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First Report of Tomato Yellow Leaf Curl Virus-Is in Spain: Coexistence of Two Different Geminiviruses in the Same Epidemic Outbreak

Epidemics of tomato yellow leaf curl have occurred annually in greenhouse- and field-grown tomato (Lycopersicon esculentum Mill.) crops in southern Spain since 1992 (2). The nucleotide sequences of two tomato yellow leaf curl virus (TYLCV) isolates from this region, TYLCV-M (GenBank accession no. Z25751) and TYLCV-Alm (L27708), have been determined and these isolates are closely related to isolates reported from Italy (X61153 and Z28390), suggesting the existence of a geographical cluster of closely related TYLCV isolates in the Western Mediterranean Basin (2). In June 1997, new and unusually severe symptoms of stunting, yellowing, and curling of leaflet margins, with a marked reduction in leaf size, were observed in some plants of a greenhouse-grown tomato crop in Almeria (southeastern Spain). Tomato plants showing milder symptoms similar to those previously described for TYLCV infection in that region (2) were also present in the same greenhouse. Total nucleic acids extracts from plants exhibiting both types of symptoms were analyzed by dot blot hybridization with a probe prepared by random priming on a 1,674-bp SalI fragment of the pSP95 clone of TYLCV-M (3). A strong reaction was obtained with the samples that showed mild symptoms, whereas a weak reaction was observed with the severely affected plants. Specific pairs of primers were prepared to amplify the complete pre-coat (V1) (MA10: 5′-ATGTGGGATCCTTTATTAAATG-3′; MA11: 5′-TCAGGGCTTCTGTACATTC-3′) and C2 (MA12: 5′-TAAAGACTCTTAAAAAATGACC-3′; MA13: 5′-AATGCAATCTTCGTCACC-3′) genes based on TYLCV-M sequence. With polymerase chain reaction (PCR), the expected fragments were amplified from extracts of both types of plants. The PCR products were submitted to single-strand conformation polymorphism (SSCP) analysis. Clearly distinguishable SSCP patterns were obtained: one for the plants with mild symptoms, identical to that of known TYLCV-M infected plants, and another for the plants with more severe symptoms. Further analyses done on tomato samples collected from the same area showed that both SSCP patterns were present simultaneously in several severely affected plants. The nucleotide sequences of the V1 and C2 PCR products from two samples differing in their SSCP pattern were obtained by direct sequencing, and compared with available TYLCV sequences. The sequences corresponding to the sample with mild symptoms were 100% identical to those previously reported for TYLCV-M. In contrast, the sequences from the sample that showed severe symptoms (GenBank accesion no. AF022219 for V1, and AF022220 for C2) were only 80 and 76% identical to TYLCV-M V1 and C2 genes, respectively, but were 99% identical to the sequence reported for an isolate of TYLCV-Is from Israel (X15656). Epidemics in tomato caused by TYLCV-Is have been recently reported from Portugal (1). Our results demonstrate that the unusually severe symptoms observed are associated with an isolate of TYLCV-Is that coexists in the field with the milder TYLCV previously reported from this area. This is the first report of the occurence of TYLCV-Is in Spain.

References: (1) D. Louro et al. Plant Dis. 80:1079, 1996. (2) E. Noris et al. Arch. Virol. 135:165, 1994.