Biological, serological and molecular characterization of two citrus tristeza virus isolates from Corsica

International audienc

Biological, serological and molecular characterization of two citrus tristeza virus isolates from Corsica

International audienc

Differentiation of <em>Citrus tristeza virus</em> (CTV) Isolates by Cleavase Fragment Lenght Polymorphism (CFLP) Analysis of the Major Coat Protein Gene

A panel of Citrus tristeza virus (CTV, genus Closterovirus, family Closteroviridae) isolates of different origins and with different biological properties were compared for polymorphisms in the major coat protein (CP) gene by cleavase fragment length polymorphism (CFLP) and single stranded conformation polymorphism (SSCP) analysis. The similarity between the CFLP patterns, which consisted of 15 to 20 bands, was estimated by the Pearson coefficient. The clustering patterns from the CFLP data were very similar to those from sequence data in an experiment with 16 cloned standards of the CP gene. By SSCP analysis on the other hand, most of the clones were not clustered in the same way. To assess the ability of CFLP to analyse biological samples, which may consist of a mixture of genomic variants, the CP gene of 12 CTV isolates was obtained directly from infected plants by immunocapture/ RT-PCR and analysed. With few exceptions, the isolates were correctly clustered according to the sequences of the variants composing the isolates. In artificial mixed infections of mild and severe isolates the patterns obtained were more closely related to the severe isolate. Thus the CFLP method was an accurate method for the identification, typing and clustering of CTV isolates. The usefulness of this technique as an alternative to SSCP analysis is suggested and discussed

Suppression of the ubiquitin E2 gene through RNA interference causes mortality in the banana weevil, Cosmopolites sordidus (Germar)

RNA interference (RNAi) is a natural defense mechanism triggered by double-stranded RNA (dsRNA) for protection of cells against foreign parasitic nucleic acids. The RNase III enzyme Dicer processes dsRNAs into short small interfering RNAs (siRNAs) that degrade the specific mRNA. RNAi has been demonstrated in a range of organisms including coleopterans and has a potential use in crop protection against insect pests and pathogens. In this study, we explored the use of RNAi for the control of the banana weevil that is not only the most important banana pest in East Africa, but has also eluded control through cultural, chemical and biological approaches. The ubiquitin E2 gene, essential for protein catabolism was identified, amplified and transcribed into dsRNA and fed to banana weevil larva in in vitro bioassays. The dsRNA significantly retarded banana weevil larval growth and caused up to 100% mortality at 21 days. Growth inhibition and mortality increased with dsRNA concentration (10 to 100 ng µL-1), though no significant differences were observed between the 50 and 100 ng µL-1 concentrations. We for the first time demonstrate RNAi in the banana weevil. Transgenic banana plants expressing siRNA or hairpin RNA could therefore potentially be used for controlling the banana weevil

Identification to the species level of the plant pathogens Phytophthora and Pythium by using unique sequences of the ITS1 region of ribosomal DNA as capture probes for PCR ELISA

The ribosomal internal transcribed spacer 1 region was sequenced for 10 species of Phythium and eight species of Phytophthora. Alignment of the sequences revealed considerable sequence microheterogeneity, which was utilized to prepare a capture probe of unique sequence for each species. The capture probes were tested by PCR ELISA, combining the sensitivity and specificity of the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The probes were entirely species specific, enabling the detection and identification of the amplified DNA of species from individual Cultures or front mixed samples of the DNAs of two different species. This approach to species identification, which provides a molecular technology to process large numbers of samples and still identify the fungi with a high level of confidence, may greatly reduce the resources and the time of highly trained specialists currently needed to identify these important species of plant pathogenic fungi. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved