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Biomolecular and serological methods to identify strains of cucumber mosaic cucumovirus on tomato

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Biomolecular and serological methods to identify strains of cucumber mosaic cucumovirus on tomato
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  Bulletin OEPP/EPPO Bulletin 25, 321-327 1995) iomolecular and serological methods to identify strains of cucumber mosaic cucumovirus on tomato’ by V. ILARDI, M. MAZZEI, S. LORETI, L TOMASSOLI and M. BARBA Istituto Sperimentale per la Patologia Vegetale, Via C.G. Bertero 22 00156 Rome Italy) Biomolecular and serological methods were used to classify 52 Italian isolates of cucumber mosaic cucumovirus CMV) into S and WT subgroups. Two synthetic oligonucleotides, specific for the coat-protein genes of the S and WT subgroups, were designed. These probes, used in Northern blot assays, were able to identify viral RNAs present in total nucleic-acid extracts from fruits and leaves of infected field tomatoes. CMV-specific primers, flanking the coat-protein gene, were used to amplify, by RT-PCR assay, a fragment of approximately 870 bp from either dsRNA replicative intermediates or purified genomic ssRNA. The restriction pattern analysis of the PCR product distinguished between the two subgroups. Monoclonal antibodies specific for CMV isolates belonging to the S and WT subgroups were produced. Five of these were able to detect and distinguish the strains in indirect ELISA using crude extracts from field samples. The three strategies gave concordant results and were useful in detecting and typing CMV subgroups on tomato. Introduction Cucumber mosaic cucumovirus is an icosahedric virus, distributed worldwide, causing severe diseases of many important crops. It has a positive-sense, tripartite RNA genome. RNAs 1 and 2 are associated with the replication of the viral genome Nitta et al. 1988) whereas RNA 3 contains the 3a gene, involved in viral cell-to-cell movement Davies Symons, 1988) and coat- protein gene. Coat protein is expressed from a subgenomic RNA, RNA 4;which is colinear with the 3’-portion of RNA 3. The virus is present as a variety of isolates that differ in host range, pathogenicity, biological and physical properties. On the basis of the difference in their coat-protein sequence Piazzolla et al. 1979; Owen Palukaitis, 1988), in virion antigenic property Devergne Cardin, 1973) or in coat-protein gene sequence Edwards Gonsalves, 1983), the isolates can be divided into two subgroups, named S (= TORS 11 and WT (= DTL I). Since 1987, the Italian tomato crop has been affected by a severe pathological complex, involving fruit necrosis, leaf malformation and lethal necrosis, according to the different symptomatologies caused by CMV Benetti, 1988; Gallitelli et al. 1988). Different approaches are being used to find suitable control measures. The production and use of transgenic plants resistant to viral attack seems to be the most promising strategy Tomassoli et al. 1995). Knowledge of the local epidemiology of CMV is a prerequisite for starting programmes for genetically engineering resistant plants. In this paper we report results obtained in developing and comparing biomolecular and serological methods able to discriminate CMV isolates. Materials and methods Basic molecular biology manipulations and solutions were carried out, unless otherwise indicated, as described by Sambrook er al. 1989). aper presented at the EPPO Conference on New Methods of Diagnosis in Plant Protection, Wageningen 995 OEPP/EPPO 321 NL), 1994-01 -25/28.  322 V Ilardi et al Field sample collection Fruit and leaf samples were collected in 1992 from 52 tomato plants grown in commercial fields located in Campania region southern Italy). All samples were frozen at -20 “C. Virus purification Some isolates of the WT and S subgroups were propagated in Nicotiana tabacum cv. White Burley and purified following Lot et al 1972). Purified viral preparations were used for production of monoclonal antibodies and for ssRNA extraction. Nucleic acid extraction Total RNA was extracted from leaves or fruits of each infected plant by a modification of White & Kaper 1989). Tissue 250mg) was powdered in a mortar with liquid nitrogen and collected in a 1.5-ml microfuge tube containing 400 p1 of extraction buffer and 400 p1 of water- saturated phenol. The mixture, vigorously shaken to form an emulsion, was maintained at room temperature until the end of sample preparation. Then, 400 p1 of chloroform was added, the aqueous phase recovered by centrifugation and RNA precipitated by filling the tubes with absolute ethanol. After centrifugation at 12,OOOg for 20min at 4”C, he pellet was resuspended in 4OOpl of extraction buffer. After another ethanol precipitation, the total RNA was resuspended in 40p1 of sterile water and stored at -80°C. CMV dsRNA replicative intermediates were extracted as described by Rizos t al 1992). garose gel electrophoresis and Northern transfer Aliquots of 0.5-1 ml approximately 2 mg) of total RNA, plus 3 volumes of loading buffer (50 formamide, lx MOPS, 5.5 formaldehyde) were heated at 80°C for 2min. Samples, loaded onto 1 agarose gel containing 1.1 formaldehyde and 0 1 pg ml ethidium bromide, were run at 80V for 2h in lx MOPS buffer. Gels were blotted onto nylon membrane HybondN, Amersham) using as transfer buffer 20x SSC; nucleic acids were fixed to filters by 5 min exposure to UV light. Probe design CMV coat-protein gene sequences were searched on the Gene Bank database and analysed using programs of the GCG Package, University of Wisconsin Devereux et al. 1984). The sequence of chosen probes was based on the analysis of 20 CMV coat-protein gene sequences, 5 of subgroup S and 15 of subgroup WT. On the basis of the comparison of the subgroup-specific consensus sequence, two strain-specific oligonucleotides, here named CMV-WT and CMV-S probes, 22 bases long, were synthesized using a Beckman DNA-SM automated DNA synthesizer: CMV-WT probe 5’ GGC GGA GCG GGA ACC ACG ACG C 3’; CMV-S probe 5’ GGA CCG AGA ACC TCT AGC CGG G 3’. Hybridization CMV-S and CMV-WT probes were 3’P-labelled using ’ labelling kit Amersham). Filters were prehybridized for 2.5 h and hybridized for 16 h at 45 “C in 5x SSC, 50 mM sodium phosphate pH 7, 5x Denhard’s solution, 0.5 SDS and 50mg ml-’ sonicated salmon sperm DNA. All samples were separately hybridized with both probes (5 lo6 counts min-’ ml-I). Filters, 995 OEPP/EPPO Bulletin OEPP/EPPO Bulletin 25 321-327  CMV strains on tomato 323 washed four times for 30 min at 45 C in 5x SSC, 1 SDS, were exposed at room temperature with intensifying screens. Hybridization tests were repeated four times. R T PCR amplification and restriction digests The procedure of Rizos t al. (1992), using dsRNA replicative intermediate as template, was followed for cDNA synthesis and PCR amplification. The procedure described in the Perkin- Elmer Cetus Gene-amp RNA PCR kit for the positive control RNA was followed when ssRNA was used as template. The primers proposed by Rizos et al (1992) were used in both experiments. Restriction digestions, with EcoRI and XhoI enzymes, were performed using 5 p of the RT- PCR solution; the products of the digestions were analysed on 1% (w/v) agarose gel cast and run in TBE buffer. Monoclonal antibody production and ELISA Two CMV isolates, belonging to S and WT subgroups, were used to produce strain-specific monoclonal antibodies. Four female 2-month old Balb/c mice (two for each strain) were immunized with two intraperitoneal injections, spaced 3 weeks apart, of 50 80 pg of purified virus, emulsified with Freund's complete adjuvant. The last injection of 5 pg antigen was given intravenously after one month. Spleens were removed from sacrificed mice 3 days after the last injection and spleen cells were fused with NS1-Ag8 and X63, for production of WT and S monoclonal antibodies respectively (Galfree & Milstein, 198 1). Two weeks after fusion, growing hybridomas, which reacted positive by DAS-ELISA, were cloned by the limiting dilution method and propagated, in pristane-primed mice, as ascites fluids. Immunoglobulins from ascites fluids were purified by Econo Pac 10 DG desalting column and Affi Gel Protein A column (BioRad). DAS-ELISA tests were performed according to the method of Clark & Adams (1977); monoclonal antibodies were used at a concentration of 0.2pg ml-'. Results Molecular hybridization with synthetic probes The extraction method was effective in obtaining non-degraded viral RNA using either leaves or fruits as infected source. No problems arose in using frozen field material when compared with fresh material. The two chosen oligonucleotides recognized a region located approxi- mately 5 bases downstream from the capsid-protein gene start site, where there is a low percentage of base homology (32%) between the subgroups. Probes reacted with both RNAs 3 and 4 n which the gene sequence for coat-protein gene is present; no cross reactions with other viral RNAs or with plant nucleic acids occurred. Northern blot assays allowed a clear-cut distinction among CMV isolates belonging to the S and WT subgroups (Fig. 1 . Among isolates, 63.5% belonged to WT subgroup (33/52), 3.8 to subgroup S (2/52) and 32.7% (17/52) seemed to be present as a mixed infection. No difference in the ratio of S and WT strains was observed between leaves and fruits collected from the same plant carrying a mixed infection. The technique was very sensitive, allowing detection of 1 pg of RNA extracted from purified virus. R T PCR amplification and restriction patterns The primers were effective, in so far as a product of expected size, approximately 780 bp long, was amplified by RT-PCR from the CMV coat-protein gene. The chosen enzymes gave 995 OEPP/EPPO, Bulletin OEPPIEPPO Bulletin 25 321 -321  324 V Ilardi et al different restriction patterns specific for the subgroups, confirming that there is an EcoRI site only in the coat-protein gene of the S strain and a XhoI site only in the gene of the WT strain. In fact, no digestion occurred in WT strains when EcoRI enzyme was used, whereas a fragment of 678 bp and another of 190 bp were obtained after digestion with XhoI (Fig. 2). Contrariwise, 600 and 270-bp restriction fragments were obtained by EcoRI digestion of strains, whereas no digestion was observed with XhoI. Serology and ELIS The fusion experiment yielded numerous actively growing hybridomas. Eighty-six of them were positive, in early screening ELISA tests, for the production of specific antibodies to CMV. Eight clones secreting a high concentration of antibodies were chosen for subcloning. Three clones specific for WT and two for S subgroups were obtained. All produced monoclonal antibodies belonging to the IgGl class. The concentration of monoclonal antibodies in purified ascites fluid reached about 2.0 mg ml- of protein. All specific monoclonal antibodies detected the virus in DAS-ELISA from CMV-infected fruit and leaf samples and no reaction with healthy control nor cross reactions between the two strains were observed. The detection level was as low as 1 ng of purified virus. When used in DAS-ELISA, monoclonal antibodies A3HI and B8C1, specific for WT and S subgroups respectively, confirmed the results obtained by hybridization and RT-PCR assays. Fig 1 Northern blot and hybridization with probes for strains WT (a) and S (b) of cucumber mosaic cucumovirus of nucleic acid extracted from control strains and from naturally infected tomato plants. Probes hybridized with viral RNAs 3 and 4. 3 CMV RNA 3; 4 CMV RNA ; WT WT control; PWT WT control from purified virus; S S control; h healthy control. Reaction. en northern blot et en hybridation avec des sondes pour les souches WT (a) et (b) du cucumber mosaic cucumovirus, d acides nucleiques extraits de souches tkmoins et de tomates naturellement infectees. Les sondes sont specifiques des ARN viraux 3 et 4 3 ARN CMV 3; 4 ARN CMV ; WT WT temoin; PWT WT temoin (virus purifie); S temoin; h temoin non infecte. c) I995 OEPP/EPPO. Bulletin OEPP/EPPO Bulletin 25 321-327   MV strains on tomato 325 Fig 2. Agarose gel electrophoresis of amplified cDNA, obtained by RT-PCR from the RNA of the WT strain of cucumber mosaic cucumovirus, together with its EcoRI and XhoI restriction enzyme patterns. Lane I molecular weight marker VII Boehringer Mannheim); lane 2 amplification product; lane 3 product restricted with EkoRI enzyme no digestion observed); lane 4 product restricted with XhoI enzyme digestion observed). Electrophortse sur gel d’agarose de I’ADNc amplifie, obtenu par RT-PCR a partir de 1’ARN de la souche WT du cucumber mosaic cucumovirus, ainsi que des produits de restriction par les enzymes EcoRI and XhoI. Voie 1, marqueur moleculaire VII Boehringer Mannheim); voie 2 produit d’amplification; voie 3 produit soumis a I’action de l’enzyme EcoRI aucune digestion); voie 4 produit soumis a I’action de I’enzyme XhoI digestion). onclusions Three strategies were tried out to distinguish the S and WT strains of CMV: specific synthetic probes for hybridization; restriction analysis of the RT-PCR-amplified coat-protein gene; ELISA with monoclonal antibodies specific for the two strains. The three strategies all gave useful and concordant results. This is not surprising since serological tests recognize differences in CMV coat proteins and molecular biology methods identify differences in the gene sequence coding or this protein. Northern blotting with a radioactive probe was a very sensitive strategy in checking the CMV subgroup. Moreover the extraction of total nucleic acids was a very fast and simple procedure, requiring only a few mg of sample fruit or leaf). It was even possible to use field-infected tissue directly, avoiding viral purification. In addition the small amount of tomato starting tissue 250 mg) allowed the use of 1.5-ml tubes and, consequently, the processing of several samples in a short time. Restriction analysis of the RT-PCR-derived cDNA fragments was the most sensitive and safest procedure, but propagation of isolates on a herbaceous host was required for ssRNA extraction from purified virus) or for dsRNA extraction directly from infected tissue). Monoclonal antibodies could be used to set up a rapid diagnostic system to type a high number of samples in a routine epidemiological survey; indeed, crude extracts were directly used as samples. The choice between the three approaches will be determined by the parameters and characteristics of the methods, such as number of samples to analyse, detection sensitivity, rapidity of execution, and research objectives. As reported by other authors Crescenzi t al. 1993), both strains of CMV are present in Italy, even if to a different extent. In fact, the WT subgroup is more widespread than the S 995 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 25 321-327
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