Standardizing Botryosphaeriaceae infection levels in experimental grapevine plant materials

Grapevine nursery plants have been reported with latent infections of grapevine trunk disease (GTD) pathogens. However, the link between these latent infections and GTD symptoms observed in vineyards is still unclear. Studies have also shown that water-stress can increase the susceptibility of young vines to GTDs. Accurate quantification of infection levels from naturally-infect- ed young vines is difficult since latent infections have no internal or external symptoms and may be ran- domly distributed within a vine. Thus, investigations on the effects of water stress on young vines artificially inoculated with three different conidial concentrations of GTD pathogen may provide insight on the infection thresholds that result in disease expression in vines. The objective of this study was to develop an inocula- tion method to standardize GTD infection levels in young vines. A published vacuum-inoculation method was evaluated for inoculating Shiraz cuttings with 300 (low), 3,000 (moderate) and 30,000 (high) conidia of Neofusicoccum luteum while Ringer’s solution was used to inoculate control vines. The vacuum-inoculation was a reliable method as evidenced by qPCR analyses, result- ing in the conidia infecting the basal, middle and apical part of the inoculated canes. The qPCR analyses further differentiated the low, moderate and high infections with the highest amount of pathogen detected from canes inoculated with 30,000 conidia and lowest from those inoculated with 300 conidia. No pathogen was detected in any of the control canes. This study showed the vac- uum-inoculation method was suitable for introducing different concentrations of N. luteum spores into dor- mant canes without significant impact on plant viability.

The method was used to standardize infection levels of N. luteum in ~400 Shiraz rootlings in newly established glasshouse and shade house experiments. Observations on the effect of water stress on Botryosphaeria dieback symptom expression is on-going.