Molecular Diagnosis of Tomato Spotted Wilt Tospovirus Infection of Peanut and Other Field and Greenhouse Crops.

Abstract

Tomato spotted wilt tospovirus (TSWV) is a major disease constraint to peanut, tomato, pepper, and tobacco production in Georgia. Rapid molecular diagnosis of TSWV infection in peanut and its molecular studies were severely hampered by the lack of practical and rapid procedures for the extraction and amplification of the genomic nucleic acid. To circumvent this technical constraint, we adapted an immunocapture-procedure (ICP) for enriching the peanut tissue extracts for TSWV, and combined the ICP with a single-buffer, one-tube reverse transcription polymerase chain reaction (RT-PCR) to achieve rapid and reliable amplification of TSWV sequences from peanut. Both leaf and root tissue of peanut provided PCR-quality templates. Immunocapture, RT, and PCR were done in the same tube, allowing higher throughput. The technique was applicable to a wide range of TSWV-susceptible crops such as tomato, pepper, tobacco, gloxinia, and impatiens. Primers derived from the nucleocapsid protein gene as well as from the large RNA of the viral genome were able to amplify the target sequences in a highly specific and reproducible manner. This approach facilitated rapid molecular typing of natural populations of TSWV in Georgia.