Abstract

RODRIGUEZ, Patricia; ROMERO DE PEREZ, Gloria  and  GUZMAN, Mónica. In situ serological, microscopy and hybridization detection of CVT. Rev. colomb. biotecnol [online]. 2009, vol.11, n.1, pp.94-106. ISSN 0123-3475.

Citrus tristeza virus (CTV), is deleterious for citriculture and cause citrus tristeza disease. CTV infects all Citrus species causing the death of millions of trees. Main symptoms are quick decline (QD) and stem pitting (SP). In this work serological, molecular and microscopical CTV diagnose was done in Citrus aurantifolia or Tahití Lime (Citrus latifolia Tanaka) (LT) and citrus madurensis (Lour) or Calamondino (Ca) isolates. Petioles were tissue printing (IMI) and exposed to 3DF1+3CA5 monoclonal antibodies; they were then Elisa buffer extracted and exposed to MCA 13 monoclonal discriminant antibody in a double-antibody sandwich indirected enzyme-linked Inmunossorbent Assay (DASI-Elisa). Immunocapture-reverse transcriptase amplification (IC-RT-PCR) with specific mayor coat protein gene (CPG) primers were used on the Elisa buffer extracts as template. Optic and electron microscopy detection was carried out on transversal sections of petiole and were stained with Azure A or uranile acetate and plumb citrate, respectively. Digoxigenine marked mayor CPG CTV probes were used for in situ hybridization of petioles printing. All IC-RT-PCR, IMI and Elisa results were positive for LT and C too indicating the presence of severe viral variants. Light microscopy cytoplasm inclusions were detected in the phloem and accompanying cells, confirmed with IMI and in situ hybridization. Electronic microscopy analysis showed cellular abnormalities with changes in ultraestructure with big vacuoles presence that are characteristic of cytoplasmic viral infection. This is the first work that integrate all different diagnostic techniques in this two exotics citrus species.

Keywords : inmunoimpression; Elisa; hibrization; microscopy; CTV; IC-RT-PCR.

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