First Report of Cotton leafroll dwarf virus Associated with Cotton Blue Disease Symptoms in Alabama

Abstract

During 2017, virus-like symptoms were observed in cotton fields in six counties in coastal Alabama infested by the whitefly Bemisia tabaci (Genn.) and cotton aphid Aphis gossypii (Glover). Symptoms consisted of foliar distortion, curling, rolling, and bluish-green discoloration, vein-clearing, and shortened internodes that resulted in dwarfing of plants, and reduced boll set. Disease incidence ranged from 3-30% per field, resulting in ~560 kg/ha loss, and affecting 25% of the 50,585 ha cotton crop in Alabama, with a loss of ~50,000 bales valued at $19 million dollars. Leaves were collected from symptomatic cotton plants, Gossypium hirsutum L., in Barbour County, Alabama during late August and mid-September 2017. Total DNA and RNA were purified and subjected to DNA and RNAseq Illumina Hi-Seq 2500 (paired-end reads, 150 bp) shotgun sequencing. The de novo-assembled DNA contigs yielded no match to viral sequences available in the GenBank database. However, one RNA de novo assembled contig (103 reads) of 1143 bp [Accession no. MK015625], located at nt coordinates 3830-4972, was annotated as Cotton leaf roll dwarf virus (CLRDV) ‘atypical isolate’ from Argentina [KF359947], based on 97.1% similarity and 98% coverage (BLASTn, GenBank). The CLRDV belongs to the genus, Polerovirus (family, Luteoviridae), which are aphid-transmitted, positive-sense single stranded RNA viruses with a genome size of ~5.8 kb (Distéfano et al. 2010). To verify the Illumina results, the PCR primer pair, AL674F-5’-CCGTAGCGGTCATCGTCTTT/AL1407R-5’-TAACCCTGACACAGTGGGGA, was designed based on a region conserved in all six available CLRDV genome sequences [GenBank Accession numbers: GU167940, HQ827780, KF359946, KF359947, KF906260, KF906261], and used in parallel with the primers, CLRDV3675F and Pol3982R (Sharman et al. 2015), for reverse transcription-PCR (RT-PCR) amplification of total RNA purified from symptomatic cotton plants (n=3). An amplicon of the expected size was obtained using the primers, AL674F/1407R and CLRDV/Pol3982, at 733 bp [Accession no. MH883237] and 310 bp [Accession no. MH883236] in size, respectively. Bi-directional Sanger sequencing confirmed CLRDV presence, based on the BLASTn score of 94% and 99% similarity for the AL674F/1407R and CLRDV/Pol amplicon sequence, having a closest match to CLRDV from South America [GU167940] and [KF906261], respectively. Pairwise distance analysis (Muhire et al. 2014) of the CLRDV fragments from Alabama cotton plants with the analogous genome fragment from the six available CLRDV isolates from South America indicated 91.5-99.3% nt sequence identity, suggesting the US isolate is closely related, but not identical, to previously reported CLRDV isolates. The CLRDV, endemic to Africa and/or Asia-Pacific (Ray et al. 2016), was introduced into Brazil and Argentina during 2005 and 2010, respectively (Corrêa et al. 2005, Distéfano et al. 2010). Although A. gossypii is the only known CLRDV vector, and it was prevalent in symptomatic cotton fields during 2017, aphid transmissibility of the Alabama isolate has not been verified. Whether the CLRDV becomes established and causes economic losses to the U.S. cotton crop remains to be determined. To our knowledge, this is the first detection of the exotic CLRDV in U.S. cotton plantings.