First Report of Cotton Leafroll Dwarf Virus Infecting Cotton (Gossypium hirsutum) in Kansas

In the United States, cotton leafroll dwarf disease, caused by cotton leafroll dwarf virus (CLRDV, Polerovirus, Luteoviridae), was first reported from cotton (Gossypium hirsutum L.) fields in Alabama (Avelar et al. 2019) and later Mississippi (Aboughanem-Sabanadzovic et al. 2019) and Georgia (Tabassum et al. 2019). During a survey of cotton fields in Barber County, Kansas, in the 2019 growing season, cotton plants showed virus-like symptoms including leaf yellowing, discoloration, reddening, and short internodes with reduced or small boll sets. Disease incidence based on virus-like symptoms was ∼1 to 15% depending on the affected fields. Eleven symptomatic and seven apparently asymptomatic leaf samples from 18 independent cotton plants (cv. PhytoGen 210) were collected in commercial cotton fields and tested for the presence of the virus in the Plant Virology Lab at the University of Tulsa, Oklahoma. Total RNA was extracted from all 18 samples by the Spectrum Plant Total RNA Kit (Sigma-Aldrich) and was synthesized into complementary DNA (cDNA) using the Superscript IV Reverse Transcription Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. The polymerase chain reaction (PCR) was performed with primers CLRDV3675F and Pol3982R as described previously (Sharman et al. 2015) to amplify a specific PCR product of 310 bp of the ORF3-5 fragment of the CLRDV. cDNA of CLRDV for the specific fragment was obtained from the Plant Disease Diagnostic Lab at Auburn University and was used as a positive control. A PCR product of the predicted size (310 bp) was obtained from seven symptomatic cotton samples and the positive control, whereas no bands were present in four symptomatic and seven asymptomatic samples, as well as the PCR negative control. RT-PCR was repeated twice with these samples and obtained the same results as above. The PCR products of all positive samples were directly sequenced, and sequence comparison showed 97 to 99% identity with the CLRDV-Alabama isolate (MN071395). To further confirm the presence of CLRDV in these samples, two additional pair of primers—AL674F and AL1407R for partial P0/P1 (Avelar et al. 2019) and RdRpF2 and RdRpR1 for partial RdRp (Aboughanem-Sabanadzovic et al. 2019)—were used to amplify the 694- and 770-bp product of the CLRDV genome, respectively. The expected PCR bands were obtained from all seven symptomatic cotton plants but none from asymptomatic samples. PCR products of 694 and 770 bp from two samples (KS-7 and KS-10 isolates) were purified, cloned into pGEM-T Easy vector (Promega), and transformed into Escherichia coli DH5α cells (New England Bio Labs). At least three clones were sequenced using the Applied Biosystem 3130 Genetic Analyzer at the University of Tulsa. Sequences obtained in this study for partial P0/P1and RdRP genes of KS-7 and KS-10 isolates were deposited in GenBank (MN809925 to 28). Nucleotide sequence comparisons and BLASTn analysis of partial P1/P0 (MN809927 to 28) showed 98 to 99% identity with corresponding sequences of Alabama isolates (MH883237 and MN071395) and 92 to 93% identity with typical and 91 to 92% identity with atypical CLRDV isolates from Argentina and Brazil. Similarly, partial RdRP genes (MN809925 to 26) showed 94 to 98% identity with the corresponding sequences of available CLRDV isolates from Mississippi (MK512759, MK512762 to 64) and Alabama (MN071395) and 93 to 96% identity with typical and 92 to 95% identity with atypical CLRDV isolates from Argentina and Brazil. This is the first report of CLRDV infection and occurrence in cotton fields in Kansas. Cotton acreage in Kansas has increased since 2018 by 40% (NASS 2018), and the presence of CLRDV could be a detrimental disease for cotton production. Therefore, further studies are needed, including other cotton-growing counties in Kansas, to determine the distribution, epidemiology, vector transmission, and population structure of this virus, which would be helpful in the management of CLRDV.