In situ serological, microscopy and hybridization detection of CTV Resumen: El virus de la tristeza de los citricos (CTV) es perjudicial para la citricultura y causa la enfermedad llamada tristeza de los citricos. Infecta las especies del genero Citrus ocasionando la muerte de millones de arboles. Los sintomas son decaimiento rapido (QD) y acanalamiento de tallo (SP). En el trabajo se diagnostico molecular y serologicamente al CTV en aislados provenientes de Citrus aurantifolia o Lima Tahiti (LT) y Citrus madurensis (Lour) o Calamondino (Ca), y se realizaron estudios preliminares de deteccion viral por medio de microscopia optica e hibridacion in situ . Se utilizo IC-RT-PCR e inmunoimpresion de tejido (IMI) expuesto a los anticuerpos monoclonales 3DF1+3CA5, y con el anticuerpo discriminante MCA 13 con tecnica de Enzyme Linked Inmunossorbent Assay Doble Sandwich ( Elisa -DAS). La deteccion por microscopia se realizo sobre secciones de peciolo de LT y C que se tineron con Azure A, y con acetato de uranilo y citrato de plomo. Para la hibridacion in situ se empleo una sonda marcada con digoxigenina dirigida hacia el gen de la proteina mayor de la capside. Los resultados de IC-RT-PCR, IMI y Elisa fueron positivos para LT y C, indicando la presencia de variantes virales de tipo severo. Con microscopia de luz se detectaron inclusiones citoplasmaticas en las celulas acompanantes y del floema, confirmado con IMI y por hibridacion in situ . Se visualizaron inclusiones de particulas virales en el tejido vegetal con microscopia electronica con cambios en la ultraestructura celular como presencia de grandes vacuolas propias de la infeccion viral. Este trabajo integra distintas tecnicas diagnosticas sobre dos especies citricas exoticas. Palabras clave: inmunoimpresion; Elisa; hibridacion; microscopia; CTV; IC-RT-PCR. Abstract: 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. Key words: immunoimpression; Elisa; hybridization; microscopy; CTV, IC-RT-PCR.