J. Romero

Incidence and characterization of bean common mosaic virus isolates in Spanish bean fields

A 5-yr survey for the presence of bean common mosaic potyvirus (BCMV) in the major bean (Phaseolus vullgaris) growing areas of Spain was conducted, and the viral isolates were characterized biologically and serologically. Both serotype A and B were found. Necrosis-inducing serotype A isolates, found for the first time in Spain, resembled NL-3. Serotype B isolates resembled NL-4 and US-5. The complex antigenic relationships of Spanish BCMV isolates, as determined with specific monoclonal and polyclonal antibodies, and the incidence and distribution of serotypes are discussed

A Bipartite Geminivirus Infecting Tomatoes in Cuba

Whitefly-transmitted geminiviruses have increased in their distribution and importance during the last decade, apparently due to a wider distribution of their vector Bemisia tabaci (Gennadius). In Cuba, symptoms of geminivirus disease have been observed since the early 1990s. A survey of geminivirus-caused diseases in tomato plants has been conducted since 1994 in the province of La Habana. The most severe symptoms were observed in the commercial varieties Campbell 28, Criollo Quivican, and HC 3880. In some plants the Israeli strain of tomato yellow leaf curl geminivirus (TYLCV) was detected (1). In other cases, a viral DNA with a genome size smaller than that of TYLCV that could not be amplified with primers specific for the Israeli strain was also detected by hybridization. In these samples a polymerase chain reaction amplification with broad-spectrum, degenerate primers designed for whitefly-transmitted bipartite geminivirus (2) was carried out. Two fragments of about 1.1 and 0.6 kb, corresponding to components A and B, respectively, were amplified, indicating that a bipartite geminivirus was present. The 1.1-kb fragment amplified from a sample showing strong leaf yellowing and slight curling was cloned and sequenced. A sequence of 1,174 nucleotides was obtained and compared with geminivirus sequences deposited in the GenBank, by multiple alignment (CLUSTAL W, European Bioinformatics Institute, Hinxton Hall, UK). Genetic distances were calculated by Kimuras two parameters method. Percentages of similarities obtained with the sequence were as follows: tomato mottle virus 78%, abutilon mosaic virus 73.8%, and tomato golden mosaic 61.8%. Partial sequences of the open reading frames AL1 and AR1 and the intergenic region were present. Percentages of similarities obtained with the intergenic region were as follows: tomato mottle virus 78.1%, sida golden mosaic virus 63%, bean dwarf mosaic virus 62.6%, abutilon mosaic virus 57.1%, and tomato golden mosaic virus 32%. These results suggest that we have detected in Cuban tomatoes a new bipartite geminivirus related to tomato mottle virus, and we propose that it be named Havana tomato virus. Frequency of distribution of viruses in commercial varieties and their relationship with the presence of whiteflies are currently being studied. References: (1) Y. Martinez et al. J. Phytopathol. 144:277, 1996. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Serotype-specific detection of bean common mosaic potyvirus in bean leaf and seed tissue by enzymatic amplification

An assay involving reverse transcription and polymerase chain reaction (PCR) is described for specific detection of serotypes A and B BCMV isolates in bean leaf and seed tissues. Three oligonucleotide primers designed according to the sequence data available allow, in appropriate combination, serotype-specific detection of BCMV. The sensitivity of the method was sufficient to detect BCMV in as little as 100 fg and 50 pg of infected leaf and seed tissues, respectively.

Virus Diseases of Broad Bean in Spain

Broad bean (Vicia faba L.) plants showing symptoms suggestive of viral infection, such as stunting, leaf roll, mosaic, chlorosis, necrosis, and yellowing, were observed in the Andalucia, Baleares, Cataluna, and Murcia regions of Spain. A 4-year field survey showed the presence of five viruses: bean leaf roll luteovirus (BLRV), beet western yellows luteovirus (BWYV), bean yellow mosaic potyvirus (BYMV), tomato spotted wilt tospovirus (TSWV), and cucumber mosaic cucumovirus (CMV). Identity of viruses was first determined by enzyme-linked immunosorbent assay and confirmed by at least one other method, such as inoculation to plant hosts, electron microscopy, molecular hybridization, or immunocapture-reverse transcriptase-polymerase chain reaction. Of the 250 samples assayed, 93 were positive for BYMV, 21 for BLRV, 10 for BWYV, 30 for TSWV, and 2 for CMV. Faba bean necrotic yellow virus (a single-strand DNA virus) and broad bean mottle bromovirus, which are widely distributed in the Mediterranean basin, were not detected in the samples analyzed. BYMV was distributed in all regions, whereas TSWV was restricted only to Mediterranean areas. To our knowledge, this is the first report of viruses infecting broad bean in Spain.

First Detection of Faba bean necrotic yellow virus in Spain

Faba bean necrotic yellow virus (FBNYV) was not detected during 1994 to 1996 field surveys of faba beans (Vicia faba L.) in Spain (1). In 1997, however, one sample with symptoms of necrosis, collected in Baleares, was tested using ELISA (enzyme-linked immunosorbent assay) and was positive for both Tomato spotted wilt virus (TSWV) and FBNYV. FBNYV is a single-strand DNA virus that is transmitted by aphids and is the main virus disease of broad bean in North Africa and West Asia (2). During 1997 to 1999, faba bean plants with symptoms of necrosis, yellowing, small leaves, and stunting were collected from several fields in the Murcia Region (Spain) and were analyzed using ELISA. To detect FBNYV, we used monoclonal 2E9 supplied by H. J. Vetten (Institute of Plant Virology, Microbiology and Biosafety, BBA, Braunschweig, Germany). Of 700 samples analyzed, 34 were positive for FBNYV. Of the 34 positive samples, 12 tested positive, using commercial antiserum from Loewe, Inc. (Munich) for mixed infections with TSWV. FBNYV was transmitted to healthy faba bean plants by aphids (Acyrthosiphon pisum) in greenhouse experiments and was confirmed by ELISA. Preliminary epidemiological data showed a gradual increase in the number of plants infected with time in the same field. Aphid transmission of FBNYV to faba beans has established the disease in Spain and is a potential threat to other leguminous crops. This is the first report of a nanovirus in Europe. References: (1) J. Fresno et al. Plant Dis. 81:112, 1997. (2) L. Katul et al. Ann. Appl. Biol. 123:629, 1993.

Use of enzymatic cDNA amplification as a method of detection of bean yellow mosaic virus

The C-terminal region of bean yellow mosaic virus (BYMV) coat protein gene was selected for the design of oligonucleotide primers. Reverse transcription of viral RNA present in sap of virus-infected plants followed by polymerase chain reaction (RT-PCR) was performed for amplification of a 449 baise pairs fragment. These primers supported BYMV amplification, but did not support amplification on both bean common mosaic virus (BCMV) and potato virus Y (PVY) templates.