Subgroup Determination of Bulgarian Isolates of Cucumber Mosaic Virus and the Presence of Satellite RNAs.

Cucumber mosaic virus (CMV) is one of the most important viruses in Bulgaria, causing severe losses to agriculture, but little is known about the occurrence and distribution of subgroups within the country or the presence of satellite RNAs (satRNAs). Samples showing typical symptoms (mild to severe mosaic, vein clearing, vein necrosis, leaf deformation, stunting, and fruit necrosis) on several important crops (tomato, cucumber, pepper, bean, courgette, and tobacco) were collected from the main agricultural regions of the country. Isolates were maintained by sap inoculation in tobacco plants. Total RNAs were isolated from 38 samples (including two from bean) and used in reverse transcription-polymerase chain reaction (RT-PCR) assay with primers corresponding to the coat protein (CP) gene of RNA3 (3). A single strong band, 870 bp in length, was produced from all these samples. Amplified products were analyzed for subgroup differentiation by digestion with the restriction endonucleases MspI (3), PvuII, and EcoRI. The MspI subgroups 2 and 1 designated by Rizos et al. (3) according to their restriction endonuclease digest data correspond to the subgroups I and II widely used in the literature and based on serology, sequence data, and other properties. In this report, the subgroups are referred to as I and II for the sake of clarity. Isolates in both subgroups were found in all the main regions of Bulgaria. A few variations in MspI and EcoRI digestion patterns were seen, indicating some variability between isolates within subgroups. Only five samples, three from tomato and two from pepper, were found to be subgroup II. Subgroup I isolates were found in all the crops sampled. The PCR product from one representative isolate of each subgroup was cloned and sequenced by standard procedures. Alignment of the nucleotide and predicted amino acid sequences with published sequences of the CMV CP gene confirmed that the amplified products were derived from CMV. A further eight samples from bean gave only weak amplification and digestion of the products suggested they were likely to be subgroup II. However, these samples were unusual in not inducing symptoms in inoculated tobacco and in being difficult to propagate. The nature of these virus isolates is therefore unclear. Only a single occurrence in Bulgaria of satRNA of CMV has been reported (4) but in this study satRNAs were detected by RT-PCR (1) in total plant RNA extracts of 21 of the 38 samples tested. Amplified products of two of them, NB and 146D, were sequenced; comparison with published sequences confirmed that they were derived from CMV satellite. As expected from the symptoms induced by these isolates, a sequence homologous to the domain of satRNA Y responsible for bright yellow mosaic on tobacco (2) was identified in satRNA NB but not in satRNA 146D. satRNAs were not detected in the eight bean samples that had given only weak amplification with the CMV CP gene primers. The results presented here clearly demonstrate the presence of both subgroups of CMV in Bulgaria. Although CMV in Bulgaria has been studied previously by serological methods, no evidence had been found for the presence of subgroup II. References: (1) F. Grieco et al. Virology 229:166, 1997. (2) C. Masuta and Y. Takanami. Plant Cell 1:1165, 1989. (3) H. Rizos et al. J. Gen. Virol. 73:2099, 1992. (4) E. Stoimenova. J. Cult. Collect. 1:45, 1995.