154, and AY056515). Pathogenicity tests included inoculation of young I. walleriana plants by spraying with a sporangial suspension. The inoculated plants were kept in experimental chambers at 20°C and 80 to 90% relative humidity, and downy mildew symptoms were observed after 13 to 15 days. To our knowledge, this is the first report of downy mildew of I. walleriana caused by P. obducens in Serbia. So far the presence of P. obducens was recorded in Bulgaria, the Czech Republic, Denmark, Finland, Lithuania, Romania, Russia, the United Kingdom (2), and recently in Norway (4). Thorough inspection would be needed to determine the distribution and incidence of P. obducens on impatiens in Serbia both indoors and outdoors. Impatiens is one of the most popular ornamentals in Serbia and intensive and increasing production may be seriously endangered by the presence of P. obducens.

First Report of Downy Mildew of Lima Bean Caused by Phytophthora phaseoli in Virginia.

Delaware, the eastern shore of Maryland, and southern New Jersey have been the center of lima bean (Phaseolus lunatus L.) production in the eastern United States for nearly 50 years (1). Downy mildew has been the most important disease of lima bean in the humid eastern United States over that period. The causal agent of downy mildew, the oomycete pathogen Phytophthora phaseoli Thaxt., was first identified on lima bean in Connecticut in 1887 by Thaxter. Signs and symptoms of lima bean downy mildew include infection, necrosis and abscission of flowers, and shepherd's crooking of racemes, shoot tips, and petioles (1). Sporangia develop on shoot tips, petioles, pins (small pods), and pods in the field and on hypocotyls in-vitro. Since 2005, approximately 50% of the baby lima beans processed in the United States have been grown in Delaware and the eastern shore of Maryland. In 2008, commercial lima bean production began on the eastern shore of Virginia in Accomack County but no downy mildew was reported in that season. In 2009, approximately 1,825 ha in Accomack and Northampton counties were planted to baby lima bean. Weather conditions in 2009, including above average rainfall, were conducive for the development of downy mildew on the Delmarva Peninsula. Downy mildew was widespread in growers' fields in August and September in butter bean in southern New Jersey and baby lima bean in Sussex County, DE. In August 2009, a home gardener in Rappahannock, VA sent samples of infected lima bean pods from baby, Fordhook, and pole lima bean plants to the Virginia Tech Plant Disease Clinic in Blacksburg. On the basis of morphometric analysis, samples were determined microscopically to be infected by a Phytophthora sp. with rather uniform sporangia averaging 39 × 22 μm and short pedicels, diagnostic for P. phaseoli (1). On October 27, 2009, field scouts in Accomack County, VA identified two lima bean fields planted to cv. C-Elite-Select exhibiting moderate symptoms of downy mildew. Samples were brought to the Plant Diagnostic Clinic at the University of Delaware under USDA-APHIS permit and determined to be P. phaseoli based on morphometric analysis. Samples were inoculated onto a lima bean cultivar differential to determine pathogenicity to complete Koch's postulates and to determine their physiological race. Samples were inoculated onto lima bean cvs. 184-185 and C-Elite-Select, which are susceptible to race F and resistant to race E, Eastland and 8-78, which are susceptible to race E and resistant to race F, and Concentrated Fordhook, susceptible to all known races (1). Three pots containing five emerging seedlings each were inoculated with sporangia (approximately 103 per ml) prepared by soaking infected pods in 500 ml of sterile distilled water for 1 min with gentle agitation. Plants were placed in a Percival dew chamber with intermittent misting and set at 19. Infection and disease development were assessed daily and signs developed 7 days postinoculation in cvs. 184-85, C-Elite-Select, and Concentrated Fordhook, but not in Eastland and 8-78. Cultivar differential tests indicated that the isolates were P. phaseoli race F. Hypocotyls of infected plants were scraped, and isolations made on lima bean pod agar confirmed the presence of P. phaseoli. To our knowledge, this is the first time that downy mildew of lima bean has been reported in Virginia. Reference: (1) T.A. Evans et al. Plant Dis. 91:128, 2007.