First Report of Cotton Leafroll Dwarf Virus in Cotton Plants Affected by Cotton Leafroll Dwarf Disease in North Carolina

Abstract

During the 2019 growing season, cotton (Gossypium hirsutum L.) plants in North Carolina were observed to have virus-like symptoms including leaf rugosity, leaf curling, and shortened upper internodes, consistent with Cotton Leafroll Dwarf Disease (CLRDD) associated with cotton leafroll dwarf virus (CLRDV, family: Luteoviridae, genus: Polerovirus) (Avelar et al. 2020). Sentinel plots planted on June 17, 2019 at the Sandhills Research Station in Moore County, NC exhibited CLRDD symptoms and disease incidence was estimated at 75% on a 0.1-ha field. Cotton aphids (Aphis gossypii Glover), the reported vector of CLRDV (Michelotto and Busoli 2007; Heilsnis et al. 2020; McLaughlin et al. 2020), were detected on plants throughout the growing season. Samples (n = 24) were collected from sentinel plots on September 20, 2019 to test for CLRDV through RT-PCR. Each sample represented five symptomatic plants collected from a single plot. Total RNA was extracted from the petiole tissue of each sample using Qiagen RNeasy Plant Mini kit (Qiagen, Germantown, MD), following the manufacturer’s recommendations. The cDNA was synthesized using 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 genome segment of ORF3-5. Seven CLRDV positive samples were further amplified with two additional primer sets specifically designed to detect CLRDV: AL674F/AL1407R (Avelar et al. 2019), targeting a 733 bp portion of the ORF0-ORF1, and CLPOF/CLPOR (Cascardo et al. 2015), amplifying a 880 bp fragment spanning the ORF0. Nucleotide BLAST searches showed that the best matches for all sequences in this study were derived from CLRDV with a range of pairwise identity of 99.2¬¬ - 100% for all genome segments. From symptomatic samples (n = 14 ), the isolated virus was confirmed as CLRDV in several cotton varieties, including Deltapine 1646 B2XF (GenBank Accessions MN958131 (ORF3-5), MN958147 (ORF0-ORF1), MN958138 (ORF0), MN958133 (ORF3-5), MN958145 (ORF0-ORF1), MN958140 (ORF0)), Phytogen 480 W3FE (MN958134 (ORF3-5), MN958144 (ORF0-ORF1), MN958141 (ORF0), Stoneville 5471 GLTP (GenBank Accessions MN958135 (ORF3-5), MN958143 (ORF0-ORF1), MN958142 (ORF0)), and Nextgen 5711 B3XF (GenBank Accessions MN958130 (ORF3-5), MN958148 (ORF0-ORF1), MN958137 (ORF0), MN958132 (ORF3-5), MN958146 (ORF0-ORF1), MN958139 (ORF0), and MN958136 (ORF3-5). CLRDD is a newly named disease of cotton in the United States that was first reported in Alabama (Avelar et al. 2019), Georgia (Tabassum et. al. 2019), Mississippi (Aboughanem-Sabanadzovic et. al. 2019), and Texas (Alabi et al. 2019). While the virus has been reported with variable impacts, losses can be extensive in some fields that are affected (Avelar et al. 2019). North Carolina produced over one million bales of cotton in 2019 and given reported losses among fields with high disease incidence, CLRDV has the potential to significantly reduce cotton yield and quality for the state if it becomes widespread.

Genome analysis of cotton leafroll dwarf virus reveals variability in the silencing suppressor protein, genotypes and genomic recombinants in the USA

“…In addition to Africa and South America, CLRDV has also been reported in Timor-Leste [ 14 ], Thailand [ 15 ], India [ 16 ], and from Soybean aphid ( Aphis glycines ) in China [ 17 ]. Recently, there have been several reports on the detection of CLRDV in the USA: Arkansas [ 18 ], Alabama [ 19 ], Florida [ 20 ], Georgia [ 21 ], Kansas [ 22 ], Louisiana [ 23 ], Mississippi [ 24 ], North Carolina [ 25 ], Oklahoma [ 26 ], South Carolina [ 27 ] and Texas [ 28 ]. Since the first report of CLRDV, it has been detected in all the major cotton-growing counties in Georgia with 0–30% of disease incidence.…”

Cotton Leafroll Dwarf Virus US Genomes Comprise Divergent Subpopulations and Harbor Extensive Variability

“…Cotton is one of the most economically important crops worldwide, and significant yield loses caused by cotton leafroll dwarf virus (the causal agent of cotton blue-and cotton blue-like diseases) have been reported in Brazil and Argentina [9,10,12,58,59], East Timor [13,14], India [15], Thailand [16], and, most recently, in the US [19,32]. Since 2016-2017, CLRDV has emerged and spread quickly from those sites in cotton-growing states in the southern and south-central US [19][20][21][22][23][24][25][26][27][28][29][30]32]. Despite the potential for economic importance of CLRDV, little is known about CLRDV genome-level variability and the forces that may be acting upon it.…”

“…Molecular characterization of partial and complete genome sequences from similarly symptomatic cotton plants revealed the presence of an aphid-transmitted polerovirus named cotton leafroll dwarf virus (CLRDV) [12,14,15,17,18]. Since the first report of CLRDV infecting cotton in Alabama, US, during 2016-2017 [19], CLRDV isolates have been reported from symptomatic cotton in Georgia, Mississippi, North Carolina, Texas, Arkansas, Florida, Louisiana, Oklahoma, and South Carolina [20][21][22][23][24][25][26][27][28][29][30]. Thus, CLRDV has become recognized as an emerging polerovirus [31,32].…”

A Guide to Grafting for Cotton (Gossypium Hirsutum L.) Virus Transmission and the Successful Transmission of Cotton Leaf Roll Dwarf Virus

“…To test for presence of the virus, three leaves were collected from the first node below the graft for a composite sample. Virus presence was confirmed via PCR (Thiessen et al 2020).…”