Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 12/19/2018
Publication Date: 6/21/2019
Citation: Yokomi, R.K. 2019. CTV vectors and interactions with the virus and host plants. In: Catara, A., Bar-Joseph, M., Licciardello, G., editors. Citrus Tristeza Virus. Methods in Molecular Biology. New York, NY: Humana Press. p. 29-53.
Interpretive Summary: Citrus tristeza virus (CTV) is comprised of a complex of strains. Therefore, citrus trees can become infected with multiple CTV strains over time. An important initial step in characterizing a CTV strain is to use aphid transmission to separate strains and eliminate other possible graft-transmissible agents. Toxoptera citricida or Aphis gossypii are efficient CTV vectors and should be used for transmission studies. CTV donor plants and healthy receptor plants must have young flush growth for virus acquisition and inoculation, respectively. Optimum virus acquisition and inoculation periods are 24h each. CTV detection and differentiation was performed by serology and sequence-specific Reverse-Transcription Polymerase Chain (RT-PCR) or Real-Time RT-PCR (RT-qPCR). Phenotype of the aphid-transmitted isolate is determined by symptom expression following graft inoculation of a citrus host range and examining symptom expression. Adherence to these methods allows determination of CTV isolate virulence free from mixtures of strains or other pathogens.
Technical Abstract: A standardized procedure using aphid transmission is presented to separate Citrus tristeza virus (CTV) from a mixture of CTV strains and/or co-infection from other citrus graft-associated pathogens. Either Toxoptera citricida (if available) or Aphis gossypii should be used as the aphid vector, as other citrus aphid species are inefficient vectors of CTV. Madam Vinous sweet orange was used as the CTV donor host for a virus acquisition access period (AAP) of 24 h and Mexican lime or Alemow was used as the receptor host for the inoculation access period (IAP) for 24 h. During IAP, a small number of aphids (1 to 10) should be used per receptor plant to optimize separation of CTV mixtures from the donor source. Enzyme-linked immunosorbent assay (ELISA) and real time Reverse-transcription Polymerase Chain Reaction (RT-qPCR) was used to detect and differentiate CTV isolates transmitted by the aphid. Phenotypes of the transmitted isolates were established by graft-inoculation in a citrus host range and by examining symptom expression. Adherence to these standardized methods will insure consistent results to define a CTV isolate free from mixtures of strains or other pathogens.