First report of downy mildew caused by Hyaloperonospora parasitica on Siberian kale in Kentucky

Kale’s widely-touted status as a “superfood” resulted in a 57% increase in U.S. acres harvested from 2007 to 2012 (USDA-NASS 2012). Downy mildew, caused by an obligate oomycete, may kill kale seedlings outright or induce undesirable chlorotic and necrotic spots, affecting transplant sales and mature leaf marketability, respectively. In July 2015, symptoms of downy mildew were identified on greenhouse transplants and field plantings of “Siberian” kale (Brassica napus L. var. pabularia) in Lexington, KY. Symptoms included irregular chlorotic lesions with necrotic flecks; sporulation was visible with a 10× hand lens. Symptoms and signs only occurred on older Siberian leaves, while other Brassica grown in the same field were asymptomatic. Hyaline sporangiophores occurred only on abaxial surfaces, frequently in groups, and bore hyaline sporangia in clusters of 10 to 20. Sporangiophores had five to 10 proximal branches which bifurcated, then terminated in curled sterigmata, each with a single, spherical to semiovoid sporangium 13 to 22 μm in diameter. Disease incidence in the field was 50% and severity was 25%. Sporangia were dislodged from field-collected leaf tissue in 0.5% Tween 20. This 105 sporangia/ml solution was infiltrated into leaves of 7-day-old Siberian kale plants with a needleless syringe; controls were infiltrated with sterile distilled water. All plants were bagged and placed in a growth chamber maintained at 22 to 25°C, 12-h photoperiod. Necrotic lesions developed in 7 days, and sparse sporulation consistent with Hyaloperonospora parasitica was observed on two of three inoculated replicates after 14 days. Water-infiltrated plants did not develop sporulation, but slight necrosis consistent with wounding was present. In order to further fulfill Koch’s postulates and obtain enough spores for DNA sequence based identification, a baiting assay was conducted within the field. Three sets of 9-day-old Siberian kale seedlings were placed directly beneath symptomatic Siberian kale, and another three sets of seedlings were placed beneath asymptomatic B. oleracea cv. Toscano (a Lacinato type) kale, located ∼10 m apart in the field for 48 h in November 2015. Seedlings were bagged on retrieval and maintained in a growth chamber at 22 to 25°C, 12-h photoperiod. After 5 days, necrotic flecking and sporulation consistent with H. parasitica were identified on seedlings placed under symptomatic plants; seedlings placed under asymptomatic kale did not develop sporulation. Sporangia were washed from the baiting assay seedling tissue and concentrated by centrifugation, then DNA was extracted using the Qiagen DNEasy Plant kit with the following modifications: incubation temperature was 68°C for 10 min and AE buffer was reduced to 50 μl. The extracted DNA was used as template for a nested PCR by conducting the first round of amplification with primers DC6 and ITS4 (Cooke et al. 2000), which yielded a 1,200-bp amplicon. This product was used as template in subsequent PCR using ITS6 and ITS4 (Testen et al. 2012), resulting in a 584-bp fragment. The sequence was deposited in GenBank (KX231682). Comparison in GenBank BLAST indicated 99% coverage with 99% identity to H. parasitica, AY210986 (specimen voucher SMK 16040) and AY210985, amplified from B. campestris (Choi et al. 2003). This is the first report of downy mildew of kale in Kentucky (Farr and Rossman 2016), being a yield-limiting disease of a vegetable crop that is increasing in popularity among health-conscious consumers.