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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Citrus and Other Subtropical Products Research » Research » Publications at this Location » Publication #342012
Title: Development of a sensitive real-time PCR detection method for citrus tatter leaf virus

Author
item KUNTA, MADHURABABU - Texas A&M University
item PARK, JONG-WON - Texas A&M University
item McCollum, Thomas
item GONZALEZ, MARISSA - Texas A&M University
item VEDASHARAN, PALLAVI - Texas A&M University
item DA GRACA, JOHN - Texas A&M University

Submitted to: Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2017
Publication Date: 3/19/2018
Citation: Kunta, M., Park, J., McCollum, T.G., Gonzalez, M., Vedasharan, P., Da Graca, J.V. 2018. Development of a sensitive real-time PCR detection method for citrus tatter leaf virus. Journal of Plant Pathology. 100:67. https://doi.org/10.1007/s42161-018-0029-7.
DOI: https://doi.org/10.1007/s42161-018-0029-7

Interpretive Summary: Citrus is plagued by a number of graft transmissible pathogens and it is essential that citrus be certified free of such pathogens before being used for propagation. Citrus tatter leaf virus (CTLV) is a graft-transmissible pathogen that causes the tatter leaf disease of citrus and graft-incompatibility between scion and trifoliate orange or some trifoliate hybrid rootstocks which can result in serious economic losses. There is a need for a highly sensitive, reliable high throughput protocol that can be used in routine diagnostic tests for citrus budwood certification programs. Based on deoxyribonucleic acid sequence (DNA) analysis of six citrus tatter leaf virus strains, a unique highly conserved region of the viral genome was identified. A new set of primers and TaqMan® probe were designed based on this nucleic acid sequence of the citrus tatter leaf virus genome a new assay was developed. This new assay provides a reliable, rapid and sensitive assay for the detection of citrus tatter leaf virus and will be of value in citrus budwood certification programs to ensure that trees are free of this virus.

Technical Abstract: Citrus tatter leaf virus (CTLV) is a graft-transmissible capillovirus that causes the tatter leaf disease of citrus and tissue incompatibility between scion and trifoliate orange or some trifoliate hybrid rootstocks which can result in serious economic losses. Although several CTLV detection protocols have been described, there is a need for a highly sensitive and high throughput protocol that can be used in routine diagnostic tests for virus-free budwood certification programs. To address this need, a TaqMan® chemistry based real time reverse transcription-polymerase chain reaction (real time RT-PCR) assay was developed to detect CTLV from citrus plants. The nucleotide sequence analysis based on six CTLV strains, whose full genomic sequences are available at NCBI GeneBank database, showed that 3' region of the viral genome is more conserved among different CTLV strains than the rest of the viral genome. A new set of primers and TaqMan® probe were designed from the 227 bp long fragment residing at the 3' end of CTLV genome for the detection of CTLV by real time RT-PCR. The newly designed primers for CTLV real time RT-PCR assay has ~98.6% amplification efficiency and showed 100% CTLV detection rate when they were tested for previously known CTLV-positive trees. The sequencing of 132 bp-long real time RT-PCR amplicon followed by BLASTn search showed that it has 94 % to 98 % sequence identity to various CTLV strains available at NCBI database, confirming the specificity of the assay to reliably detect CTLV.