First Report Of Downy Mildew Research Articles

Pseudoperonospora cubensis Causing Downy Mildew on Luffa aegyptiaca in Korea

In November 2021, sponge gourd (<i>Luffa aegyptiaca</i>) plants with typical downy mildew symptom were observed in Korea. Based on morphological characteristics of sporangiophores and sporangia, combined with molecular phylogenetic analysis of the internal transcribed spacer rDNA and <i>cox</i>2 mtDNA regions, the causal pathogen was identified as <i>Pseudoperonospora cubensis</i> (Oomycota). This is the first report of downy mildew caused by <i>P. cubensis</i> on <i>L. aegyptiaca</i> in Korea. Given that <i>P. cubensis</i> exhibits high disease incidence and severity on many Cucurbitaceae crops, it could pose a significant threat to sponge gourd cultivation.

First global report of Hyaloperonospora brassicae in commercial broccoli and cabbage microgreens.

Microgreens are a nutrient-dense enhancement to modern diets (Choe et al. 2018), whose small production footprint in protected systems facilitates rapid crop turnover and distribution to population centers. Eleven of the 25 most broadly grown microgreens are brassicas (Choe et al. 2018). In November 2023, kale, broccoli (H009B), and cabbage (H009C) microgreen crops in Michigan were observed with downy mildew, at disease severities of 3%, 40%, and 20% foliage on 10 x 16 cm seeded blocks of plants, respectively. These crops shared a germination chamber for at least three days, which was maintained at approximately 22℃ in very humid, dark conditions. Chlorosis and grayish, sunken necrosis characterized symptoms on cotyledon surfaces (Fig. 1). In humid conditions, thick, white-light gray sporulation was present on adaxial cotyledon surfaces, accompanied by sparse sporulation on abaxial surfaces and hypocotyls. Severely diseased plants were stunted and approximately 50% gradually succumbed to downy mildew. On microscopic examination, a Hyaloperonospora spp. was tentatively identified, with long sporangiophores that dichotomously branched 3 to 6 times and hyaline sporangia borne singly on flexuous terminal sterigmata (Fig. 2). Sporangia were round to oval, with average length of 23.1 (range 16.0 to 28.3) µm; width of 20.0 (15.0 to 25.6) µm; and average length:width of 1.2 (1.0 to 1.4); (n = 97 for all). Sporangia dislodged rapidly if disturbed or as humidity decreased. Two pathogenicity tests were initiated on two sequential days. Two cotyledons from originally infected broccoli and cabbage were suspended, abaxial-side down, on coarse mesh over an open 60-mm plate of pregerminated brassica seeds on a water-saturated filter, inside a sealed, clear plastic box. Boxes contained only one type of originally diseased host, with 15 to 20 seeds of transfer varieties in unique dishes. Boxes were incubated in the dark for 2 days at 19°C with a wet paper towel atop the cotyledons. Before removal, cotyledons were lightly brushed across the surfaces of the seedlings they were just suspended above. Seedlings were grown in boxes in the presence of indirect, ambient light for 9.5 hr/day for an additional 5 days before pathogen sporulation was apparent. Filter paper was resaturated as needed. Noninoculated control plants, maintained separate from inoculated plants, were asymptomatic throughout the experiments. Total disease incidence in transfer varieties was 43.5% of 'Graffiti' cauliflower, 18.7% and 15.7% of 'Nixon' and 'Blue Vantage' cabbage; 11.8% of 'Red Russian' kale, and 6.0% of 'Ironman' broccoli, combined from two experiments. All varieties listed had at least one plant successfully infected in both pathogenicity tests. Sporulation on transfer hosts was morphologically identical to originally affected crops. Sporangiophores and sporangia were removed from H009B broccoli and H009C cabbage plants using surface sterilized forceps, placed directly into DNA extraction tubes containing buffer CD1 (Qiagen PowerSoil Pro), then kit instructions were followed. Extracts were utilized as template for ITS and cox1 PCR amplification, using DreamTaq Mastermix and ITS4/6 (45 cycles; White et al. 1990) and Levup/Levlo primers (30 cycles; Robideau et al. 2011). Cycling conditions were as published, with the number of cycles indicated by primer set. Each reaction yielded a single amplicon of approximately 1000 and 700 bp, for ITS and cox1, respectively,. Amplicons were cleaned using ExoSap-IT and submitted for Sanger sequencing, using ITS6 and Levup as sequencing primers (Robideau et al. 2011; White et al. 1990). After quality trimming, amplicons shared >98.5% identity with H. brassicae (NCBI Genbank accession MG757792 or reference genome CANTFL010000892.1). Sequences were submitted to Genbank (PP093830, PP093831, PP776812, PP776813). This is the first report of downy mildew, caused by H. brassicae, in commercial brassica microgreens, crops with vast nutritional value and expanding production.

Morphological and molecular characterization of downy mildew on sweet basil (Ocimum basilicum) caused by Peronospora belbahrii in Turkiye.

Sweet basil (Ocimum basilicum) is one of the most significant aromatic plants in Turkiye. Recently, a new pathogen induced symptoms were discovered and identified as basil downy mildew caused by Peronospora belbahrii Thines. The pathogen has been introduced into the country and it has quickly become the most damaging disease in basil cultivation. The purpose of this study was to investigate the molecular and morphological properties of the causal organism of downy mildew observed on sweet basil and determine the disease incidence and prevalence in Antalya province. According to morphological characteristics (conidia, conidiophores) disease was determined as downy mildew caused by P. belbahrii. Pathogenicity tests were performed by spraying with a sporangial suspension of P. belbahrii (1 × 105 sporangia/mL). After 1week, all inoculated plants exhibited characteristic downy mildew symptoms on their leaves, whereas non-inoculated control plants remained disease-free. All molecular analyses involving the internal transcribed spacer region were amplified using Nested PCR with primer pairs ITS4 and ITS6 for the first round and ITS4 and DC6 for the second round. Resulting sequences of all the nested PCR products had 99% similarity with P. belbahrii isolates. Disease incidence was 22.4-70.2% of sweet basil cultivation area in Antalya province. Based on the molecular analysis, morphological characteristics and pathogenicity tests the pathogen was identified as P. belbahrii. To our knowledge, this is the first report of downy mildew caused by P. belbahrii on sweet basil in Turkiye.

First Report of Downy Mildew on Sweet Basil Caused By Peronospora belbahrii in India.

Sweet basil (Ocimum basilicum L.; Family Lamiaceae) is an annual aromatic and medicinal plant grown in tropical and subtropical regions of the world. In India, it is cultivated as a commercial crop on ~8,000 ha. Aerial plant parts and essential oil of sweet basil are used in pharmaceutical, perfumery, food industries and in different formulations of traditional Ayurvedic and Unani medicines (Shahrajabian et al. 2020). The leaves have the highest concentrations of secondary metabolites such as terpenes and phenylpropanoids which provide the distinctive aroma (Viuda-Martos et al. 2011). During October 2020, severe foliar disease was observed in experimental fields of sweet basil at Council of Scientific and Industrial Research (CSIR)-Central Institute of Medicinal and Aromatic Plants (CIMAP) in Lucknow, India. Initial symptoms included large, interveinal chlorotic lesions on the adaxial surface of the leaves and black sporulation on the abaxial surface. Within a few days, the abaxial side of leaves turned necrotic, and leaf senescence and defoliation occurred on plants with severe symptoms. Disease incidence was 20 to 30% of plants. The pathogen was characterized morphologically using a light microscope. Sporangiophores were hyaline, dichotomously branched, 186.9 to 423.07 × 6.85 to 9.06 µm and, branched 3 to 5 times with each branch, terminating in two slightly curved branchlets, the longer one 7.05 to 25.31 µm and the shorter one 4.98 to 15.92 µm. Each branchlet had a single sporangium at the tip. Conidia were ellipsoidal to sub-globose, olive-brown in color, and typically measured 25.21 to 33.86 × 17.92 to 26.24 µm, each, without a pedicel. Based on these morphological characteristics, the foliar disease was identified as downy mildew was caused by Peronospora belbahrii (Thines et al. 2009). Eight symptomatic and two asymptomatic plant samples were collected from different locations in the field, and genomic DNA was extracted from the conidia of the eight naturally infected tissues of sweet basil samples as well as leaf tissues from two asymptomatic plants, using the CTAB method. The internal transcribed spacer region was amplified using ITS1 and ITS4 primers. Only eight infected samples amplified products of expected size (~ 700 bp) and two asymptomatic samples showed no amplification. Only five amplified PCR products were sequenced (White et al. 1990). All five sequences were identical and were a 98.1% match with five P. belbahrii isolates (MN450330.1, MN308051.1, MH620351.1, KJ960193, and MF693898). The consensus sequence was deposited into the NCBI database (GenBank Accession No. MW689257). Downy mildew caused by P. belbahrii previously has been reported on sweet basil from several countries (Wyenandt et al. 2015). To confirm the pathogenicity of these isolates on sweet basil (cv. CIM-Saumya), 25 - day-old sweet basil plants were sprayed with a suspension (1 × 105 sporangia/ml) of P. belbahrii. All plants were kept in a growth chamber with a 23/18°C diurnal cycle with 65 to 85% relative humidity for 24 h. Non-inoculated plants treated with sterile water served as a control treatment. After 8 days, typical symptoms of downy mildew appeared on all the inoculated plants while non-inoculated plants remained asymptomatic. Inoculated leaves with symptoms consistent of downy mildew were collected and the causal agent again identified as P. belbahrii on the basis of microscopic examination and ITS rDNA sequence data. To our knowledge, this is the first report of downy mildew caused by P. belbahrii on sweet basil in India. The pathogen has a broad host range and may pose a serious threat to the cultivation of this valuable crop in India. Thus, it is pertinent to develop effective control measures to avoid further spread and mitigate economic loss.

Plasmopara viticola causing downy mildew on Vitis davidii in Korea.

Vitis davidii (Rom.Caill.) Foëx, commonly known as spine grape, is a deciduous climber native to China. Its fruits are consumed fresh or used to make wine in South and Central China. In recent years, spine grape has been cultivated in Korea. In July 2020, downy mildew was detected on spine grape vines in Jeongeup (35°42'17″N, 126°54'02″E), Korea, with a disease incidence of 70%. The symptoms appeared as yellowish, brownish, or reddish, vein-limited, poly-angular adaxial leaf spots, correspond to dense, white downy growth abaxially. A representative specimen was deposited in the Kunsan National University Herbarium (KSNUH679). Sporangiophores were tree-like, hyaline, mostly straight, and monopodially branched in orders of three to six; they measured (219.4-)273.2 to 435.1(-546.6) × (4.8-)6.7 to 9.0(-10.0) μm (n = 50). Ultimate branchlets were bi or trifurcate, straight to slightly curved, with truncate or, rarely, a swollen tip and measured 2.9 to 9.7 μm long and 0.8 to 2.5 μm wide at the base (n = 50). Sporangia were hyaline, ovoidal or lemon-shaped; they measured (16.8-)20.0 to 28.8(-34.2) × (11.4-)13.1 to 17.0(-20.1) μm with a length to width ratio of (1.28-)1.46 to 1.78(-2.07) (n = 50). This morphology was as described for Plasmopara viticola (Berk. & M. A. Curtis) Berl. & De Toni (Hall, 1989). Genomic DNA was extracted directly from infected V. davidii leaves. Three regions were PCR-amplified and sequenced: cox2 mtDNA with primers cox2F and cox2-RC4 (Choi et al., 2015), actin with primers pve04815-F and pve04815-R, and beta-tubulin with primers pvc389-F3 and pvc389-R4 (Rouxel et al., 2013). The resulting sequences were deposited in GenBank (accession nos. MT834527 for cox2, MT834525 for actin, and MT834526 for beta-tubulin). A BLASTn search revealed that the Korean sample was identical to P. viticola clade aestivalis originating from Vitis species: MK215072 for cox2 sequence, KY933800 for actin, and MK358393 for beta-tubulin. In all phylogenetic analyses of the three genes (cox2, actin, and beta-tubulin), KSNUH679 came out as phylogenetically place within P. viticola clade aestivalis, which has recently been reported on V. coignetiae and V. ficifolia var. sinuata in Korea (Kim et al., 2019). A pathogenicity test was performed twice by inoculating the leaves of 10 healthy spine grape plants with a sporangial suspension (~1 × 106 sporangia·mL-1) and incubating them in a growth chamber at 25 °C, 12-h day/night cycle, and 90% relative humidity; five non-inoculated plants served as controls. After two weeks, all inoculated plants developed typical downy mildew symptoms could be observed, whereas the controls remained symptomless. Morphology and molecular features confirmed the identity of the pathogen of spine grape to be P. viticola. To the best of our knowledge, this is the first report of downy mildew caused by P. viticola on V. davidii in Korea. Recently, downy mildew outbreaks caused by P. viticola have been recorded in spine grape plantations in southern China (Yi et al., 2019). Considering the potential of spine grape as a novel crop for Korea, P. viticola appears to represent a significant threat to this industry.

First Report of Downy Mildew Caused by Plasmopara viticola on Vitis coignetiae in Korea

Vitis coignetiae Pulliat ex Planch., known as crimson grapevine, is a deciduous climbing vine native to the temperate climate zone of East Asia, including Korea and Japan. Edible fruits of crimson grapevine are used for wine production and medicinal purposes in Korea. In November 2018, downy mildew infections were observed on the vines growing at a farm of the Grape Research Institute of Chungcheongbuk-do Agricultural Research and Extension services (36°19′55″N, 127°45′39″E), Korea. Symptoms appeared as reddish or dark brownish, vein-limited, angular spots on the upper surface of leaves, with a white downy oomycete growth developed on the corresponding lower surface. A representative specimen was deposited in the Kunsan National University Herbarium (KSNUH376). Sporangiophores were hyaline, straight or slightly curved, (220–) 420 to 690 (–770) (n = 50), tree-like, and monopodially branched in three to six orders. Ultimate branchlets in pairs or three, straight to slightly curved, 2.5 to 7.0 μm long, 0.9 to 1.6 μm wide at the base (n = 50), with truncate or rarely swollen tips. Sporangia were hyaline, ovoidal or lemon-shaped, measured (17.7–) 20.9 to 24.4 (–26.9) × (12.7–) 13.9 to 16.6 (–18.3) μm, with a length/width ratio of (1.18–) 1.37 to 1.59 (–1.72) (n = 50). The morphological characteristics closely resembled those reported for Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni (Hall 1989). To confirm the morphological identification, genomic DNA was extracted from a diseased leaf of KSNUH376. Three DNA regions were sequenced: internal transcribed spacer I (ITS1) using primers DC6 and ITS2, large subunit (LSU) rDNA using primers LROR and LR6-O (Choi et al. 2011), and cytochrome c oxidase II (cox2) mtDNA using primers cox2F and cox2-RC4 (Choi et al. 2015). The resulting sequences were deposited in GenBank (accession nos. MK557845 for ITS1, MK517538 for LSU, and MK519373 for cox2). A BLASTn search revealed that the Korean sample is identical to the sequences of P. viticola parasitic to V. vinifera (DQ665668 for the ITS1, HM628762 for the LSU, and MH468768 and DQ365760 for the cox2). Pathogenicity was confirmed through inoculation by putting four discs (diameter 3 mm) of the infected leaves onto the lower leaf surface of five healthy V. coignetiae. They were incubated in a growth chamber at 25°C under a relative humidity of 80%, and sterilized dH₂O was sprayed daily on the inoculated leaves to induce zoospore formation and infection. Five noninoculated leaves served as controls. After 10 days, downy mildew symptoms developed on the lower surface of the inoculated leaves, whereas the control leaves remained symptomless. Based on morphological characteristics, molecular sequencing data, and the pathogenicity test, the pathogen was identified as P. viticola. Previously, P. viticola has been recorded on V. coignetiae in the United Kingdom and Japan (Farr and Rossman 2019) and under a laboratory condition by Jurges et al. (2009). To our knowledge, this is the first report of downy mildew caused by P. viticola on V. coignetiae in Korea. Our field observations suggest that downy mildew would be a potential threat to crimson grapevine cultivation.

First report of downy mildew of statice caused by Peronospora statices in Japan

In June 2013, plants of hybrid statice (Limonium spp.) cultivar Misty Blue in a greenhouse in Hokkaido Prefecture, Japan, were observed to have yellowish leaves with gray to white downy growth on the surface, mainly on the abaxial side. The present report describes the identification of the causal pathogen as Peronospora statices based on its morphology and the internal transcribed spacer of its ribosomal DNA (rDNA-ITS) sequences. After inoculation of statice seedlings with the isolate, the original symptoms were reproduced. This is the first report of downy mildew of statice in Japan.

First Report of Downy Mildew on Basil (Ocimum basilicum) in China

HomePlant DiseaseVol. 99, No. 11First Report of Downy Mildew on Basil (Ocimum basilicum) in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Downy Mildew on Basil (Ocimum basilicum) in ChinaX. Y. Kong, S. Wang, S. L. Wan, C. L. Xiao, F. Luo, and Y. LiuX. Y. KongSearch for more papers by this author, S. WangSearch for more papers by this author, S. L. WanSearch for more papers by this author, C. L. XiaoSearch for more papers by this author, F. LuoSearch for more papers by this author, and Y. LiuSearch for more papers by this authorAffiliationsAuthors and Affiliations X. Y. Kong S. Wang S. L. Wan C. L. Xiao F. Luo Y. Liu , Sanya Science & Technology Academy for Crop Winter Multiplication, Winter Multiplication Crop and Plant Protection Research Center, Sanya Key Laboratory for Pest Management of Winter Cucurbits and Vegetables, Sanya 57200, Hainan, China. Published Online:26 Aug 2015https://doi.org/10.1094/PDIS-01-15-0077-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In December 2014, Sanya City (18°N) of Hainan Province, the southernmost city of China, suffered an onslaught of downy mildew over large basil-growing areas. According to our investigation, almost 40% of the plants in the region (70 km2) were infected by the disease, which spread very rapidly. Given favorable conditions, the disease rate could reach 100% three to five days after the attack, making prevention and control extremely difficult. The seedling and adult plant stages of basil are subject to infection by the disease. In the early stages of the disease, the infected parts of the green leaf blades turn yellow, and then brown spots develop at the leaf margins. In the later stages of the disease, the spots spread along the margins or main veins of the leaves into larger areas, amassing as irregular, brown, necrotic spots that can consequentially wither the blades, and more seriously, cover more than half of the leaves. Meanwhile, the leaf back can be completely covered with a grayish-black layer of mildew. In the final stages of the disease, all the leaves of the plant will be infected, whereupon they will turn brown. When examined under a microscope, large amounts of sporangiophores and sporangia were found in the samples of the mildew layer collected from the infected leaves. Sporangiophores were hyaline, 197.0 to 595.3 μm (average 370.6 μm) in length, branched two to seven times, and ending with two slightly curved branchlets having a single sporangium at the tip of each branchlet. The longer branchlets measured 14.3 to 38.3 μm in length (average 23.2 μm), while the shorter ones were 2.4 to 22.1 μm in length (average 8.1 μm). Sporangia were oval or circular, ranged from brown to dark brown, measured 19.8 to 33 μm in length (average 27.6 μm) and 15.1 to 29.3 μm in width (averaging 23.0 μm) (a length/width ratio of 1.2), and produced germ tubes by direct germination. Based on these morphological characteristics, the causal agent was identified as Peronospora belbahrii Thines (Belbahri et al. 2005; Thines et al. 2009). Furthermore, genomic DNA was extracted from infected plant tissue according to Dellaporta et al. (1983). The complete ITS rDNA region was amplified and sequenced using primers ITS5 and ITS4 (Nagy and Horvath 2011). The consensus sequences were deposited in GenBank (Accession No. KP657570), and a BLAST analysis in the NCBI database revealed 99% similarity to all of the P. belbahrii sequences previously reported in regard to basil (EU863410 and KJ960193). For the preparation of the inoculum, infected leaves were flooded with sterile distilled water to collect the sporangia. The sporangia were washed once with sterile distilled water and the suspension was diluted to 1 × 105 sporangia/ml. The sporangia suspension was sprayed onto 15 healthy leaves grown in the same period for inoculation, and sterilized distilled water was sprayed on 15 healthy leaves serving as controls. The experiment was repeated 10 times. Every replicate was placed in a separate petri dish under 12 h of light at 20°C, and 90 to 95% relative humidity. Five days after inoculation, the leaves inoculated with the suspension showed typical symptoms of the disease while the control leaves showed no symptoms. Infected leaves under the microscope showed large amounts of P. belbahrii fungi. Therefore, all of the results suggest that the downy mildew on basil was caused by the fungus P. belbahrii. To our knowledge, this is first report of downy mildew on basil in China.

Downy mildew of coleus caused by Peronospora belbahrii in Japan

In April 2007, coleus (Solenostemon scutellarioides) plants cultivated in a greenhouse in Chiba Prefecture, Japan were observed to have poor growth, with yellowing of the adaxial leaf surface and a gray to black downy growth on the abaxial leaf surface. The causal pathogen was identified as Peronospora belbahrii based on its morphology and rDNA-ITS sequences. Uninfected coleus was inoculated with the pathogen, the original symptoms were reproduced, and the isolate was reisolated. This is the first report of downy mildew of coleus in Japan.

The First Report of Downy Mildew Caused by Peronospora belbahrii on Sweet Basil in Greenhouses in the Czech Republic.

Sweet basil (Ocimum basilicum L.) is an aromatic plant that is cultivated as a pot plant in greenhouses or in fields in the Czech Republic. The plants are intended for direct consumption or for drying. In April of 2012, the first large chlorotic from the middle necrotic spots occurred gradually on leaves of pot plants O. basilicum cv. Genovese in greenhouses in Central Bohemia. The characteristic gray to brown furry growth of downy mildew appeared on abaxial surfaces of leaves in the place of chlorotic spots within 3 to 4 days. The infested leaves fell off in the late stages of pathogenesis. The infestation gradually manifested itself in ever-younger plants and in July, cotyledons and possibly the first true leaves were already heavily infected and damaged and these plants rapidly died. The plant damage reached 80 to 100%, so it was necessary to stop growing the plants in the greenhouse at the end of July. The causal agent was isolated and identified as Peronospora belbahrii Thines by means of morphological and molecular characters (2,3). Conidiophores were hyaline, straight, monopodial, 280 to 460 μm, branched three to five times, ended with two slightly curved branchlets with a single conidia on each branchled tip. The longer branchlets measured 13 to 24 μm (average 18.2 μm), the shorter one 4 to 15 μm (average 9.7 μm). Conidia were rounded or slightly ovoid, from brownish to dark brownish, measured 22 to 31 × 20 to 28 μm (length/width ratio 1.2). A pathogen-specific sequence was detected with the help of the pathogen ITS rDNA specific primers in symptomatic leaves (1). DNA from plant tissues was isolated using the DNeasy plant Mini Kit (Qiagen, Germany) following the standard protocol. PCR was performed using KAPA2G Robust HotStar kit (Kapa Biosystems, United States) according to the conditions recommended in Belbahri et al. (1). The specific products were visualized by electrophoresis through 1.5% agarose gels. Leaves of 20-day-old potted plants O. basilicum 'Genovese' were inoculated by spraying with 5 × 105 conidia/ml of the pathogen. Each pot contained 10 plants. Sterilized distilled water was applied to control plants. Plants were covered with polyethylene bags during the entire incubation period to maintain high humidity, and kept at a temperature of 22 to 24°C. Typical disease symptoms appeared on leaves 5 to 9 days after inoculation. Control plants were symptomless. P. belbahrii was re-isolated from the lesions of inoculated plants, thus fulfilling Koch's postulates. Downy mildew on sweet basil was reported in countries in Africa, Europe, and South and North America (4). To our knowledge, this is the first report of downy mildew on sweet basil in the Czech Republic.

First Report of Downy Mildew Caused by Hyaloperonospora camelinae on Camelina sativa in Slovenia.

Camelina or false flax (Camelina sativa), of the Brassicaceae, is an annual flowering plant native to Europe and Central Asia where it is grown commercially as an oilseed crop. At the end of May 2012, symptoms of downy mildew were observed on camelina plants grown in the Savinja Valley in Slovenia. The disease was found in four monitored fields (total area 3 ha), and the incidence ranged from 2 to 38% depending on the variety. Symptomatic plants showed whitish, abundant, and fluffy mycelia covering the stems, flowers, seed pods, and undersides of the leaves. The disease mainly affected the upper half of the plants, and the stems were reduced and distorted. During disease progression, the mycelium turned from gray to black. Microscopic observations revealed hyaline, straight conidiophores that were branched monopodially (3 to 4 times) with 6 to 12 re-curved tips/branch, and measured 140 to 300 × 12 to 20 μm. Conidia were hyaline, oval to broadly ellipsoidal, 24 to 29 × 18 to 24 μm. Oospores formed in necrotic stem and leaf tissues were dark brown and measured 30 to 38 μm in diameter. Based on these morphological characteristics, the causal agent was identified as Hyaloperonospora camelinae (1,3,4,5). DNA was extracted from mycelium and conidia collected from infected plants in two fields in the Savinja Valley (1HpC and 2HpC). Nuclear internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) were amplified by PCR assay from two isolates using the universal primers ITS4 and ITS5, and sequenced. Both samples yielded a 781-bp sequence, which showed 100% identity to H. camelinae ITS sequence JX445136 in GenBank. The nucleotide sequence was assigned to GenBank Accession No. KJ768405. Pathogenicity was confirmed by spraying 25 3-week-old plants of C. sativa cv. Ligena planted in pots (5 plants/pot) with a conidial suspension (105 conidia/ml) obtained from 10 infected plants of the same variety collected from the field 1HpC. Inoculated plants were covered with polyethylene bags for 2 days to maintain high humidity, and incubated at 20°C with a 12-h photoperiod/day in a growth chamber. Downy mildew symptoms first developed on leaves 6 days after inoculation. An additional 25 control plants sprayed with sterilized distilled water and otherwise treated similarly to the inoculated plants developed no symptoms. The identity of the pathogen on the inoculated plants as H. camelinae was confirmed based on the morphological features described above. Downy mildew of false flax caused by H. camelinae has been reported in Europe from Austria, Bulgaria, Germany, Poland, Portugal, Spain, and Switzerland (2); and in the United States from Florida, Oregon, Minnesota, Montana, Nebraska, and Washington (1,3,4,5). To the best of our knowledge, this is the first report of downy mildew caused by H. camelinae on C. sativa in Slovenia. The representative samples were deposited in the phytopatological herbarium of the Slovenian Institute of Hop Research and Brewing.

Downy mildew of alyssum caused by Hyaloperonospora lobulariae in Japan

In September 2012, alyssum (Lobularia maritima) plants cultivated in a greenhouse in Nagano Prefecture, Japan were observed with yellowing of the adaxial surface of leaves and a white downy growth on the abaxial surface. We identified the causal pathogen as Hyaloperonospora lobulariae based on the morphology and rDNA-ITS sequences. Seedlings of alyssum were inoculated with the pathogen, and the original symptoms were reproduced. This is the first report of downy mildew of alyssum in Japan.

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

During spring 2012, potted impatiens (Impatiens walleriana Hook.f.) plants with symptoms of a foliar disease were found in several commercial greenhouses in Mobile County, Alabama. Symptomatic leaves were chlorotic with no distinct lesions, and quickly wilted and abscised from erect green stems. In summer 2012 and 2013, numerous landscape impatiens plants with similar symptoms were observed in a large area from Mobile County north to Lee County, Alabama. A downy mildew was observed on the lower surfaces of symptomatic and abscised leaves from all locations. It consisted of hyaline, monopodial sporangiophores and ovoid, hyaline sporangia. Sporangiophores, which emerged from stomata, consisted of apical branches arranged at right angles to the supporting branches; they measured 69 to 90 μm long with individual branches measuring 7 to 14 μm long. Sporangia were borne on the tips of sporangiophore branches and measured 10 to 16.5 × 17 to 22.5 μm. No oospores were observed. In 2013, symptomatic plants were obtained from two separate locations in Alabama (Mobile and Tallapoosa counties). Total genomic DNA was extracted directly from symptomatic plant tissue and the large ribosomal subunit DNA was amplified by PCR using primers NL-1 and NL-4 (1). From both isolates, amplicons of 600 and 775 bp were obtained. DNA from each amplicon of both isolates was purified, sequenced, and the sequences were deposited in GenBank (Accession Nos. KF956518 to 21). The sequences of the 600-bp amplicons were 99% similar to that of I. walleriana (JX142135); the sequences of the 775-bp amplicons were 99% similar to Plasmopara obducens isolates from Florida (JX217746), Ohio (JX142134), Serbia (HQ246451), and the United Kingdom (AY587558). In pathogenicity tests, 10 potted impatiens plants, I. walleriana'Super Elfin,' were inoculated with a sporangial suspension (1 × 105 sporangia/ml washed from infected leaves) from the Mobile County isolate, by spraying until runoff. Controls were inoculated with sterile water. Plants were incubated in a moist chamber at 21°C for 48 h and then maintained in a greenhouse at 22 to 25°C until symptom development. All inoculated plants developed symptoms of downy mildew within 10 days. Microscopic examination of the symptomatic tissue revealed sporangiophores and sporangia similar to those observed in naturally infected plants. Control plants showed no symptoms. To our knowledge, this is the first report of downy mildew caused by P. obducens on impatiens in Alabama. This disease has been reported to have a significant economic impact for growers throughout the United States (2,3). Impatiens downy mildew is likely to be a recurring problem in Alabama.

First Report of Impatiens Downy Mildew Outbreaks Caused by Plasmopara obducens Throughout the Hawai'ian Islands.

Downy mildew of impatiens (Impatiens walleriana Hook.f.) was first reported from the continental United States in 2004. In 2011 to 2012, severe and widespread outbreaks were documented across the United States mainland, resulting in considerable economic losses. On May 5, 2013, downy mildew disease symptoms were observed from I. walleriana 'Super Elfin' at a retail nursery in Mililani, on the Hawai'ian island of Oahu. Throughout May and June 2013, additional sightings of the disease were documented from the islands of Oahu, Kauai, Maui, and Hawai'i from nurseries, home gardens, and botanical park and landscape plantings. Symptoms of infected plants initially showed downward leaf curl, followed by a stippled chlorotic appearance on the adaxial leaf surfaces. Abaxial leaf surfaces were covered with a layer of white mycelia. Affected plants exhibited defoliation, flower drop, and stem rot as the disease progressed. Based on morphological and molecular data, the organism was identified as Plasmopara obducens (J. Schröt.) J. Schröt. Microscopic observation disclosed coenocytic mycelium and hyaline, thin-walled, tree-like (monopodial branches), straight, 94.0 to 300.0 × 3.2 to 10.8 μm sporangiophores. Ovoid, hyaline sporangia measuring 11.0 to 14.6 × 12.2 to 16.2 (average 13.2 × 14.7) μm were borne on sterigma tips of rigid branchlets (8.0 to 15.0 μm) at right angle to the main axis of the sporangiophores (1,3). Molecular identification of the pathogen was conducted by removing hyphae from the surface of three heavily infected leaves using sterile tweezers, then extracting DNA using the QIAGEN Plant DNA kit (QIAGEN, Gaithersburg, MD). The nuclear rDNA internal transcribed spacer was sequenced from each of the three samples bidirectionally from Illustra EXOStar (GE Healthcare, Piscataway, NJ) purified amplicon generated from primers ITS1-O and LR-0R (4). Resultant sequences (GenBank KF366378 to 80) shared 99 to 100% nucleotide identity with P. obducens accession DQ665666 (4). A voucher specimen (BPI892676) was deposited in the U.S. National Fungus Collections, Beltsville, MD. Pathogenicity tests were performed by spraying 6-week-old impatiens plants (I. walleriana var. Super Elfin) grown singly in 4-inch pots with a suspension of 1 × 104 P. obducens sporangia/ml until runoff using a handheld atomizer. Control plants were sprayed with distilled water. The plants were kept in high humidity by covering with black plastic bags for 48 h at 20°C, and then maintained in the greenhouse (night/day temperature of 20/24°C). The first symptoms (downward curling and chlorotic stippling of leaves) and sporulation of the pathogen on under-leaf surfaces of the inoculated plants appeared at 10 days and 21 days after inoculation, respectively. Control plants remained healthy. Morphological features and measurements matched those of the original inoculum, thus fulfilling Koch's postulates. To our knowledge, this is the first report of downy mildew on I. walleriana in Hawai'i (2). The disease appears to be widespread throughout the islands and is likely to cause considerable losses in Hawai'ian landscapes and production settings.

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 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.

Severe Outbreak of Downy Mildew Caused by Plasmopara obducens on Impatiens walleriana in Florida.

Impatiens, Impatiens walleriana Hook.f., are grown as an ornamental crop in greenhouse and shade house production in Florida and other regions of the United States. Downy mildew on impatiens was detected from numerous landscapes (Manatee, Hillsborough, Collier, Hendry, Broward, Palm Beach, and Miami-Dade counties) in the winter of 2012. Incidence reached nearly 100% on many affected landscape plantings. Symptoms initially appeared as yellowing on the lower leaves and were typically vein-delineated, although in some cases the entire leaf was affected. Diseased plants later wilted and infected leaves abscised from the stem. A white, downy growth was apparent on the abaxial leaf surface. Microscopic observation revealed coenocytic mycelium with sporangiophores that were hyaline, thin-walled, and had slightly swollen bases. Branches of sporangiophores were monopodial and formed right angles to the supporting branches. Sporangia were hyaline and obvoid with a single pore on the distal ends that was mostly flat. Sporangia measured 19 to 22.5 × 13 to 17 μm. Oospores were observed in stem and leaf tissue. Leaves of 10 potted impatiens plants, I. walleriana 'Super Elfin XP Coral' and 'Super Elfin XP White,' were inoculated with a suspension containing 1 × 105 sporangia/ml and sprayed till runoff (approximately 20 ml per plant) with a handheld pressurized Ulva sprayer. Plants were maintained outside in a shade house under 73% shade where the daytime temperatures averaged 24°C and RH averaged 74% and nighttime temperature averaged 18°C with an average of 91% RH. Ten non-inoculated impatiens plants served as controls. After 10 days, symptoms typical of downy mildew occurred on 100% of the inoculated impatiens plants and sporulation was confirmed microscopically. The non-inoculated control plants remained healthy. The 5' end of the large ribosomal subunit gene (762 bp) from two isolates, one collected in Hillsborough County and one from Miami-Dade County, was amplified by PCR (primers NL1-GCATATCAATAAGCGGAGGAAAAG and NL4-GGTCCGTGTTTCAAGACGG) and sequenced bi-directionally (1,2,3). The consensus sequence from both isolates was identical and it was deposited into GenBank (Accession No. JX217746). Sequence data matched (99% homology) with Plasmopara obducens reported on I. walleriana in Europe and Australia (1,2). To our knowledge, this is the first report of downy mildew on I. walleriana in Florida (4). The disease has made a major impact on impatiens in landscapes throughout Florida and will likely continue to affect future production.

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

Impatiens downy mildew (Plasmopara obducens (J. Schröt.) J. Schröt. (syn Peronospora obducens) was first reported in the United States in 2004, but widespread outbreaks were observed throughout North America in 2011 (5). In June 2012, symptoms, including severe defoliation while plants retained upright stems, were observed on approximately 100 landscape impatiens (Impatiens walleriana Hook.f.) in Franklin County in central Kentucky. All plants in the landscape were affected. Plants were primarily defoliated and remaining leaves were stunted, mottled, and chlorotic with edges curled downward; no flowers were present. Under examination with a dissecting microscope, white downy fungal growth was observed. Closer examination confirmed that the growth consisted of colorless sporangiophores that were mainly unbranched, straight, and rigid (1,3). Sporangiophores consisted of apical branches attached at right angles to main axes, ranging from 67.2 to 89.9 μm long (1). Sporangia were ovoid and hyaline, measuring 11.2 to 13.3 μm × 8.2 to 10.7 μm (3). No oospores were observed. Pathogenicity tests were performed by inoculating 20 to 40 leaves on three plants each of the cvs. Dazzler and Super Elfin with suspensions of 1 × 105 sporangiophores per ml in sterile distilled water. Sporangia were obtained by washing infected leaves with sterile distilled water, and inoculations were completed by spraying leaves until runoff. Plants sprayed with sterile water served as controls. Plants were covered with black plastic bags for 48 h and then maintained under fluorescent lights for 10 days at room temperature (22 to 25°C). Sporangiophores were recovered from inoculated plants after 10 days, and morphology matched original inoculum; symptoms included chlorotic, downward curling leaves with sporulation on the undersides. Non-inoculated plants did not develop symptoms after 21 days. Molecular identification of the pathogen was conducted using three leaves from one plant from each cultivar. PCR was conducted by amplifying the large ribosomal subunit DNA using primers NL-1 and NL-4 (2). Amplicons of 762 to 691 bp were produced from diseased plant tissue that contained visible sporangiophores, and the bands were extracted from the gel and purified. Sequence results confirmed 100% similarity to accessions from Florida (GenBank Accession No. JX217746.1) and Ohio (JX142134.1) and 99% similarity to amplicons reported from Serbia (HQ246451.1) and UK (AY587558.1). This is believed to be the first report of downy mildew infecting impatiens in Kentucky.

First Report of Downy Mildew (Hyaloperonospora camelinae) on Camelina sativa in Florida.

Camelina sativa (L.) Crantz, Brassicaceae, whose common name is Crantz-large-seeded false flax, is an annual oilseed species. It is grown as a forage and biofuel crop in Europe and North America. False flax is an ideal low-input crop for biodiesel production, because of its low requirements for nitrogen fertilizer and pesticides. Production costs of this crop are substantially lower than those of many other oilseed crops such as rapeseed, corn, and soybean. It is an excellent rotation crop and can reduce disease and insect and weed pressure in wheat fields. During the spring of 2011, commercial and research plantings of C. sativa cultivar Calena in Liberty and Gadsden counties in north Florida developed symptoms typical of downy mildew. In spring of 2012, the same symptoms were observed in experimental plantings of false flax. A white downy mold was observed on the upper third portion of the plants, on the upper stem internodes, and on the developing seed. The affected stems exhibited a twisted growth. Conidiophores had main trunks with dichotomous branches terminating in slender curved tips. Conidia were ovoid and 28 to 45 (mean 36) μm long and 22 to 38 (mean 30) μm wide. Conidiophores were branched (three to four branches, each with six to eight curved tips) and ranged from 107 to 236 μm long and 5 to 14 μm wide. Mycelium was obtained directly from diseased plants for DNA extraction (Qiagen DNeasy Plant Mini Kit, Valencia, CA). Primers ITS1 and ITS4 were used for PCR amplification (4). The PCR product was sequenced bidirectionally with the PCR primers. A consensus nucleotide sequence (Accession JQ997103) was compared to those in the NCBI GenBank database using a BLAST search. The sequence was 99% similar to sequence from Hyaloperonospora camelinae (Gäum.) Göker, Voglmayr, Riethm, M. Weiss & Oberw. (Accession AY198249.1) with a 95% query coverage (1). Pathogenicity was established by applying white conidial masses of downy mildew from field samples to stems of 4-week-old plants grown in pots in a greenhouse maintained at 25 ± 2°C. Noninoculated plants maintained under the similar conditions served as control. Symptoms and signs of downy mildew developed after 14 days only on inoculated plants. Downy mildew constitutes a serious threat to the cultivation of C. sativa in Florida because of the humid climate favoring disease development. Diseased plants may reduce yield and disease management would increase production costs. H. camelinae was previously reported on C. sativa in Oregon, Minnesota, Montana (3), and Nebraska (2). To the best of our knowledge, this is the first report of downy mildew caused by H. camelinae on C. sativa in Florida.