Anomalous PCR results to Grapevine leafroll associated closterovirus type 3 in South Africa

Abstract

Grapevine leafroll-associated virus type 3 (GLRaV-3) is the major causative agent of grapevine leafroll disease. The disease has a major negative impact on grape production for wineries, and can cause up to 62.8% loss in production. Despite the negative impact of GLRaV-3 on the grapevine industry worldwide, knowledge on the variability of the virus, which is essential for developing effective control measure of the virus in vineyards, is surprisingly scarce. To test this, six primers sets used in a one tube, one step polymerase chain reaction (PCR) protocol, together with ELISA were used to detect GLRaV-3 virus in 135 plant samples collected from a single vineyard. As expected the more sensitive PCR detected more infected samples than ELISA. However, some samples yielded positive results with the ELISA, but negative results using PCR. This might suggest that strain variants exist. Amongst PCR results of the different primer sets, anomalous results occurred, as often a plant will yield an amplicon with one primer set, but not with another primer set. Using the entire set of 7 PCR results per sample, each plant was assigned a PCR ‘fingerprint’. This yielded 24 different fingerprints in the vineyard. Mapping the spatial distribution of given fingerprints supported the possibility that strain variants exist. However, sequencing areas incorporating the primer binding sites showed no nucleotide sequence differences, indicating that the anomalous PCR results were not due to variants, but rather to protocol error. The PCR protocol used initially was adapted to obtain more optimal detection of virus. Different extraction methods and PCR protocols were tested. It was found that using the two step RT-PCR and using a less dilute plant macerate in ELISA extraction buffer (1:5), yielded amplicon of the expected size from all known infected plant samples. The protocol was further optimized with regards the RT step and subsequent PCR. Since the modified protocol did detect all known infected plant samples it can be concluded that in the 135 plant samples tested no significant sequence variation in strains at the primer binding sites occurred and that the anomalous PCR results initially obtained were due to a sub-optimal extraction method.