First report of areolate mildew of cotton, caused by in Mississippi.

In August of 2022, cotton (Gossypium hirsutum L.) growing in several north central Mississippi counties was observed to exhibit yellowish lesions on the adaxial leaf surface with white powdery fungal growth on the corresponding abaxial surface. By the end of the 2022 growing season, 19 Mississippi counties were observed to have infected cotton. Symptomatic leaves were collected from affected plants, sealed in plastic freezer bags, stored on ice in a cooler, and transported to the laboratory. Prior to isolation, the pathogen was microscopically examined and determied to be morphologically similar to the description of Ramulariopsis spp. (Ehrlich and Wolf 1932). Using a sterile needle, conidia were transferred to V8 medium amended with chloramphenicol (75 mg/liter) and streptomycin sulfate (125 mg/liter) and incubated in the dark at 25°C. After 14 days, the colony diameter was measured, and morphological characteristics were consistent with previous descriptions (Videira et al. 2016; Volponi et al. 2014). On V8 medium, the 7 mm diameter colonies grew raised, lumpy, and lobed, with iron-grey coloration. The mycelia were hyaline, septate, branched, and 1 to 3 μm in diameter. Conidia ranged from 2.8 to 25.6 μm in length and 1.0 to 4.9 μm in width (x = 12.8 × 3.1 μm; n = 20). Pure cultures were obtained on V8 medium, and DNA was extracted from a 14-day-old-culture. TW098-22, a representative isolate, was amplified, and sequenced targeting the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF 1-α), and actin (ACT) genes as described by Videira et al. (2016). The consensus sequences were deposited in GenBank (accession no. OQ653427, OR157986, OR157987). BLASTn query of the NCBI GenBank showed 100% identity of the 483-bp (ITS) and 706-bp TEF 1-α sequences from TW098-22 with Ramulariopsis pseudoglycines CPC 18242 (type culture; Videira et al. 2016). Koch's postulates were performed after multiplying individual colonies by streaking on V8 medium as above. Culture plates were subsequently incubated at 25°C for 14 days in the dark. Colonies were aseptically transferred to 50 ml centrifuge tubes containing 50 ml of autoclaved reverse osmosis (RO) water amended with Tween 20 (0.01%). The resulting inoculum suspension was adjusted to 13.5 × 105 conidia/ml using a hemocytometer. The foliage of five, 25-day-old cotton plants were sprayed with 10 ml of the suspension and a plastic bag was placed over each plant to maintain humidity for 30 days. Five plants were sprayed with sterilized RO water to serve as controls. Plants were incubated in a growth chamber at 25°C and ~70% relative humidity with 16:8 h of light:dark. Thirty days post-inoculation, foliar symptoms and signs were observed on all inoculated plants including small necrotic lesions and white powdery growth. Control plants remained asymptomatic. The trial was repeated. When reisolated, the colony and conidia morphology and DNA sequence (ITS) were consistent with the description of the original field isolate. Areolate mildew of cotton can be caused by two species of Ramulariopsis: R. gossypii and R. pseudoglycines (Videira et al. 2016). The two species have been reported in Brazil (Mathioni et al. 2021); however, this is the first report of R. pseudoglycines in the United States. In addition, even though areolate mildew has previously been reported from much of the southeastern U.S. (Anonymous 1960), the report herein serves as the first description of R. pseudoglycines in Mississippi and U.S. cotton.