REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (RT- PCR) FOR DIRECT DETECTION OF CUCUMBER MOSAIC VIRUS (CMV) IN GLADIOLUS

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1 Vol. 44, No. 1, January 1998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Poges REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (RT- PCR) FOR DIRECT DETECTION OF CUCUMBER MOSAIC VIRUS (CMV) IN GLADIOLUS S.K. Raj, Sangeeta Saxena, Vipin Hallan and B.P. Singh Plant Virus Laboratory, National Botanical Research Institute, Lucknow , India Received October 10, 1997 Received after revision October 28, 1997 SUMMARY A diagnostic method based on Reverse Transcription - Polymerase Chain Reaction (RT-PCR) was performed for detection of CMV genome directly in crude extracts of both healthy and infected gladiolus tissue using primers from the conserved sequences of CMV RNA-3. RT-PCR resulted in the amplification of 540 bp long fragment of CMV coat protein gene (CMV-CP), as expected in most of the plant parts of infected gladiolus samples. Positive signals in Southern hybridization of amplified fragments with cloned CMV-CP cdna probe also confirmed the presence of CMV genome in gladiolus. Keywords Gladiolus psittacinus, Cucumber mosaic virus (CMV), Reverse Transcription- Polymerase Chain Reaction (RT-PCR), Southern hybridization, CMV coat protein (CP) cdna probe. INTRODUCTION Gladiolus psittacinus is an important ornamental plant for floriculture industry. Various cultivars of gladiolus viz. Friendship White, My Love, Snow Princess, Tambri, Oscar, Fidelio and Kajal 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 to Feb., 1997 at NBRI gardens and adjacent areas of Lucknow. Natural infection of cucumber mosaic virus (1-3) and bean yellow mosaic virus (4-9) has been repbrted on gladiolus by earlier workers. Diagnosis of CMV infection in gladiolus has usually been done by serological detection tests like: double diffusion (3), enzyme linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM)(2) l 2/98/ /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved.

2 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Serological procedures sometimes are confusing to differentiate among the strains of cucumoviruses due to the cross reactivity between CMV, tomato aspermy virus (TAV) and peanut stunt virus (PSV) (10). Polymerase chain reaction assay for CMV in Lupin seeds was developed (11) and detection of CMV using RT-PCR in infected Nicotiana glutinosa was done (12). Detections based on combination of reverse transcription (RT) and PCR amplification have also been published by various workers for BYMV (5-6,13) and other RNA viruses (14-18). Wetzel et al., (16), Borja and Ponz (17) and Bias et al., (18) developed the procedure for performing RT-PCR directly in crude plant extract to detect CMV genome. We performed RT-PCR for specific detection of CMV genome directly from crude extracts of different gladiolus plant parts. MATERIALS AND METHODS Samples from floret, leaf, stem, root, corm and cormule of gladiolus were collected and stored at -70~ Detection of CMV by RT-PCR was carried out using the primers as, described (18), based om the nucleotide sequences of conserved region of CMV-RNA3 (19-20). A 19-mer upstream primer (5'-GTAGACATCTGTGACGCGA-3') and a 21-met downstream primer (5'-GCGCGAAACAAGCTTCTTATC-3') were selected resulting in a 540 bp amplification product as described earlier (18). RT-PCR was performed as described (18), with slight modification. Infected tissues of gladiolus were homogenized in extraction buffer (0.1 g/ml) containing 20 mm Tris-HCI, ph 8.3, 5.0 mm MgCI 2, 0.1% Tween 20, 0.02% gelatin and 100 mm KCI. For reverse transcription reaction 20 p tool downstream primer, 2 pl of 10 mm dntps, 25 units ribonuclease inhibitor and 200 units of M-MLV reverse transcriptase (BRL, USA) were added in 10 pl of extracted samples and reaction tubes were incubated at 37~ for 1 h. Further, the amplification was carried out in Robocycler (Stratagene) by adding 80 pl of amplifcation mixture (20 p tool upstream primer and 10 pl of 10 x buffer) and overlayed with mineral oil. The reaction mixture was heated to 94~ for 2 rain, cooled to 72~ and then three units of Taq DNA polymerase (Genei, India) were added. Each cycle consisted of a denaturation step of 1 rain at 94~ an annealing step of 1 rain at 54~ and an elongation step of 1 min at 72~ Each test consisted of 35 cycles and the final cycle had an elongation step of 5 min/72uc. After amplification, the DNA was precipitated with 2.5 volumes of absolute ethanol. A part of the reaction mixture was electrophorased in an agarose gel (21) for the detection of the amplified DNA products after staining with Ethidium bromide. After electrophoresis, PCR products were transferred to Zetaprobe membrane (Bio - Rad) and hybridized to the radiolabelled probe. CMV Coat protein cdna was used as the probe (22). Probe was prepared by random primer extension method (23) using hexanucleotide primers and Klenow fragment (Pharmacia). RESULTS AND DISCUSSION Naturally infected gladiolus plants exhibited severe mosaic, dark green stripes on leaves and colour breaking of petals. The severity of disease 90

3 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL symptoms was variable among various hybrids and cultivars. Sap inoculation test revealed necrotic local lesions on Nicotiana tabacum cv. White Burley, systemic mosaic, green blisters and leaf deformations in Nicotiana rustica and systemic mosaic in Lycopersicon esculentum but no symptoms appeared in Nicotiana glutinosa even after thirty days of virus inoculation. In aphid trasmission studies Myzus persicae transmitted the virus in non-persistant manner from gladiolus to Nicotiana tabacum cv White Burley only upto an extent of 12.5% (1/8). Our data thus support the findings (3) which state that CMV from gladiolus showing stripe symptoms could infect N. glutinosa, Gomphrena globosa systemically and Chenopodium amaranticolor, faba bean, cow pea locally and could not be transmitted by Myzus persicae. In electron microscopic study a few virus particles of nm could be seen in leaf dip preparations and ISEM (3). Stein et al.,(2) observed that CMV can be easily detected in gladiolus corms by ELISA or ISEM. However, we could not detect CMV infection in gladiolus by ELISA and western immunoblot assay using antisera to CMV-T and chrysanthemum aspermy virus (CAV) (data not incorporated). RT-PCR was performed as described earlier (18) to detect CMV infection in gladiolus plants. 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 (Fig. 1 and Table 1) M Fig. 1 RT-PCR amplification of 540 bp fragment from the crude extract of healthy tobacco (lane 1, negative control ), infected tobacco (lane 2, positive control ), infected chrysanthemum (lane 3, positive control ), root (lane 4), stem (lane 5), healthy tobacco (lane 6, negative control), corm (lane 7), cormule (lane 8), floret (lane 9) and leaf (lane 10). 91

4 BIOCHEMISTRYancl MOLECULAR BIOLOGY INTERNATIONAL Table1 RT-PCR amplification of 540 bp fragment of CMV-CP and signals of Southern hybridization using cloned CMV-CP as probe. Sample description RT-PCR I RT-PCR II Southern, hybridization * Healthy tobacco **Infected tobacco **Infected Chrysanthemum Root Stem Corm Cormule Floret 4- Leaf * = Negative control = Heavy signals, = No signal ** = Positive control + ++ &++ = Moderate, -t- = weak In RT-PCR of gladiolus corm, cormule and floret samples the band (540 bp) was not seen, which may be due to low concentration of the 540 bp fragment or presence of some interfering substances to PCR reaction as earlier evidenced (2). Extracts of RNA from corm tissues were shown to inhibit the amplification of viral fragment when added to the PCR reaction (5). However on removal of interfering substances as suggested earlier (5) we could amplify above mentioned 540 bp fragment in corm, cormule and floret as well (Fig.2 and table 1). 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, cormule and floret (Fig.3 and table 1). 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 92

5 BiOCHEMISTRYondMOLECULAR BIOLOGY INTERNATIONAL M Fig. 2 RT-PCR amplification of 540 bp fragment from the purified extract ( after removal of interfering substances) of healthy tobacco (lane 1, negative control ), infected tobacco (lane 2, positive control ), infected chrysanthemum (lane 3, positive control ), root (lane 4), stem (lane 5), healthy tobacco (lane 6, negative control), corm (lane 7), cormule (lane 8), floret (lane 9) and leaf (lane 10) M Fig. 3 Southern hybridization of the agarose gel shown in Fig. 1 using CMV- CP cdna probe. using CMV-CP (cdna probe) we may correlate the relationship of the present isolate with the members of CMV and CAV. However, using the same strategy Bias et al., (18) could not detect CAV and CMV in pepper plants. Presence of CMV infection in gladiolus was also confirmed by sap transmission, aphid transmission and electron microscopic observations of leaf dip preparations. PCR is a quick and reliable technique of 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 ng of CMV 93

6 BIOCHEMISTRYclnd MOLECULAR BIOLOGY INTERNATIONAL particles per 10 mg of fresh leaf weight (12). This technique may also be adopted for quarantines i.e., detection of virus in cut flowers to be exported or imported. It is also very useful for indexing of nuclear materials (corms) to be used for mass propagation or industrial production in floriculture industry. Indexing of corms and weeds is also essential when a breeding programme is to be undertaken because weeds have a certain role in the epidemiology of gladiolus viruses (1). 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. REFERENCES. Vicchi, V. and Bellardi, M.G.(1988) Acta Horticulturae, 234, Stein, A., Levy, S. and Loebenstein, G. (1988) Acta Hort. 234, Kim, J.S., Choi, G.S. and Lee, K.H. (19.92) Research Report of the Royal development Administration, Crop Protection 34, Raizada, R.K., Zaidi, A.A., Srivastava, K.M., Shreni, V.C.D. and Singh, B.P.(1989) Ind. J. Plant Path Vunsh, R., Rosner, A. and Stein, A. (1991) Annals of Appl. Biol. 119, Rosner, A., Stein, A. and Levy, S. (1992) Annals of Appl. Biol. 121, Rosner, A., Stein, A., Levy, S. and Liliau-kipnis, H. (1994) J. Virol. Methods 47, Stein, A., Rosner, A. and Hammond, J. (1994) Acta Hort. 377, Bellardi, M.G. and Vicchi, V. (1995) Sementi Elette, 41, Palukaitis, P., Roosinck, MoJ., Dietzgen, R.G. and Francki, R.I.B. (1992). Cucumoviruses. Advances in virus research 41, Vylie, S., Wilson, C.R., Jones, R.A.C. and Jones, G.K. (1993) Aust. J. Agri. Res 44, Takamatsu, S., Tsuchiya, T. and Makara, K. (1994) The Bulletin of the faculty of Bioresources. Mie Univ. Tsu, Japan, No. 13,

7 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Vunsh, R., Rosner, A. and Stein, A. (1990) Annals of AppI.Biol 117, Jones, T.D., Buck, K.W. and Plumb, R.T. (1991) J. Virol. Methods 35, Robertson, N.L., French, R. and Gray, S. (1991) J. Gen. Virol. 72, Wetzel, T., Candresse,T., Ravelonandro, M. and Dunez, J. (1991) J. Virol. Methods 33, Borja, M.J. and Ponz, F. (1992) J. Virol. Methods 36, Bias, C. De, Borza, M.J., Saiz, M. and Romero, J (1994) J. Phytopath. 141, Quemada, H., Kearny, C., Gonsalves, D. and Slightom, J.L. (1989) J. Gen. Virol. 70, Owen, J., Shintaku, M., Aeschleman, P., Tahar, S.B. and Palukaitis, P. (1990) J. Gen. Virol, 71, Sambrook, J. Fritsch, E.F. and Maniatis, T. (1989). Molecular cloning A laboratory Manual, 2nd Edn. New York, Cold Spring Harbor Laboratory. Raj, S.K., Haq, Q.M.R., Srivastava, K.M. and Singh, B.P. (1995) J. Plant Biochem. and Biotech 4, Feinberg, A.P. and Vogelstein, B. (1983) Analytical Biochem. 132,