DETECTION AND DISTRIBUTION OF SWEET-POTATO FEATHERY MOTTLE VIRUS IN SWEET-POTATO BY INVITRO-TRANSCRIBED RNA PROBES (RIBOPROBES), MEMBRANE IMMUNOBINDING ASSAY, AND DIRECT BLOTTING

An in vitro-transcribed RNA probe (riboprobe) system was developed to detect sweetpotato feathery mottle virus (SPFMV) in infected plants. The essential components of the system include selection of an SPFMV cDNA clone that reacts with all known strains of SPFMV, optimization of assay procedures, and modified hybridization conditions. Additionally, a direct blotting technique on nitrocellulose membrane was developed to detect SPFMV by either riboprobe or membrane immunobinding assay (MIBA). The riboprobe system provided greater sensitivity of detection of virus in symptomless tissues than MIBA. Reconstitution experiments showed that the limit of detection of SPFMV using the riboprobe was 0.128 pg of RNA per sample, whereas MIBA only detected 170 pg of capsid protein per sample. Experiments that measured the accumulation of viral RNA and capsid protein of SPFMV in naturally infected sweetpotato plants cv. Jewel demonstrated that the riboprobe assay was nearly as effective as grafting to detect the virus, whereas the MIBA was only effective in symptomatic leaves.