First report of cotton leafroll dwarf virus in Florida

Authors: IRIARTE, FANNY - University Of Florida; DEY, KISHORE - Florida Department Of Agriculture And Consumer Services; SMALL, IAN - University Of Florida; CONNER, KASSIE - Auburn University; O'BRIEN, G - University Of Florida; JOHNSON, L - University Of Florida; SAVERY, C - Anchor Ag Solutions, Llc; CARTER, E - University Of Florida; SPRAGUE, D - University Of Florida; NICHOLS, ROBERT - Cotton, Inc; WRIGHT, DAVID - University Of Florida; MULVANEY, MICHAEL - University Of Florida; PARET, MATTHEW - University Of Florida

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/17/2020
Publication Date: 7/27/2020
Citation: Iriarte, F., Dey, K., Small, I., Conner, K., O'Brien, G.K., Johnson, L., Savery, C., Carter, E., Sprague, D., Nichols, R., Wright, D., Mulvaney, M., Paret, M. 2020. First report of cotton leafroll dwarf virus in Florida. Plant Disease. 104(10):2744. https://doi.org/10.1094/PDIS-10-19-2150-PDN.
DOI: https://doi.org/10.1094/PDIS-10-19-2150-PDN

Interpretive Summary: Cotton leafroll dwarf virus (CLRDV) was previously reported in Alabama, Mississippi, Georgia, Kansas, North Carolina, and Texas. Three CLRDV genotypes have been reported throughout the world: “typical”, “atypical”, and North American CLRDV-AL. A survey was conducted in FL for CLRDV following detection of the virus in sentinel plots by Auburn University. 232 symptomatic and asymptomatic cotton samples were collected from 12 FL counties. RNA was extracted from the samples, cDNA was synthesized, and PCR was performed targeting the P0-P1 region of the genome. 102 symptomatic samples tested positive for CLRDV and 130 asymptomatic samples tested negative. 19 representative isolates were sequenced and analyzed with BLASTn for identification. Sequences were 100% identical to CLRDV isolates from Alabama, 94% identical to “typical” isolates from Argentina and Brazil, and 92% identical to “atypical” isolates from Argentina and Brazil. Phylogenetic analysis was conducted and three genotypes were observed. Three representative sequences from this analysis were submitted to GenBank. 21 of the isolates were further analyzed with an additional set of primers targeting the coat protein of the virus for confirmation. All 21 samples tested positive and the products were sequenced. BLASTn analysis of resulting sequences revealed 100% identity to the CLRDV Alabama isolates and 99% identity to a Brazilian “typical” isolate. Phylogenetic analysis of these 21 CLRDV isolates resulted in two genotypes. Three representative sequences from the two genotypes were submitted to GenBank. CLRDV was detected in all 12 counties surveyed in Florida with high prevalence in Gadsden, Jefferson, and Santa Rosa counties. This is the first report of CLRDV in FL.

Technical Abstract: Cotton leafroll dwarf disease, caused by the Cotton leafroll dwarf virus (CLRDV), is an emerging disease of cotton (Gossypium hirsutum L.) in the United States. It was previously reported in Alabama, Mississippi, Georgia, Kansas, North Carolina, and Texas. To date, three CLRDV genotypes have been reported throughout the world: “typical”, “atypical”, and North American CLRDV-AL. During the 2019 growing season in several counties in north Florida, cotton seedlings and mature plants exhibited stunting, leaf distortion, yellowing, and/or reddening. A total of 232 symptomatic and asymptomatic plant samples from 12 Florida counties were tested for the presence of CLRDV. Total RNA was purified from lower stems, petioles, and/or leaves using the Zymo Quick-RNA Microprep kit. Complementary DNA was obtained using GoScript Reverse Transcription Mix. Polymerase chain reaction (PCR) was performed using CLRDV primers AL674F/AL1407R, to amplify the partial P1 (ORF1) and P0 (ORF0) region of the genome. CLRDV cDNA provided by K. N. Conner at Auburn University was used as the positive control. No cDNA was used as the negative control. An amplicon of the expected size, 733 bp, was obtained from all 102 symptomatic samples but not from 130 asymptomatic samples. Purified reverse transcription PCR products of 19 representative Florida isolates from the above-mentioned counties were subjected to Sanger sequencing. NCBI BLASTn analysis of the resulting sequences revealed 100% identity to CLRDV isolate from Alabama (MH883237), 94% identity to “typical” isolates from Argentina (GU167940) and Brazil (HQ827780), and 92% identity to “atypical” isolates from Argentina (KF359946) and Brazil (KF906261). Phylogenetic analysis using MEGA X was also conducted to support previous results. Three genotypes were observed. Three representative sequences from this analysis were submitted to GenBank: Escambia County (Co.) isolates ES312A (MT036373) and ES229 (MT036374), and Santa Rosa Co. isolate SR225 (MT036375). For 21 CLRDV isolates, an additional set of primers, CLRDV3675F/Pol3982R, was used to amplify the partial ORF3 of the virus encoding the coat protein. An amplicon of the expected size, 310 bp, was obtained from all these isolates. PCR products were purified and directly sequenced. NCBI BLASTn analysis of resulting sequences revealed 100% identity to the CLRDV Alabama isolate (MH883236) and 99% identity to a Brazilian “typical” isolate (HQ827780). Phylogenetic analysis using MEGA X of these 21 CLRDV isolates resulted in two genotypes. Three representative sequences from two genotypes were submitted to GenBank: Gadsden Co. isolate GA219 (MT036376), Jefferson Co. isolate JE223 (MT036377), and Santa Rosa Co. isolate SR225s (MT036378). CLRDV was detected in 12 counties in Florida with high prevalence in Gadsden, Jefferson, and Santa Rosa counties. More research is needed on the transmission, epidemiology, virulence, and host tolerance to this virus, which will help establish best management practices.