Comparative analysis of genetic diversity and killer sensitivity of Brettanomyces bruxellensis and Pichia guilliermondii from Patagonian grapes and wines

SÁEZ J.S.; LOPES C.A.; CONSTANZO M.; SANGORRÍN M.P.

Rosario

Congreso; V Congreso Argentino de Microbiología General; 2008

The yeasts of the genus Dekkera/Brettanomyces are known because of their volatile phenols production that lead off flavours wine defects. Recently, it has been demonstrated that the speciePichia guilliermondii is also able to produce these compounds.P. guilliermondii is a common yeast of grapes, fermentation musts and surfaces of Patagonian wineries, although any isolation of B. bruxellensis had been found until this moment inthe same substrates. In this work, B. bruxellensis indigenous isolates were detected for the first time in Patagonian disturbed wines. The intraspecific diversity of this specie as well as thediversity among P. guilliermondii indigenous isolates kept in the culture collection of the laboratory were also analyzed. Finally, the sensibility of both species to killer toxins produced byreference and indigenous yeasts was evaluated as a first step in the development of an efficient biocontrol strategy against these spoilage yeasts.The taxonomic identification of the yeasts was carried out by ITS1-5.8S-ITS2 PCR-RFLP and sequence analysis of the D1/D2 26S-ADNr region. The intraspecific characterization was carried out by RAPD-PCR and mtDNARFLP with the endonuclease HinfI. Killer sensitivity was evaluated against 20 indigenous and three reference yeasts belonging to the species Metschnikowia pulcherrima, Pichia anomala and Torulaspora delbrueckii usingthe grown inhibition method in YPD-MB agar plates.From a total of 10 different primers tested, only OPA 3 and OPA 10 evidenced polymorphism among indigenous B. bruxellensis and P. guilliermondii indigenous isolates respectively. Fivedifferent patterns were detected among B. bruxellensis and six among P. guilliermondii. On the other hand, the mtDNA-RFLP analysis evidenced eight different patterns among B.bruxellensis, and a common pattern for all the P. guilliermondii isolates. The combined use of both RAPD-PCR and mtDNARFLP characterization methods allowed us to discriminate ahigher number of different profiles (different strains). The intraspecific diversity detected among B. bruxellensis isolates was larger than that observed among P. guilliermondii isolates,even though the former were obtained from the same cellar and the latter from different cellars and vintages. A differential sensitivity against the three killer species tested was detected among the spoilage species analyzed; evidenced a high sensitivity against P. anomala killer yeasts but theisolates belonging to the B. bruxellensis species also showed a high sensitivity against M. pulcherrima killer yeasts. Only one isolate belonging to T. delbrueckii species was able to kill, in a high percentage, the B. bruxellensis indigenous isolates. Some of these indigenous killer isolates could be taken into account as future wine spoilage yeasts biocontrol tools.