Nucleic Acid Probes for Detection and Strain Discrimination of Cucurbit Geminiviruses

Abstract

A nuclei acid spot hybidization assay was developed for detection of curcurbit geminiviruses in plant tissue extracts. With a probe composed of four unique full-length DNA geminiviral genome components cloned from squash infected with two variants of squash leaf curl virus (SLCV), viral nucleic acid could be detected in various infected host representing nine genera from four plant families. The least amount of virus that could be detected by the spot hybridization assay was 300-400-fold less than that by enzyme-linked immunosorbent assay (ELISA). The limit of detection of purified SLCV was 3.2 pg of viral nucleic acid (17 pg of virus). A unique restriction fragment from each of the four genome components was identified (19). Two biologically different strains could be differentiated with these cloned fragments probes and mixtures of these two strains could be detected in experimentally infected plants. All four SLCV genome components were detected in field-collected squash plants. The genome components of both strains could be found in the same plant; however, in some plants the two genome components of one strain and only one of the other strains could be detected.