Investigation of bacteria associated with Australian wine grapes using cultural and molecular methods

Abstract

This thesis presents a systematic investigation of bacterial species associated with wine grapes cultivated in Australian vineyards during 2001-2004. Grapes, sampled throughout cultivation, were analysed for bacterial species using a combination of cultural and molecular methods. Red (Shiraz, Cabernet Sauvignon, Merlot) and white (Chardonnay, Semillon, Sauvignon Blanc) grape varieties were examined. Factors affecting the bacterial ecology of grapes were considered. The bacterial populations of mature undamaged grapes at harvest were consistently low at 102-103 CFU/g. Higher populations (103-106 CFU/g) were found on grapes at earlier stages of maturity and correlated with application of Bacillus thuringiensis as a biological pesticide. B. thuringiensis was the most prevalent bacterial species on wine grapes throughout cultivation, as determined by plate culture, enrichment culture and PCR-DGGE. B. thuringiensis carried over into wine processing but did not grow in juice or wine and did not adversely affect the growth of Saccharomyces cerevisiae or Oenococcus oeni in liquid culture. B. thuringiensis inhibited the growth of several spoilage and mycotoxigenic fungi found on grapes. Curtobacterium flaccumfaciens was the second most prevalent species detected on wine grapes. Its populations rarely exceeded 103-104 CFU/g. Other bacteria (Arthrobacter, Bacillus, Microbacterium, Pantoea, Pseudomonas, Sphingomonas) were sporadically found on grapes. Lactic acid bacteria and acetic acid bacteria were rarely detected on undamaged grapes by culture and PCR-DGGE methods. A greater incidence of lactic acid bacteria was detected by specific enrichment procedures, especially on damaged grape berries. Species found were Lactobacillus plantarum, Lactobacillus mali, Lactobacillus lindneri and Lactobacillus kunkeei. The malolactic organism, O. oeni, was never isolated from any grape sample, raising questions about its enological origin. Enrichment cultures also revealed the presence of other bacteria (e.g. Sporolactobacillus inulinus, Asaia siamensis) not previously found on wine grapes. Atypical, hot and dry conditions during cultivation may account for the low populations of bacteria found on wine grapes. This factor combined with the overwhelming presence of B. thuringiensis prevented meaningful comparisons of data to determine influences of vineyard location, grape variety, grape maturity, climate and viticultural practices on the bacterial ecology of grapes. More systematic and controlled studies of these variables are required.