Technical Yearbook 2023

FIGURE 4. Graph of ELISA results showing the effect of mixing one grapevine fleck virus-infected vine with increasing ratios of healthy vines in pools, to determine how many vines at a time can be tested and still detect the virus. Four GFkV infected sources were tested using two different means of preparing the extracts.

FIGURE 5. ELISA values obtained for 232 individual vines of Vitis accessions selected to reflect the genetic variability of GFkV. Blue bars = individual vines containing GFkV as determined by high-throughput sequencing (HTS), grey bars = did not contain GFkV reads as determined by HTS, and black bars = contained grapevine rupestris vein feathering virus (GRVFV) as determined by HTS.

detect variants of the virus) of each in reciprocal tests against their supplied positive controls (virus-infected material) and a limited number of South African GFkV sources. No differences were observed in the specificity of three of the four supplied ELISAs to GFkV and only minor differences in sensitivity were observed between the same three kits. All three of these could therefore be utilised within the VIA scheme. The remaining commercial ELISA kit yielded very poor results and is not recommended for routine use.

The most affordable GFkV ELISA was selected for further study. We demonstrated that GFkV was a stable virus, still reacting in ELISA seven days after storage at 4 ° C of a macerate in a phosphate-based buffer with various additives. This buffer proved to be more effective than a Tris-based buffer to extract and stabilise GFkV for ELISA and is recommended for general use in all the ELISA systems.

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TECHNICAL YEARBOOK 2023