Diagnostics of bean common mosaic virus in common bean (Phaseolus vulgaris L.) seeds
2008
Trajkova, A., Plant Protection Institute, Kostinbrod (Bulgaria) | Khristova, D., Plant Protection Institute, Kostinbrod (Bulgaria)
Phaseolus vulgaris L. is an important crop in Bulgaria. Bean common mosaic virus (BCMV) is one of the most widely spread viruses in bean in Bulgaria as well as in the world. It is easily transmitted by sap, by several aphid species (in a non-persistent manner) and through seeds (up to 83%). This virus individually or together with other seed transmitted viruses can reduce the bean yield up to 30%. The aim of our investigation was to develop rapid methods for diagnostics of BCMV directly in matured bean seeds. In order to achieve this, different diagnostic methods were compared: DAS-ELISA, DOT-ELISA and RT-PCR. DAS-ELISA and DOT-ELISA were applied on dry and soaked bean seeds from fifteen Phaseolus vulgaris L. cultivars, collected from different Bulgarian fields. Ten seeds were taken. from each location in DOT-ELISA there were no differences between positive and negative samples, therefore this method is not suitable for BCMV detection in nietter nor soaked seeds. DAS-ELISA is not a 100% reliable method for the BCMV detection in bean seeds (dry and soaked). Sometimes the positive reaction in ELISA is negative in RT-PCR but there is not sample which is positive in RT-PCR and negative in DAS-ELISA. Thereby it is not possible to miss a BCMV positive sample in DAS-ELISA so the method could be used for mass testing. For RT-PCR, total RNA from dry and soaked seeds was isolated by phenol/chloroform extraction. The nucleotide sequences of the coat protein (CP) for four BCMV strains (NL-1, NL-2, Nl-4 and NL-7) were aligned and primers were chosen to amplify a 404bp fragment. In our opipion, the most reliable technique for BCMV detections directly in common bean seeds is RT-PCR. From all available evidence presented here we may conclude that the RT-PCR assay is a powerful and reliable method for diagnostic of BCMV not only in seeds. The PCR method described in this work could be useful in a very early diagnostic of this seed transmitted virus and could be helpful for testing bean seeds in germplasm collection.
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