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Complete Genome Sequence of Cotton Leafroll Dwarf Virus Isolated from Cotton in Texas, USA

Authors:
Akhtar Ali at University of Tulsa
Akhtar Ali
Samira Mokhtari at University of Tulsa
Samira Mokhtari
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Abstract

Cotton leafroll dwarf virus (CLRDV; genus, Polerovirus; family, Luteoviri-dae) was first described in Alabama. In this study, we present the complete genome (5,865 nucleotides) sequence of a CLRDV isolate (CS4) that was collected from cotton during the 2019 growing season in Texas.
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Complete Genome Sequence of Cotton Leafroll Dwarf Virus
Isolated from Cotton in Texas, USA
Akhtar Ali,
a
Samira Mokhtari
a
a
Department of Biological Science, The University of Tulsa, Tulsa, Oklahoma, USA
ABSTRACT Cotton leafroll dwarf virus (CLRDV; genus, Polerovirus; family, Luteoviri-
dae) was first described in Alabama. In this study, we present the complete genome
(5,865 nucleotides) sequence of a CLRDV isolate (CS4) that was collected from cot-
ton during the 2019 growing season in Texas.
Cotton (Gossypium hirsutum L.) is one of the leading cash crops in the United States
and is grown in more than 20 states (1). Cotton leafroll dwarf disease, caused by
Cotton leafroll dwarf virus (CLRDV; genus, Polerovirus; family, Luteoviridae), was first
reported from cotton in Alabama during the 2017 to 2018 growing season (2). Since
then, it has been reported in Mississippi (3) and Georgia (4). The virus causes leaf
yellowing and cupping, discoloration, reddening, and short internodes with reduced or
small boll sets. CLRDV has a single-stranded RNA (ssRNA) genome, and the first
complete genome from Alabama (GenBank accession number MN071395) has been
reported to be 5,865 nucleotides (nt) long (5).
Texas is one of the leading states for cotton production (1). To our knowledge, no
complete genome of any CLRDV isolate from Texas has been reported so far. In this
work, we report the complete genome sequence of a CLRDV isolate collected from a
cotton field in College Station, Texas.
During a survey in the 2019 growing season, leaf samples were collected from
cotton plants showing virus-like symptoms in College Station, Brazos County, Texas.
Total RNA was extracted from leaf samples using the Spectrum plant total RNA kit
(Sigma-Aldrich, St. Louis, MO) and subjected to reverse-transcription PCR (RT-PCR) with
the primers CLRDV3675F and Pol3982R as described previously (6) to amplify a specific
PCR product of 310 bp of the ORF3-5 fragment of the CLRDV. The expected PCR band
was obtained from symptomatic samples but not from the asymptomatic healthy
control. The PCR products from the positive samples were cleaned with Exosap-IT
(Affymatrix) and directly sequenced in both directions using an Applied Biosystems
3130 instrument.
Nucleotide BLAST searches showed that the sequences from all PCR products
obtained in this study had 96 to 98% nucleotide identity with the available CLRDV
isolates in the GenBank database.
One of the PCR-positive samples, labeled as College Station isolate 4 (CS4), was
selected for further molecular characterization of CLRDV. Total RNA extracted from the
CS4 isolate was submitted to the genomics facility at Oklahoma State University for
Illumina sequencing. A plant RNA library kit with Ribo-Zero (Illumina, USA) was used to
obtain an indexed plant ribosome-subtracted sequencing library according to the
manufacturer’s instructions, followed by shotgun sequencing using the NextSeq 500/
550 high-output kit v2.5 (Illumina, USA). The paired-end reads were cleaned of low-
quality or adapter sequences using the Illumina bcl2fastq software v2.2. A total of
24,425,349 trimmed paired-end reads with an average sequence length of 74.37 bp
were assembled using CLC Genomics Workbench v12.0 (Qiagen), resulting in 36,809
Citation Ali A, Mokhtari S. 2020. Complete
genome sequence of Cotton leafroll dwarf virus
isolated from cotton in Texas, USA. Microbiol
Resour Announc 9:e01587-19. https://doi.org/
10.1128/MRA.01587-19.
Editor Jelle Matthijnssens, KU Leuven
Copyright © 2020 Ali and Mokhtari. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Akhtar Ali,
akhtar-ali@utulsa.edu.
Received 9 January 2020
Accepted 2 April 2020
Published 16 April 2020
GENOME SEQUENCES
crossm
Volume 9 Issue 16 e01587-19 mra.asm.org 1
contigs. The resulting contigs were compared in a BLAST search using BLAST⫹v2.8.1
against the CLRDV reference sequence (GenBank accession number MN071395). De-
fault parameters were used for all software.
A 5,821-nt-long contig was assembled with an average coverage of 67⫻, represent-
ing the near-complete genome of CLRDV isolate CS4, missing only 44 nt at the 5=end,
compared to the CLRDV isolate AL reference sequence (GenBank accession number
MN071395). To obtain the complete genome of this isolate, the 5=and 3=ends of isolate
CS4 were also sequenced using the rapid amplification of cDNA 5=and 3=ends (RACE)
procedure according to the manufacturer’s instructions (TaKaRa Bio, Inc.), followed by
Sanger sequencing, which confirmed the missing 44 nt at the 5=end. Thus, the total
length of the CLRDV isolate CS4 genome was 5,865 nt. The G⫹C content of the
complete genome of CLRDV isolate CS4 is 49%. Nucleotide BLASTn searches showed
that CLRDV isolate CS4 shared 96% nucleotide and 97 to 98% amino acid sequence
identity with CLRDV isolate AL (MN071395). Based on the sequence comparison, CLRDV
isolate CS4 is closely related to CLRDV isolate AL.
Data availability. The complete genome sequence of CLRDV isolate CS4 was
deposited in GenBank under accession number MN872302. The raw data have been
deposited in the Sequence Read Archive (SRA) under BioProject accession number
PRJNA597539, part of BioSample number SAMN13677091.
ACKNOWLEDGMENTS
This work was supported by Cotton Incorporated through award 12-991. We thank
the Department of Biological Science and Office of Research and Sponsored Programs
at The University of Tulsa for supporting the publication charges for this work.
The Illumina sequencing work was carried out at the genomics facility of Oklahoma
State University (Stillwater, OK).
REFERENCES
1. Ali A, Haker K. 2017. Early warning and remedies for emerging pests in
cotton with emphasis on viruses. ICAC Rec 35:17–19.
2. Avelar S, Schrimsher DW, Lawrence K, Brown JK. 2019. First report of
Cotton leafroll dwarf virus associated with cotton blue disease symptoms
in Alabama. Plant Dis 103:592–592. https://doi.org/10.1094/PDIS-09-18
-1550-PDN.
3. Aboughanem-Sabanadzovic N, Allen TW, Wilkerson TH, Conner KN, Sikora
EJ, Nichols RL, Sabanadzovic S. 2019. First report of Cotton leafroll dwarf
virus in upland cotton (Gossypium hirsutum) in Mississippi. Plant Dis
103:1798. https://doi.org/10.1094/PDIS-01-19-0017-PDN.
4. Tabassum A, Bag S, Roberts P, Suassuna N, Chee P, Whitaker JR, Conner
KN, Brown J, Nichols RL, Kemerait RC. 2019. First report of Cotton leafroll
dwarf virus infecting cotton in Georgia, U.S.A. Plant Dis 103:1803. https://
doi.org/10.1094/PDIS-12-18-2197-PDN.
5. Avelar S, Ramos-Sobrinho R, Conner K, Nichols RL, Lawrence K, Brown JK.
2020. Characterization of the complete genome and P0 protein for a
previously unreported genotype of Cotton leafroll dwarf virus, an intro-
duced polerovirus in the USA. Plant Dis 104:780 –786. https://doi.org/10
.1094/PDIS-06-19-1316-RE.
6. Sharman M, Lapbanjob S, Sebunruang P, Belot J-L, Galbieri R, Giband
M, Suassuna N. 2015. First report of Cotton leafroll dwarf virus in
Thailand using a species-specific PCR validated with isolates from
Brazil. Australas Plant Dis Notes 10:24. https://doi.org/10.1007/s13314
-015-0174-1.
Ali and Mokhtari
Volume 9 Issue 16 e01587-19 mra.asm.org 2
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... 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. ...
... The first CLRDV complete genome sequence was reported five years later [17]. Most recently, several genomes of a new CLRDV variant, CLRDV-AL, were reported in the US [21,25,32,57]. In this study, eight CLRDV full-length genome sequences were determined by HTS and Sanger sequencing of isolates collected from commercially grown cotton plants in AL, FL, and TX. ...
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... The closest relatives of the 5870-nt viral sequences are the nine CLRDV isolates previously identified from Argentina, Brazil, and USA [14,[20][21][22][23]. All the nine available CLRDV complete genome sequences were retrieved from the GenBank Database, and pairwise sequence identity was analyzed using SDT1.2. ...
... Phylogenetic analysis of the complete genome sequences to reported typical species of the family Solemoviridae also revealed that the identified virus grouped with poleroviruses, but separated from members of the Enamovirus, Polemovirus, and Sobemovirus genera. A detailed BLASTn and phylogenetic analysis showed that this virus was closely related to CLRDV, a polerovirus that has been described to cause cotton blue disease in Argentina, Africa, Asia, Brazil, and the United States [14,20,21,[23][24][25]. The species demarcation criterion for the Polerovirus genus utilizes a >10% amino acid sequence difference in any of the viral products as a new species threshold [26]. ...
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... Cotton leafroll dwarf virus (CLRDV), a phloem-limited virus, is associated with the emerging cotton leafroll dwarf disease (CLRDD) in the United States. It was first reported from Alabama in 2019 (2) and subsequently from the major cotton-growing regions in the United States, including Florida (3), Georgia (4), Louisiana (5), Mississippi (6), South Carolina (7), and Texas (8). Symptoms of the disease include reddening of the leaves and petioles and drooling, crinkling, and deformation of the leaves ( Fig. 1A and B), and it has the potential to cause significant yield and economic losses. ...
... The CLRDV genome from Georgia characterized in this study was 5,868 bp long and encoded seven ORFs, as reported earlier for isolates from North and South America (8)(9)(10). It was 95 to 98% identical to the genome of other CLRDV isolates from the United States (Alabama, GenBank accession number MN071395; Texas, MN872302) and South America (KF359947, KF906261, KF906260, NC_014545, GU167940, and HQ827780) (Fig. 1C). ...
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... CLRDV is considered an emerging disease in cotton in the USA. Although the viral genome of individual isolates each from Alabama [29], Georgia [30], and Texas [31] have been sequenced, no information is available on the genetic diversity and population structure of the virus. In this study, the nearly complete genome of six isolates from Georgia and one isolate from Alabama were sequenced, representing diverse symptoms, plant growth stage, and geographical locations to understand the genome variability of CLRDV in the USA. ...
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Cotton leafroll dwarf virus (CLRDV) is an emerging virus in cotton production in Georgia and several other Southeastern states in the USA. To better understand the genetic diversity of the virus population, the near complete genome sequences of six isolates from Georgia and one from Alabama were determined. The isolates sequenced were 5,866 nucleotides with seven open reading frames (ORFs). The isolates from Georgia were >94% identical with other isolates from the USA and South America. In the silencing suppressor protein (P0), at amino acid position 72, the isolates from Georgia and Alabama had a valine (V), similar to resistant-breaking ‘atypical’ genotypes in South America, while the Texas isolate had isoleucine (I), similar to the more aggressive ‘typical’ genotypes of CLRDV. At position 120, arginine (R) is unique to Georgia and China isolates, but absent in Alabama, Texas and South American isolates. Ten potential recombinant events were detected in the isolates sequenced. An increased understanding of CLRDV population structure and genetic diversity will help develop management strategies for CLRDV in the USA cotton belt.
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Abstract Background: Cotton leafroll dwarf disease is caused by the Cotton leafroll dwarf virus (CLRDV),which belongs to the genus Polerovirus and family Luteovridiae. CLRDV was first reported from Alabama in 2018 and poses a new challenge to cotton production in the United States (US). The purpose of this work was to detect and determine the distribution of CLRDV in three mid-southwestern states (Kansas, Oklahoma, and Texas) during the 2019 growing season. Results: During the first surveys, more than 104 leaf samples showing virus-like symptoms were collected from cotton plants in different areas located in the above three states. Symptoms observed on cotton plants included leaf yellowing, discoloration, reddening, and short internodes with reduced or small boll sets. Total RNA was extracted from individual samples and tested by reversetranscription polymerase chain reaction (RT-PCR) using virus-specific primers of CLRDV. Nearly 50% of the samples were positive which confirmed the presence of CLRDV in samples collected from all three states. However, the disease incidence varied from field to field. Conclusions: These Results indicate the occurrence and distribution of CLRDV in different regions of Kansas, Oklahoma, and Texas and the virus could pose a threat to the production of cotton in the future. Keywords: Cotton, Etiology, Cotton blue disease, Epidemiology, Polerovirus, Virus etiology, Cotton leafroll dwarf virus
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The identification of alternate hosts that can act as virus inoculum sources and vector reservoirs in the landscape is critical to understanding virus epidemics. Cotton leafroll dwarf virus (CLRDV) is a serious pathogen in cotton production and is transmitted by the cotton/melon aphid, Aphis gossypii, in a persistent, circulative, and non-propagative manner. CLRDV was first reported in the United States in Alabama in 2017, and thereafter in several cotton-producing states. CLRDV has since established itself in the southeastern United States. The role of alternate hosts in CLRDV establishment is not clear. Fourteen common plant species in the landscape, including crops, weeds, and ornamentals (cotton, hollyhock, marshmallow, country mallow, abutilon, arrowleaf sida, okra, hibiscus, squash, chickpea, evening primrose, henbit, Palmer amaranth, and prickly sida) were tested as potential alternate hosts of CLRDV along with an experimental host (Nicotiana benthamiana) via aphid-mediated transmission assays. CLRDV was detected following inoculation in hibiscus, okra, N. benthamiana, Palmer amaranth, and prickly sida by RT-PCR, but not in the others. CLRDV accumulation determined by RT-qPCR was the highest in N. benthamiana compared with cotton and other hosts. However, aphids feeding on CLRDV-infected prickly sida, hibiscus, and okra alone were able to acquire CLRDV and back-transmit it to non-infected cotton seedlings. Additionally, some of the alternate CLRDV hosts supported aphid development on par with cotton. However, in a few instances, aphid fitness was reduced when compared with cotton. Overall, this study demonstrated that plant hosts in the agricultural landscape can serve as CLRDV inoculum sources and as aphid reservoirs and could possibly play a role in the reoccurring epidemics of CLRDV in the southeastern United States.
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First report of Cotton leafroll dwarf virus in Thailand using a species-specific PCR validated with isolates from Brazil
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Full-text available
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Partial virus genome sequence with high nucleotide identity to Cotton leafroll dwarf virus (CLRDV) was identified from two cotton (Gossypium hirsutum) samples from Thailand displaying typical cotton leaf roll disease symptoms. We developed and validated a PCR assay for the detection of CLRDV isolates from Thailand and Brazil.
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Characterization of the complete genome and P0 protein for a previously unreported genotype of cotton leafroll dwarf virus, an introduced polerovirus in the USA
Article
  • Oct 2019
  • PLANT DIS
Virus-like disease symptoms consisting of leaf cupping, shortened internodes, and overall stunting were observed in commercial cotton fields in Alabama in 2017 to 2018. To determine the complete genome sequence of the suspected causal polerovirus, symptomatic leaf samples were collected in Macon County, Alabama, and subjected to Illumina RNA sequencing. Based on BLASTn analysis, the Illumina contig of 5,771 nt shared the highest nucleotide identity (approximately 95%) with members of the species Cotton leafroll dwarf virus (CLRDV) (genus Polerovirus; family Luteoviridae) from Argentina and Brazil. The full-length viral genome sequence was verified by reverse transcription (RT)-PCR amplification, cloning, and Sanger sequencing. The complete CLRDV genome of 5,865 nt in length shared 94.8 to 95.2% nucleotide identity with six previously reported CLRDV isolates. The genome of the CLRDV isolate amplified from Alabama samples (CLRDV-AL) has seven predicted open reading frames (ORFs). Viral proteins 1 to 5 (P1 to P5) shared 91.9 to 99.5% amino acid identity with the six CLRDV isolates from Argentina and Brazil. However, P0, the suppressor of host gene silencing, shared 82.4 to 88.5% pairwise amino acid identity with the latter CLRDV isolates. Phylogenetic analysis of the seven full-length CLRDV genomes resolved three sister clades: CLRDV-AL, CLRDV-typical, and CLRDV-atypical, respectively. Three recombination events were detected by the recombination detection program among the seven CLRDV isolates with breakpoints occurring along the genome. Pairwise nucleotide identity comparisons of ORF0 sequences for the three CLRDV-AL field isolates indicated that they were >99% identical, suggesting that this previously unknown CLRDV genotype represents a single introduction to Alabama.
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