Biological and molecular differentiation of subgroup III geminiviruses

Abstract

The biological and molecular properties of Sinaloa tomato leaf curl virus (STLCV) were investigated to test the hypothesis that STLCV is a previously uncharacterized whitefly-transmitted geminivirus from North America. STLCV causes leaf curling and yellowing in tomato plants. STLCV was transmissible to N. benthamiana by sap inoculation, and to Solanaceous and Malvaceous species by the whitefly vector. STLCV has transmission characteristics like other persistent viruses, and was not transovarially passaged. PCR fragments containing the large intergenic region (IR) of the STLCV A and B components and coat protein gene (AR1) were cloned from STLCV-infected tomato, and their DNA sequences obtained. Regions 174 nt in length containing diagnostic sequences present in the IR of geminiviruses, and a putative ORF AR1 of 756 nt were identified. A and B component IR sequences were 97.9% identical, suggesting a homogeneous, bipartite viral quasi-species. Pairwise alignment (Wilbur-Lipman) of STLCV AR1 and those of subgroups I, II, and III geminiviruses indicated 22-81% similarity, whereas STLCV AR1 was 36-61% similar to subgroup III viruses, collectively, suggesting STLCV is a unique viral quasi-species (>90% = same virus). Multiple sequence alignment (Clustal) and parsimony analysis (PAUP) of IR or AR1 sequences supported placement of STLCV with Western Hemisphere subgroup III viruses. Both A and B types of the whitefly vector transmitted tomato yellow leaf curl (TYLCV-Th) and chino del tomate (CdTV) geminiviruses, and transmission frequencies increased with greater AAPs. TYLCV-Th was transmitted by both vectors at a higher frequency than was CdTV. The B type, indigenous to the Eastern Hemisphere, transmitted the Old World TYLCV-Th (87%) more effectively than the New World A type vector (63%). The Western Hemisphere CdTV, was transmitted more often by the A type whitefly (50%), also from the New World, than by B type (27%). PCR detection of geminiviruses in single whiteflies indicated virus ingestion occurred after a 0.5 h AAP. Detection frequencies increased in both whiteflies given longer AAPs (0.5-72 h), irrespective of virus tested. PCR primers were designed that effectively discriminate between Old and New World geminiviruses, and between monopartite and bipartite genomic organizations.