First Report of Areolate Mildew of Cotton caused by Ramulariopsis pseudoglycines in Alabama

In September of 2024, green to yellowish, irregular lesions (3-4 mm wide) were observed on the adaxial leaf surface, with a dense white powdery fungal growth on the abaxial side of the leaf in commercial and research cotton ( Gossypium hirsutum L.) fields in central and south Alabama. Symptomatic leaves were collected from ‘Deltapine 2127 B3XF’ cotton, placed in plastic bags, and transported to the laboratory on ice. Small pieces (~1 cm) of infected tissue were placed in a sterile Eppendorf tube with 1% Tween 20, macerated using a sterile micropestle, and serially diluted seven-fold. The sixth- and seventh-fold dilutions were plated on modified V8 agar supplemented with chloramphenicol (75 mg/L) and streptomycin sulfate (125 mg/L) (Conner 2023) and incubated in dark at 28°C. Microscopic examination of white fungal growth revealed morphology consistent with Ramulariopsis spp. (Videira et al. 2016). Gray fungal colonies (7–10 mm diameter) with raised, lumpy surfaces and dark margins were observed within 14 days. Conidia were 9.9 – 26.5 µm long and 1.8 – 4.3 µm wide. Occasionally, fungal cultures developed pinkish-white growth after 45 days of storage at 4°C. Colonies were aseptically transferred using hyphal-tip culturing to modified V8 medium twice to obtain a pure culture and transferred to a 70% glycerol solution at -80°C for long-term storage. DNA was extracted using the CTAB method (Saghai-Maroof et al. 1984) from a four-week-old culture by lyophilizing fungal growth cut from modified V8 agar. A representative isolate ALWN1-2024 was selected for species identification using the internal transcribed spacer (ITS1F and ITS4) (White et al. 1990), translation elongation factor 1-α ( tef1-α ), histone H3 ( his3 ) and RNA polymerase II second largest subunit ( rpb2 ) regions (Videira et al. 2016). Consensus sequences were deposited in GenBank (accession nos.: ITS, PZ378719; TEF1-α, PZ158317; HIS3, PZ158314; RPB2, PZ158316). A BLASTn query of NCBI GenBank showed 100% identity of the 559-bp (ITS), 99.71% identity of the 696-bp ( tef1-α ), 100% identity of the 353-bp ( his3 ) and 99.9% identity of the 998-bp ( rpb2 ) sequences from ALWN1 2024 with R. pseudoglycines CPC 18242, CPC 20036 and CPC 18241 (type culture; Videira et al. 2016). Koch’s postulates were completed by growing individual colonies of ALWN1-2024 on V8 medium incubated at 28°C for four weeks in dark. Fungal colonies were aseptically cut from V8 medium and smeared onto leaves of 45-day-old cotton plants previously sprayed with 1% Tween20 solution. Three plants sprayed with distilled water and 1% Tween20 were used as inoculated controls. Plants were covered with plastic bags to maintain humidity and incubated in the greenhouse at 28°C under 80% relative humidity with a 12-h/12-h light/dark cycle. Plants inoculated with ALWN1-2024 developed characteristic necrotic lesions and white powdery growth within 14 days, whereas control plants remained asymptomatic. When re-isolated, colony and conidial morphology and DNA sequence (ITS, tef1-α , his3 , and rpb2 ) matched the original field isolate. R. gossypii and R. pseudoglycines cause areolate mildew on cotton (Videira et al. 2016). Although R. gossypii was first reported in Auburn, Alabama in 1890 (Atkinson 1890), this is the first report of R. pseudoglycines in Alabama. Both species have been reported in Brazil (da Silva et al. 2023) and in the United States R. pseudoglycines has recently been identified in Mississippi and Georgia (Conner et al. 2023; Madrid et al. 2025).