First Report of Cotton Leafroll Dwarf Virus in Cotton Fields of South Carolina

Cotton leafroll dwarf virus (CLRDV, family Luteoviridae, genus Polerovirus) was recently reported in cotton (Gossypium hirsutum L.) plants affected by cotton leafroll dwarf disease (CLRDD) in the United States (Alabama, Georgia, Kansas, Mississippi, and Texas) (Aboughanem-Sabanadzovic et al. 2019; Alabi et al. 2020; Ali and Mokhtari 2020; Avelar et al. 2019; Tabassum et al. 2019). From July to September 2019, a CLRDV survey was conducted in 10 counties (Aiken, Anderson, Barnwell, Calhoun, Chester, Clarendon, Darlington, Florence, Sumter, and York) representing the cotton-growing areas in South Carolina. The CLRDD-like symptoms observed in the fields included pronounced rugosity, curling, and distortion of leaves, reddening of the petioles and leaves, brittle leaves when handled, shortened internodes and stunting, and a reduction in boll set. The disease incidence across 17 fields was estimated to range from 5 to 25% based on visual assessment of symptoms. From each county, leaf/petiole samples from two to four cotton plants showing CLRDD-like symptoms were collected from one to two fields and tested in the lab for the presence of CLRDV by RT-PCR. RNA was extracted from each plant sample using Qiagen RNeasy Plant Mini kits (Qiagen, Germantown, MD) following the manufacturer’s recommendations. The cDNA was synthesized using a SuperScript IV first-strand synthesis system (ThermoFisher Scientific, Waltham, MA) and amplified with CLRDV-specific PCR primers CLRDV3675F/Pol3982R (Sharman et al. 2015) targeting a 310-bp segment of ORF3 to ORF5. Positive (cDNA of CLRDV, Elmore County, AL) and negative (nontemplate) controls were included in each run, and PCR products were examined by gel electrophoresis. Twenty-six samples (78.8%) out of 33 tested produced the expected PCR bands. The positive samples were derived from cultivars DeltaPine 1646 B2XF and Phytogen 430 W3FE, and they spanned all 10 surveyed counties. All positive samples were further amplified with three additional primer pairs specifically designed to detect CLRDV: CLRDV-RdRpF2/CLRDV-RdRpR1 (Aboughanem-Sabanadzovic et al. 2019), amplifying a 770-bp region of ORF1 to ORF2; AL674F/AL1407R (Avelar et al. 2019), amplifying a 733-bp portion of ORF0 to ORF1; and CLPOF/CLPOR (Cascardo et al. 2015), amplifying a 783-bp fragment spanning ORF0. DNA bands of the expected sizes were obtained with each primer pair as examined by agarose gel electrophoresis. The PCR products for one Barnwell County sample (all four primer pairs) and the CLPOF/CLPOR-specific products from one sample each from Anderson and Sumter Counties were directly sequenced. After removing the primer sequences, the partial CLRDV sequences from the three samples were deposited in GenBank (MN651102 to MN651107). All six sequences showed very high nucleotide identities (98.9 to 100%; 98 to 100% query coverage) to corresponding sequences of CLRDV isolates from Alabama (MN071395 and MN883237) based on BLASTn analysis. To our knowledge, this is the first report of CLRDV infecting cotton in South Carolina. CLRDD could negatively impact the cotton industry in South Carolina and other cotton-producing states due to its potential to reduce yield. Future research is needed to quantify the impact of the virus on cotton production in the United States and to identify strategies for minimizing the potential economic losses.