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

摘要

This thesis presents a systematic investigation of bacterial species associated with winegrapes cultivated in Australian vineyards during 2001-2004. Grapes, sampledthroughout cultivation, were analysed for bacterial species using a combination ofcultural and molecular methods. Red (Shiraz, Cabernet Sauvignon, Merlot) and white(Chardonnay, Semillon, Sauvignon Blanc) grape varieties were examined. Factorsaffecting the bacterial ecology of grapes were considered.The bacterial populations of mature undamaged grapes at harvest were consistently lowat 102-103 CFU/g. Higher populations (103-106 CFU/g) were found on grapes at earlierstages of maturity and correlated with application of Bacillus thuringiensis as abiological pesticide. B. thuringiensis was the most prevalent bacterial species on winegrapes throughout cultivation, as determined by plate culture, enrichment culture andPCR-DGGE. B. thuringiensis carried over into wine processing but did not grow injuice or wine and did not adversely affect the growth of Saccharomyces cerevisiae orOenococcus oeni in liquid culture. B. thuringiensis inhibited the growth of severalspoilage and mycotoxigenic fungi found on grapes.Curtobacterium flaccumfaciens was the second most prevalent species detected on winegrapes. Its populations rarely exceeded 103-104 CFU/g. Other bacteria (Arthrobacter,Bacillus, Microbacterium, Pantoea, Pseudomonas, Sphingomonas) were sporadicallyfound 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 wasdetected by specific enrichment procedures, especially on damaged grape berries.Species found were Lactobacillus plantarum, Lactobacillus mali, Lactobacillus lindneriand Lactobacillus kunkeei. The malolactic organism, O. oeni, was never isolated fromany grape sample, raising questions about its enological origin. Enrichment cultures alsorevealed the presence of other bacteria (e.g. Sporolactobacillus inulinus, Asaiasiamensis) not previously found on wine grapes.Atypical, hot and dry conditions during cultivation may account for the low populationsof bacteria found on wine grapes. This factor combined with the overwhelmingpresence of B. thuringiensis prevented meaningful comparisons of data to determineinfluences of vineyard location, grape variety, grape maturity, climate and viticulturalpractices on the bacterial ecology of grapes. More systematic and controlled studies ofthese variables are required.