3.3.63
RT-PCR BASED DIAGNOSIS OF CUCUMBER MOSAIC VIRUS IN GLADIOLUS
RT-PCR BASED DIAGNOSIS OF CUCUMBER MOSAIC VIRUS IN GLADIOLUS SK RAJ, S SAXENA, V HALLAN and BP SINGH Plant Virus Laboratory, National Botanical Research Institute, Lucknow 226 001, India < b > Background and objectives < /b > < i > Gladiolus psittacinus < /i > is an important ornamental plant for floriculture industry. Various cultivars of gladiolus are grown in India for cut flowers. Natural infection of a severe mosaic and leaf stripe disease in gladiolus culitivars with a variable degree of severity was observed during survey from Dec. 1996 to Feb., 1997 at NBRI gardens and adjacent areas of Lucknow. A procedure was developed for performing RT-PCR directly in crude plant extract to detect CMV genome [1]. We performed RT-PCR with the objective of specific detection of CMV genome directly from crude extracts of different gladiolus parts. bmaterials and methods < /b > Detection of CMV by RT-PCR was carried out in floret, leaf, stem, root, corm and cormule of gladiolus using the primers based on the nucleotide sequences of conserved region of CMV-RNA3 [1]. 19-mer upstream (5'-GTAGACATCTGTGACGCGA-3') and 21-mer downstream primers (5' G,CGCGAAACAAG,CTTCTTATC-3') were selected to yield a 540 bp amplification product. A part of the reaction mixture was electrophorased in an agarose gel and stained with Ethidium bromide. For Southern hybridization tests, PCR products were transferred to Zetaprobe membrane and hybridized to the radiolabelled probe derived from cloned CW coat protein. < b > Results and discussion < /b > Naturally infected gladiolus exhibited severe mosaic, dark green stripes on leaves and colour breaking of petals. The severity of disease symptoms was variable among various hybrids and cultivars. CMV can be easily detected in gladiolus corms by ELISA or ISEM [2]. However, we could not detect CMV in gladiolus by ELISA, ISEM and Western immunoblot assay using antisera to CMV-T and chrysanthemum aspermy virus (CAV). RT-PCR followed by gel electrophoresis of PCR product revealed a band of 540 bp in most of the samples of gladiolus except corm, cormule and floret. Southern hybridizations using CDNA clone of CMV-CP as probe gave positive signals in all the plant parts of the gladiolus samples. The intensity of signals was highest in leaf followed by root and stem while it was lowest in corm, corinule and floret. On the basis of PCR amplification of 540 bp fragment in good amount from tissues of tobacco and chrysanthemum infected with CMV and CAV respectively (positive control) and strong signals in Southern hybridizations using CMV-CP (CDNA probe) we may correlate a relationship of the isolate with the members of CMV and CAV. PCR is a quick and reliable technique for virus detection in gladiolus corms where virus persists either in latent form or in a very low concentration and is not detectable by conventional serological methods. The detection limit of CMV by RT-PCR ranged from 10-100 ng of CMV particles per 10 mg of fresh leaf weight [3]. Since cucumber mosaic and bean yellow mosaic viruses persist latently in corms of gladiolus, they spread by cultivation of infected corms and ultimately affects the floriculture trade. Development of such a sensitive diagnostic tool is highly useful for indexing of gladiolus corms and cormules to be used for breeding or mass propagation. < b > References < /b > 1 . Blas C De, Borza MJ, Saiz M, Romero J, 1994. Journal of Phytopathology 141, 323-329. 2 . Stein A, Levy S, Loebenstein G, 1988. Acta Horticulturae 234, 275-280. 3 . Takamatsu S, Tsuchiya T, Makara K, 1994. The Bulletin of the faculty of Bioresources. Mie Univ. Tsu, Japan, 13, 16.