Identification and characterization of RB isolates of citrus tristeza virus

Authors: SAPONARI, MARIA - Consiglio Per La Ricerca In Agricoltura E L'Analisi Dell'economia Agraria, Unita Di Ricerca Per I S; GIAMPETRUZZI, ANNALISA - Bari University; SELVARAJ, VIJAY - Foreign Agricultural Service (FAS, USDA); MAHESHWARI, YOGITA - Foreign Agricultural Service (FAS, USDA); Yokomi, Raymond - Ray

Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 12/21/2018
Publication Date: 6/21/2019
Citation: Saponari, M., Giampetruzzi, A., Selvaraj, V., Maheshwari, Y., Yokomi, R.K. 2019. Identification and characterization of RB isolates of citrus tristeza virus. In: Catara A., Bar-Joseph M., Licciardello G., editors. Citrus Tristeza Virus. Methods in Molecular Biology. New York, NY: Humana Press. p. 105-126.

Interpretive Summary: Citrus tristeza virus (CTV) has caused decline and death of hundreds of millions of citrus trees on sour orange rootstock worldwide. This disease is controlled by grafting citrus on rootstocks of Poncirus trifoliata (trifoliate orange) or its hybrids. Resistance-Breaking (RB) strains of CTV are now found in multiple countries. The RB strain is a unique sequence group (genotype) that replicates and move systemically in P. trifoliata, a citrus species resistant to other known strains of CTV. A detailed step-by-step procedure is provided to identify and fully characterize the RB strain. The protocol involves: a) collecting tissue from a field source; b) conducting molecular and biological characterization; and c) determining full-length genome sequence and phylogeny. These methods provide a uniform procedure to describe and identify RB isolates, as well as other CTV isolates and strains. This information improves understanding of genetic diversity, strain interactions, cross-protection, diagnosis, and control of CTV.

Technical Abstract: Resistance-Breaking (RB) strains of Citrus tristeza virus (CTV) constitute a unique genotype named for the ability to replicate and move systemically in Poncirus trifoliata (trifoliate orange), a citrus species resistant to other known strains of CTV. This is important because P. trifoliata and its hybrids are used as rootstocks to control CTV. This book chapter provides a step-by-step procedure to identify and characterize CTV RB strains. Methods are provided on how to collect and homogenize tissue to perform molecular characterization by real time Reverse-Transcription Polymerase Chain Reaction (RT-qPCR), RT-PCR, and amplicon sequencing. To characterize disease symptom phenotype, a full description is provided on soil mixtures, fertigation, phytosanitation and greenhouse operation to grow and inoculate citrus plants in a standard biological indicator plant series. For confirmation, a putative RB isolate is graft-inoculated in trifoliate orange and virus is shown to replicate in leaves and bark. RB infectivity should be confirmed by inoculating other citrus species with tissue from infected P. trifoliata and evaluating for virus replication and systemic movement. Next Generation Sequencing of CTV small interfering RNAs is described to obtain full-length genome sequence. These methods provide a uniform procedure to identify RB CTV isolates as well as other CTV isolates. This information is important for understanding genetic diversity, strain interactions, cross-protection, diagnosis, and control of CTV.