Detección del virus de la tristeza de los cítricos por serología, microscopía e hibridación in situ

Citrus tristeza virus (CTV) is deleterious for citriculture and causes citrus tristeza disease. CTV infects all citrus species thereby causing the death of millions of trees. Its main symptoms are quick decline (QD) and stem pitting (SP). Serological, molecular and microscopy techniques were used in this work for diagnosing CTV in Citrus aurantifolia or Tahiti Lime (Citrus latifolia Tanaka) (TL) and Citrus madurensis (Lour) or Calamondin (Ca) isolates. Petioles were tissue printed (IMI) and exposed to 3DF1+3CA5 monoclonal antibodies; they were then ELISA buffer extracted and exposed to a discriminant MCA 13 monoclonal antibody in a double-antibody sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA). Immunocapture reverse transcriptase-polymerase chain reaction (IC-RT-PCR) amplification, using specific major coat protein gene (CPG) primers, was used on the ELISA buffer extracts as template. Optical and electron microscopy were used for detection on transversal sections of petiole and stained with azure A, uranyl acetate or lead citrate. Digoxygenin-labelled major CPG CTV probes were used for in situ hybridisation of petioles printing. All IC-RT-PCR, IMI and ELISA results were positive for both LT and C, indicating the presence of severe viral variants. Light microscopy cytoplasm inclusions were detected in the phloem and accompanying cells, confirmed by IMI and in situ hybridisation. Electron microscopy analysis revealed cellular abnormalities with changes in ultrastructure and the presence of big vacuoles which are characteristic of cytoplasmic viral infection. This is the first work integrating all available diagnostic techniques on these two exotic citric species.