Macroptilium yellow mosaic virus, a New Begomovirus Infecting Macroptilium lathyroides in Cuba.

Macroptilium lathyroides (L) is a weed that is widely distributed in Cuba. Frequently, leaves show bright yellow mosaic symptoms, which suggest the incidence of a viral disease. Since begomovirus occurrence in Macroptilium lathyroides has been previously reported in other islands of the Caribbean (1,3), symptomatic plants from three distant places in Cuba (Havana, Villa Clara, and Camaguey), were collected and tested for the presence of begomoviruses. Plant DNA extracts were analyzed by Southern blot hybridization and polymerase chain reaction with two sets of degenerate primers (2). The presence of a bipartite begomovirus was evident through strong hybridization signals obtained with the DNA-A and DNA-B of Taino tomato mottle virus as probes at low stringency. Furthermore, 1.4-kb and 1.2-kb PCR amplified fragments were obtained with DNA-A degenerate primers, PAL1v1978-PAR1c715 and PAL1c1960-PAR1v722, respectively. Both PCR fragments from the samples from the three locations were cloned, and restriction fragment length polymorphism analysis of the 1.4-kb fragments were performed using PstI, EcoRI, HincII, XbaI and BglII. Restriction fragment patterns were the same for the three clones. The DNA-A sequence (GenBank Accession No. AJ344452) of the isolate from Villa Clara was compared with sequences available for other geminiviruses using CLUSTAL program. For the coat protein (CP) gene, the comparisons had the highest percentage of identity with various strains of Bean golden yellow mosaic virus (BGYMV, GenBank Accession Nos. AF173555, M91604, and L01635) (85 to 87% and 93 to 94%, nucleotide and amino acid sequences, respectively). For Rep gene (1,044 nt), the best percentages of identities were with BGYMV (81 to 82% and 80 to 82% nucleotide and amino acid sequences, respectively), Tomato leaf crumple virus (GenBank Accession No. AF101476) (78 and 81%, nucleotide and amino acid sequences, respectively), and Sida golden mosaic virus from Florida (GenBank Accession No. AF049336) (78 and 79%, nucleotide and amino acid sequences, respectively). Finally, the comparative analysis of the intergenic region (i.e. the common region plus the CP gene promoter) had the highest identity with BGYMV (56 to 55%) and Tomato severe rugose virus (GenBank Accession No. AY029750) (49%). Interestingly, this virus has in this region the three G-box elements that are characteristic of BGYMV but it differs in the Rep protein-binding iterative motif that is GGTGA instead of GGAGA, for BGYMV. These data indicate that this virus is a new begomovirus and the name of Macroptilium yellow mosaic virus (MaYMV) is proposed. References: (1) A. M. Idris et al. Plant Dis. 83:1071, 1999. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) M. E. Roye et al. Plant Dis. 81:1251, 1997.

Molecular Characterization of Tobacco leaf rugose virus, a New Begomovirus Infecting Tobacco in Cuba.

Tobacco (Nicotiana tabacum L.) is the main raw material for the cigar industry and one of the most important crops in Cuba comprising 49,654 ha. During the past 20 years, foliar rugosity and stunting symptoms have been observed in several tobacco producing areas. These symptoms were correlated with the presence of typical geminivirus nuclear inclusions and the transmission of the causal agent by whiteflies (Bemisia tabaci Genn) (1). To identify the suspect geminivirus, diseased leaf samples were collected in Havana province in 2000 and 2001. Sap extracts or leaf pieces were used to inoculate healthy tomato and tobacco plants by mechanical and graft inoculation procedures. Characteristic symptoms were reproduced in tobacco plants only by grafting (8 to 10 plants). DNA extracts from symptomatic plants were analyzed by Southern blot and polymerase chain reaction. The presence of a bipartite begomovirus was supported by the observation of hybridization signals (1.6 kb to 3 kb) at low stringency to probes derived from DNA-A and DNA-B of Taino tomato mottle virus. Furthermore, typical begomovirus amplicons of approximately 1.4 kb and 1.2 kb were amplified using the primer sets PAL1v1978-PAR1c715 and PAL1c1960-PAR1v722 (2), respectively. Amplicons were cloned, and their nucleotide sequences (nt) obtained from two clones each. Sequence for component A was assembled, and some fragments were compared with those for other begomoviruses using CLUSTAL W. For the CP gene (756 nt) (GenBank Accession No. AJ488768), the comparison revealed the highest percentages of nt identity with Sida golden mosaic virus from Florida (SiGMV-F, GenBank Accession No. AF049336) (86%), Tomato mottle virus (GenBank Accession No. L14460) (83.5%), and the yellow vein strain of Sida golden mosaic virus from Honduras (GenBank Accession No. Y11099) (83.3%). In addition, the percentages of nt identity obtained using the core region (a 540-nt fragment located between positions 147 and 687) of the CP gene from the tobacco virus were calculated. The best scores were as follows: SiGMV-F, 87.8%; Jatropha mosaic virus (JMV) from Puerto Rico (GenBank Accession No. AF058025), 86.9%; and Tomato rugose mosaic virus (GenBank Accession No. AF291705), 86.3%. Finally, comparisons of the common region (CR, 144 nt) revealed the highest values with JMV from Jamaica (JMV-JM) DNA-A and DNA-B (GenBank Accession Nos. AF324410 and AF324411; 89% and 91.1%, respectively). Interestingly, the CR analysis revealed the presence of the Ori-associated iterative motif GGGGT, which is the same in the CR of JMV-JM. Although the data suggest that the tobacco begomovirus is related to the JMV-JM isolate, it is a new species, and the name of Tobacco leaf rugose virus (TbLRV) is proposed. References: (1) S. Quintero and J. Santiesteban, Agrotec. Cuba 11(1), 1979. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Cuban Isolate of Bean golden yellow mosaic virus is a Member of the Mesoamerican BGYMV Group.

In Cuba, the emergence of bean golden mosaic was associated with high populations of Bemisia tabaci in common bean (Phaseolus vulgaris L.) plantings in the 1970s (1). During the last two decades, the disease has caused significant economic losses, forcing some growers to abandon bean production. In Holguín, one of the main bean producing provinces of the country, about 2,000 ha of beans were abandoned in 1991 due to the high incidence of this whitefly-transmitted virus. At that time, yield losses associated with this disease reached 90 to 100% in farmer's fields. In spite of various control measures, the disease affected 33, 28, and 6.5% of the total area planted in Cuba to common bean in 1990, 1992, and 1996, respectively. For this investigation, common bean leaves showing systemic yellowing symptoms were collected in fields located in the provinces of Havana, Matanzas, and Holguín during 1998-1999. Sap and total DNA leaf extracts were used to inoculate healthy bean plants by manual and biolistic procedures, respectively. Characteristic yellowing symptoms were more efficiently reproduced using a particle gun device than by manual inoculation (18/20 plants and 5/20 plants, respectively, for a Holguín virus isolate). DNA extracts were further analyzed by polymerase chain reaction using two degenerate primer sets: PAL1v1978-PAR1c715 and PAL1c1960-PAR1v722 (2). Fragments of approximately 1.4 and 1.2 kb were amplified and cloned. Restriction fragment length polymorphism analysis of the cloned 1.4-kb fragments was performed with BglII, HincII, SalI, EcoRI, PstI, and XbaI, indicating that selected isolates from the three Cuban provinces shared identical restriction patterns. The nucleotide sequence obtained from two clones of a virus isolate from Holguín, was compared to sequences available for other begomoviruses using BLAST. The Cuban isolate shared up to 94% nt sequence identity with various strains of Bean golden yellow mosaic virus (BGYMV) in the first 250 nt of the rep gene. For the common region (CR), scores were 93% for BGYMV-GA (Guatemala), 92% for BGYMV-MX (southern Mexico) and BGYMV-PR (Puerto Rico), and 91% for BGYMV-DR (Dominican Republic). The iterative sequence ATGGAG was identified in the CR of the Cuban BGYMV isolate, as reported for other BGYMV isolates. Finally, the Cuban begomovirus, hereafter referred to as BGYMV-CU, shared nt and aa sequence identities of 94 and 100%, respectively, with the coat protein gene of BGYMV-MX. We conclude that the begomovirus isolated from mosaic-affected common bean plants in the province of Holguín is a member of the Mesoamerican BGYMV group (3). References: (1) N. Blanco and C. Bencomo. Cienc. Agric. 2:39, 1978. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) Morales and Anderson, Arch. Virol. 146:415, 2001.