Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes

• Published in: FEMS yeast research 2004-09, Vol.4 (8), p.865-877
• Main Authors: Prakitchaiwattana, Cheunjit J., Fleet, Graham H., Heard, Gillian M.
• Format: Article
• Language: English
• Subjects: Aureobasidium pullulans; Cryptococcus; Denaturing gradient gel electrophoresis; DNA, Fungal - genetics; Ecology; Electrophoresis, Polyacrylamide Gel - methods; Fungi - growth & development; Fungi - physiology; Gel electrophoresis; Grape; Hanseniaspora; Metschnikowia; Mixed culture; Polymerase Chain Reaction; Rhodosporidium; Rhodotorula; Species; Species Specificity; Vitaceae; Vitis; Vitis vinifera; Wine; Wine - microbiology; Yeast; Yeasts - growth & development; Yeasts - physiology
• ISSN: 1567-1356; 1567-1364
• DOI: 10.1016/j.femsyr.2004.05.004

The performance of denaturing gradient gel electrophoresis (DGGE) for analysing yeasts associated with wine grapes was compared with cultural isolation on malt extract agar (MEA). After optimisation of PCR and electrophoretic conditions, the lower limit of yeast detection by PCR-DGGE was 102 cfuml−1, although this value was affected by culture age and the relative populations of the species in mixed culture. In mixed yeast populations, PCR-DGGE detected species present at 10–100-fold less than other species but not when the ratio exceeded 100-fold. Aureobasidium pullulans was the main species isolated from immature, mature, and both damaged and undamaged grapes. It was not detected by PCR-DGGE when present at populations less than 103 cfug−1. When approaching maturity, damaged grapes gave a predominance of Metschnikowia and Hanseniaspora species (105–107 cfug−1), all detectable using PCR-DGGE. However, various species of Rhodotorula, Rhodosporidium and Cryptococcus were not detected by this method, even when populations were as high as 104 cfug−1. PCR –DGGE was less sensitive than culture on MEA for determining the yeast ecology of grapes and could not reliably detect species present at populations less than 104 cfug−1. However, this method detected a greater diversity of species than agar plating.