Annual Report: CIRAD 2008

Banana is naturally infertile and therefore propagated vegetatively as micropropagated plantlets. This intensive propagation method facilitates the exchange of disease-free plant material, but it can also lead to widespread dissemination of the virus when the source plant is infected. Most viruses are actually not eliminated by conventional pathogen cleaning techniques, such as heat treatment, shoot-tip micropropagation or in vitro culture. It is thus essential to have ready access to effective detection methods when plant material is being introduced in a new area, or exchanged between areas or countries.

Banana bract mosaic virus

Prior to the introduction of micropropagated banana plantlets in the French West Indies, routine ELISA tests carried out by the crop protection services revealed the presence of banana bract mosaic virus (BBrMV). This potyvirus causes a serious banana disease, especially in plantain, and prevails in a few Asian and Pacific countries, but not in the Caribbean or Latin American regions. This virus is transmitted by aphids that are all present in the French West Indies. Infected cv Cavendish bananas generally do not display any symptoms of disease until the onset of typical mosaic symptoms on bracts.

CIRAD confirmed the presence of BBrMV on micropropagated plantlets using molecular detection methods complementary to the serological approach used by the crop protection services. CIRAD also highlighted that the molecular detection tests available were not very powerful and thus, in response to an urgent request from the French Ministry of Agriculture, developed a reliable and sensitive BBrMV molecular detection method.

A reliable and sensitive detection method

The aim was to propose a suitable detection technique tailored to meet the requirements of quarantine and plant material exchanges, for which the virus rate in the tested samples is mostly relatively low. The developed method involves a BBrMV-specific immunocapture (IC) step followed by two molecular reactions, ie reverse transcription (RT) and polymerase chain reaction (PCR), to amplify the RNA viral genome. The entire assay is simultaneously performed in the same tube. Molecular primers are located on the central and C-terminal parts of the capsid protein gene, which is a highly conserved region in all isolates of the same potyvirus. In order to obtain a broad virus detection spectrum, primers were selected from the multiple sequence alignment for 23 isolates available in GenBank. The developed method is based on commercial assay kits using reliable and efficient enzymes.

This method enables BBrMV detection in single samples of ground banana leaf extracts at up to 1 x 10^–10 dilution, and in bulked samples of 10 plants, for which the virus concentrations are low and undetectable by ELISA.

This technique, which was developed in less than a year, now provides crop protection service laboratories with a reliable and sensitive BBrMV molecular detection method that is effective even for low virus concentrations. Experiments are under way to validate the performance criteria of this method so as to be able to propose it as a new international BBrMV-detection standard.

Contact

Marie-Line Caruana
Biology and Genetics of Plant-Pathogen Interactions (UMR BGPI)
E-mail

Partners

• Ministry of Agriculture and Fisheries (France)
• Laboratoire départemental d’analyses de Mâcon (France)
• Laboratoire de la protection des végétaux de Martinique (France)
• Laboratoire national de la protection des végétaux d’Angers (France)

For further information

• Iskra-Caruana M.L., Galzi S., Laboureau N., 2008. A reliable IC one-step RT-PCR method for the detection of BBrMV to ensure safe exchange of Musa germplasm. Journal of Virological Methods, 153 : 223-231.