Occurrence of Killer Yeasts in Spontaneous Wine Fermentations from the Tuscany Region of Italy

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1993, p /93/ $2./ Copyright C 1993, American Society for Microbiology Vol. 59, No. 12 Occurrence of Killer Yeasts in Spontaneous Wine Fermentations from the Tuscany Region of Italy PAOLA VAGNOLI, ROSA ANNA MUSMANNO, STEFANIA CRESTI, TIZIANA DI MAGGIO, AND GRAZIETTA CORATZA Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Universita degli Studi di Siena, Via Laterina 8, 531 Siena, Italy Received 1 June 1993/Accepted 7 September 1993 The occurrence of killer yeasts in an area of Tuscany (central Italy) was studied. Killer yeasts were found in 88% of spontaneous wine fermentations from 18 wineries. The incidence of killers varied with respect to fermentation stage and vintage period, increasing from the first vintage to successive ones and from the commencement to the end of fermentation. At the end of fermentation, the proportion of killer strains relative to total yeast population was below 25% in 15 cases, above 75% in 6 cases, from 25 to 5%o in 5 cases, and from 5 to 75% in 3 cases. Karyotype analysis also showed a mixed killer population in the fermentations in which the killers dominated. The killer phenomenon of yeasts was originally observed in certain strains of the genus Saccharomyces (3), and killer yeasts were subsequently found in several other genera (4, 15, 18, 19, 21, 29). Among the yeasts, killer, sensitive, and neutral strains have been described. Killer strains are able to kill sensitive cells by secreting a proteinaceous toxin to which they are immune (27). On the other hand, killer strains may be sensitive to a toxin produced by other killer strains, showing the phenotype killer/sensitive (KS) (28). Neutral strains are resistant to the killer toxin and do not kill sensitive cells. Depending on the spectrum of killing activity against sensitive strains or the cross-reactivity of the killer yeast (17, 3), killer yeasts have been classified into at least 11 groups (Kl to Kl1). Five Saccharomyces cerevisiae killer classes, Kl (3), K2 (3), K3 (3), KT28 (14), and K3GR1 (8), have been reported. However, Wingfield et al. (26) recently showed that the K3 killer yeast is a mutant K2 killer strain, suggesting that the K3 killer type should be included within the K2 class. In S. cerevisiae, the genetic determinants coding for the killer toxin and for the factor conferring immunity reside in virus-like particles present in the yeast cell cytoplasm (4, 29, 3). Killer strains have been isolated from a great variety of sources, including wine cellar equipment, grape skins, and must fermentations (6, 7, 9, 1, 24). It has been suggested that the killer activity is one of the mechanisms of antagonism among yeasts during spontaneous fermentations and that, because of this mechanism, killer strains could dominate at the end of wine fermentation (2, 5, 11, 12, 16, 22). The K2 killers could be important in fermenting grape musts, as their toxin is stable at the ph values of musts (14). Many surveys have been conducted to verify the incidence of killer yeasts in spontaneous must fermentations, with killer yeasts found differently distributed in various wine-producing areas. Thus, in certain areas killer yeasts Corresponding author. 437 were not detected, while in others only killer yeasts were isolated (6, 7, 9, 22, 24). If selected sensitive strains are used in wine fermentations, a serious problem such as lagging or stuck fermentation may occur because of contamination by wild killer strains (25), and this could have an adverse effect on the quality of the wine. To avoid such a phenomenon, it has been suggested that killer or neutral strains be used as starters (11). In Italy, industrial winemaking is carried out by using commercially selected wine yeasts, but until now spontaneous fermentations have made up the highest percentage of all fermentations of well-known wines. However, considering the advantages of the use of specifically selected strains, several efforts are being made to select yeast strains to be used as starters for the production of quality wines. In selecting yeast strains to use as starters, the first selection is usually carried out by taking into consideration characteristics such as alcohol tolerance, H2S production, volatile acid production, and fermentation rate, with toxin production or toxin resistance as a secondary criterion for selection. We believe that determining the presence of the killer factor and resistance to killing activity are also important criteria, especially if the selected strains are to be used in areas in which killer yeasts are widespread. Therefore, in such research, it is essential first of all to known how widespread the killer yeasts are in the wine production area where the strain specifically selected for fermentation is intended for use. Brunello di Montalcino is a prestigious wine produced in a restricted area of Tuscany, a region in central Italy. Brunello wine production has been generally carried out by spontaneous fermentation. However, a plan to select strains with improved enological characteristics from the natural wine yeast population is in progress. In this paper, we report results concerning the occurrence of killer yeasts in the Brunello di Montalcino production area, the dynamics of killer yeasts during spontaneous fermentations, and the interactions between killer and nonkiller isolates with Kl and K2 reference strains. When fermentations were found to be carried out by killer yeasts, the

2 438 VAGNOLI ET AL. electrophoretic karyotypes of the strains were also examined. MATERIALS AND METHODS Musts. The occurrence of killer yeasts in 33 spontaneous wine fermentations was studied. All musts, from Sangiovese variety grapes, came from several vineyards of the Brunello di Montalcino producing area during the 1991 vintage. We distinguished first, middle, and end vintages to differentiate between the musts according not only to the vintage period but also to the series of fermentations in the same cellar. The musts were naturally fermented by traditional winemaking techniques in 18 selected wineries. For this study, we chose only cellars which either had not been using selected yeast strains for at least 2 years or had not used them at all. The number of fermentations examined for each winery varied from one to four depending on the size of the winery. Samples (5 ml; at least two for each fermentation) were taken from the middle of fermentation vessels and transported to the laboratory for microbiological examination. Diluted samples were spread on WL nutrient medium (Difco) plates to count yeasts. The plates were incubated at 25 C for 3 days to allow colony development. The various colony types were counted and tested for killing activity as specified below. Representative colonies were purified on WL medium and identified by the procedures of Barnett et al. (1). Strains. As a sensitive reference strain, S. cerevisiae Kandell SC2 (J. Kandell, California State University, Fullerton) was used. We received this strain from the original source via L. Polonelli (Universita di Parma). ICV KlM, a commercial wine strain (Lallemand Inc., Montreal, Quebec, Canada) and NCYC 232 were used as K2 and Kl reference killer strains, respectively. Procedures to detect killer yeasts. To screen killer isolates, YPDA medium (Difco), buffered at ph 4.5 and containing methylene blue (.3% [wt/vol]), was used. The sensitive strain was incorporated into the medium at a concentration of about 15 CFU/ml. The colonies grown on WL agar were tested for killing activity by replica plating on YPDA medium. Yeasts were scored as killers when the replicated colony was surrounded by a region of blue cells or by a clear zone in which no growth of the seeded sensitive strain had occurred, bounded by a dark blue zone of dead cells. To assay interaction between the strains, a drop of the killer reference strain was streaked on an uninoculated yeast extract-peptone-dextrose (YEPD)-methylene blue plate, and subsequently each isolate was streaked on the plate perpendicularly to the seeded killer reference strain, so that the end of each streak was close to the reference strain but did not overlap it. Sensitivity to killer activity was shown by a zone of inhibition surrounded by a dark blue zone on the streak of the sensitive strain. If only one zone (either blue or clear) (indicative of sensitivity) appeared where the streaks crossed, one of two strains was sensitive to the killing activity of the other; the sensitive strain was the one in which the zone appeared. If two zones were present, the two strains were both killers and each of them was sensitive to the killing activity of the other. If no zone appeared, the strains were mutually resistant to killing activity. Karyotype analysis. Chromosomal DNAs were prepared essentially as described by Schwartz and Cantor (2). The electrophoresis was performed with a model 215 PULSA- PHOR apparatus (LKB-Pharmacia Biotechnology, Uppsala, Sweden). Samples were electrophoresed through 1% (wt/ vol) agarose gels in.5 x Tris-borate-EDTA (TBE) buffer at 9 C. Electrophoresis was carried out for 18 h with a 65-s pulse time and then for 7 h with a 1-s pulse time at 2 V/cm. The gels were stained with ethidium bromide (.5,ug/ml) for 4 min, destained in distilled water, and photographed under UV light. RESULTS APPL. ENvIRON. MICROBIOL. Prevalence of killer yeasts. Figure 1 shows the geographical locations of the wineries examined in this study. Killer yeasts were found in samples from 15 of the 18 wineries. Killer yeasts were found in 29 out of 33 spontaneous wine fermentations (88%) (Table 1). The prevalence of killer yeasts varied according to the stage of fermentation and the period of vintage. At the first vintage and at the commencement of fermentation, the percentage of tanks containing killer yeasts was lower than those observed at later vintages and at the end of fermentation, respectively. At the first vintage, killer strains were detected in 8 of 19 (42%) and 15 of 19 (78.9%) vats at the commencement and at the end of alcoholic fermentation, respectively. At the later vintages, on the other hand, we detected killer strains in 13 of 14 (92.8%) vats at the commencement and in 14 of 14 (1%) vats at the end of fermentation. In 14 of 15 wineries, all tanks examined contained killer strains at the end of fermentation, suggesting an environmental spread. As a whole, the proportion of killer yeasts relative to total yeast population in the samples varied from to 95.6%. In the samples at the end of fermentation, the proportions of killer strains were below 25% in 14 cases, above 75% in 6 cases, from 25 to 5% in 6 cases, and from 5 to 75% in 3 cases. All but two killer strains, identified as Candida valida, were S. cerevisiae. All killer strains were tested for differences in both their abilities to kill K2 and Kl reference strains and in their immunities to K2 and Kl toxins. All but the two C. valida killer strains showed the K2 phenotype; i.e., they were resistant to K2 toxin and did not kill the K2 reference strain but were sensitive to Kl toxin and were killed by the Kl reference strain. Further studies are necessary (and are in progress) to determine whether killer isolates belong to the K2 group. The two C. valida strains did not kill either the Kl or the K2 reference strain and were resistant to Kl and K2 toxins. Evolution of killer yeasts during fermentation. The dynamics of the presence of killer yeasts throughout fermentation were studied with 18 fermentations (marked by asterisks in Table 1). The samples were taken at different stages: at the commencement of fermentation (stage I), when the musts were in tumultuous fermentation (stage II), at the end of the tumultuous fermentation (stage III), and at the end of the alcoholic fermentation (stage IV). At stage I, killer strains were usually absent or present in very low concentrations; in only one sample was their concentration relatively high (18%). The concentration of killer strains usually increased in stage II, while no further increases in killer concentrations were observed in later stages (Fig. 2 to 4). With respect to the ability of killer strains to dominate the fermentation process, three trends were found. (i) The killer strains did not dominate the fermentation at any stage; in fact, their number was less than 1% of the total yeast population. This behavior was observed in all of the fermen-

3 VOL. 59, 1993 KILLER YEASTS IN SPONTANEOUS FERMENTATIONS 439 M_ -B +M +F -N +G +.. LCINO C.M ' +I. +E s U. S. ANGELO. +A IN COLLE S.ANGELO SCALO ~~~~~~.CASTELNUOVO.l- Om- FIG. 1. The Brunello di Montalcino producing area and geographical distribution of the wineries examined. Symbols:, wineries;, villages; capital letters, wineries examined; +, presence of killer yeasts; -, absence of killer yeasts. Scale, 1:141,. Montalcino is about 3 km south of Siena. tations during three vintage periods at wineries A (Fig. 2) and S (data not shown). (ii) The killer strains were present in all stages of fermentation; however, despite their relatively high concentration at stage I (from 11.1 to 36%), they did not dominate the fermentation completely, reaching at most 65% of the total yeast population in stages II, III, and IV. This trend was observed for the fermentations at wineries (Fig. 3) and I (data not shown). (iii) The killer strains were not detectable at stage I of the fermentations of the first vintage, but they became quickly dominant beginning in stage II, the proportions of killer yeasts relative to total yeasts being higher than 9%. This trend was observed for the fermentations at wineries F (Fig. 4) and E (data not shown). It should be noted that in the fermentations of successive vintages, the killer strains dominated once, at stage I. Phenotypes of nonkilier yeasts. In order to analyze the relationship between the three trends observed and the susceptibility of the nonkiller yeast population to the various toxins, the nonkiller strains isolated were examined to verify their sensitivity or resistance to the killing activities of Kl and K2 reference strains and of the killer strains present in the same samples. Two different resistance patterns were observed: resistance to both Kl and K2 toxins and resistance to K2 toxin only, the latter being the resistance pattern of killer isolates.. The non-saccharomyces strains showed only the former phenotype, while the Saccharomyces strains showed only the latter. The non-saccharomyces Kl- and K2-resistant strains were mostly found at the initial stage of fermentation and then decreased, persisting throughout fermentation at a low concentration. In the fermentations in which the killer yeasts were present as only a small proportion of the total yeast population, a large majority of the nonkiller yeasts were sensitive to all toxin types. In the fermentations in which the killer yeasts did not clearly dominate, a high proportion of nonkillers were sensitive to all toxins and a low proportion showed a resistance pattern similar to that of the killer strains. In the fermentations in which the killer yeasts dominated, a very low proportion of yeasts was sensitive to all killer toxins, while the strains showing the resistance pattern of the killer strains were the majority of the nonkiller yeast population. Karyotypes of killer strains. To analyze the homogeneity of the yeast population in fermentations conducted by killers, 1 independent isolates from each of the six fermentations from wineries E and F were tested by pulsed-field gel electrophoresis to examine their karyotypes. Figure 5 shows the patterns observed among the killer isolates of the fermentations at wineries F and E. The results indicated a heterogeneous killer population, both in the same

4 44 VAGNOLI ET AL. TABLE 1. Occurrence of killer yeasts in wine fermentations Presence of killer yeasts Winery Vintage Fermentation period tank no.' at stageb: A First" (1.9) First 2 Middle" (4) End" 8 + (2.5) + (4.5) B First 1 C First 1 D First (3.4) Middle 2 + (5.2) + (2.6) End 3 + (5) + (11.6) E First' 1 _ + (92.5) Middle' 2 + (4) + (87.3) End' 3 + (2.8) + (86) F First' 1 _ + (95.6) Middle" 2 + (86.6) + (89.7) End' 5 + (87.5) + (88) G First 1 + (16) + (31) H First (4.5) I First' (45) Middle" 2 + (24) + (44) End" 3 + (15.5) + (46.8) L First 1 + (5.6) + (18.4) M First 1 + (1) + (13) N First 1 First" 1 + (18) + (52.8) Middle" 2 + (11.1) + (64) End" 3 + (36) + (66) P First (7.6) Q First 1 + (.41) + (34) R First 1 + (2.2) + (13.2) S First' 1 + (2.2) + (2.5) Middle' 2 + (2) + (3.4) End" 3 + (.5) + (4) T First 1 + (8.3) + (25.5) a Each tank corresponds to an independent fermentation. b Stage I, commencement of fermentation; stage IV, end of alcoholic fermentation; -, killers absent, +, killers present. Numbers in parentheses refer to the percentage of killer yeasts relative to total yeast population. c Fermentations in which the evolution of killer yeasts was examined. fermentation and in the same winery. In particular, among killer strains isolated from winery F, two different patterns were observed at the first vintage (Fig. 5A, lanes b and c), while four different patterns were observed both at the middle (lanes d to g) and at the end (lanes h to m) vintages. One pattern (lanes b, e, and h) was present in all fermentations, and the strain showing this pattern was the prevalent one in each fermentation. Among killer strains isolated from winery E, three different patterns were observed at the first vintage (Fig. 5B, lanes b to d), while four different patterns were observed both at the middle (lanes e to h) and at the end (lanes i to n) vintages. In this winery, one pattern (lanes b, f, and i) was present in all fermentations, while another pattern (lanes h and n) was present in the fermentations of the middle and end vintages. DISCUSSION The results obtained show that in the area where Brunello di Montalcino wine is produced, killer yeasts are widespread, the high incidence of the killer factor being evident from the number of fermentations and the number of wineries where the killer yeasts are present. Almost all the killer isolates apparently belong to the K2 group. I IV. 2 8 IIii III IIV 6U.U APPL. ENvIRON. MICROBIOL. Ii III IV FIG. 2. Evolution of killer yeasts at different stages during fermentations in three vintage periods at winery A. Abbreviations: F, first vintage; M, middle vintage; E, end vintage. : I, commencement of fermentation; II, tumultuous fermentation; III, end of tumultuous fermentation; IV, end of alcoholic fermentation. Symbols: U, killer; X, sensitive; El, neutral. The killer strains isolated from the fermentations carried out by killers have different electrophoretic patterns, indicating the presence of different strains in the fermentations. In fact, the number of generations occurring during the fermentation is too low to justify a chromosomal rearrangement of one strain (13). The data prove that, notwithstanding the homogeneity of the population in one characteristic (in this case, the killer factor), the population is still mixed, with one strain becoming prevalent. The same electrophoretic pattern was found in the fermentations carried out in a cellar during different vintage periods. This fact suggests that one strain could circulate in a winery. This strain could have been already present in the winery, or, more realistically, it could have reached the winery with the first collected grapes. It is likely that the strain, having multiplied in the course of the first fermentation, spread to the other fermentation vats via the use of

5 VOL. 59, 1993 KILLER YEASTS IN SPONTANEOUS FERMENTATIONS F ii III Iv III 1 M 8 a. a- 1 E Downloaded from t11 II IV FIG. 3. Evolution of killer yeasts at different stages during fermentations in three vintage periods at winery. Abbreviations: F, first vintage; M, middle vintage; E, end vintage. : I, commencement of fermentation; II, tumultuous fermentation; III, end of tumultuous fermentation; IV, end of alcoholic fermentation. Symbols: U, killer;, sensitive; 8, neutral. winery tools and implements. However, other hypotheses, such as widespread occurrence on most grapes or common insect vectors, cannot be excluded. Concerning the dominance of killer yeasts in the fermentations, our data point out to at least three different situations: (i) the majority of fermentations are conducted by sensitive strains despite the presence of killer strains, occasionally in relatively high proportions, in the early phases of fermentation; (ii) fermentations are conducted by a mixed population of sensitive and killer strains, as if some sort of balance had come about between the two components, with one or the other attaining a slight prevalence; and (iii) fermentations are conducted by killer strains, which attain a clear prevalence over the sensitive strains starting in the early stages of fermentation. The lack of dominance of killer strains in the fermenta- 1 III IV FIG. 4. Evolution of killer yeasts at different stages during fermentations in three vintage periods at winery F. Abbreviations: F, first vintage; M, middle vintage; E, end vintage. : I, commencement of fermentation; II, tumultuous fermentation; III, end of tumultuous fermentation; IV, end of alcoholic fermentation. Symbols: U, killer; X, sensitive; El, neutral. tions of types i and ii does not seem to be due to the resistance of nonkiller strains to the toxin produced by the killers, for the vast majority of nonkillers were apparently sensitive to the toxin. In this regard, however, we must point out that, since our aim was to verify the occurrence of the killer factor in the production area of Brunello di Montalcino, the technique used allowed us to produce a qualitative evaluation of killer activity and of sensitivity. Using such a technique, we have noticed a certain variability in the dimensions of the inhibition halo, both when it is induced by various killer strains on the reference sensitive strain and when it is induced by reference strains Kl and K2 on the nonkiller sensitive strains. However, the nature of the assay was different from the nature of the fermentation: the ph in the assay medium was 4.5, versus 3.4 in the fermentation, and the medium of the assay was a synthetic solid medium, versus a liquid must in the fermentation. The latter differ- on April 15, 219 by guest

6 442 VAGNOLI ET AL. APPL. ENvIRON. MICROBIOL. B a b c d e f g h i I m -n I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~, FIG. 5. Electrophoretic patterns of the killer strains isolated from three vintages (first, middle, and end) at wineries F (A) and E (B). (A) Lanes: a, S. cerevisiae YNN295; b and c, first vintage; d to g, middle vintage; h to m, end vintage. (B) Lanes: a, S. cerevisiae YNN295; b to d, first vintage; e to h, middle vintage; i to n, end vintage. ence may explain the greater production or activity of the toxin in the assay than in the fermentation (27); on the other hand, the different ph values should not affect the production or the activity of K2 toxin, since it has proved completely stable in the range of phs from 2.8 to 4.8 (24). Quantitative tests of musts are in progress to determine whether the three situations referred to above can depend on the degree of production or activity of toxins and/or on the sensitivity level of nonkillers or whether some factors other than the killer factor intervene in the competition among yeasts (23). According to Jacobs and Van Vuuren (11), the amounts and activities of toxins produced by the killers and the degrees of sensitivity of nonkillers are variables which may explain the discrepancies in the data available in the literature concerning the initial concentration necessary for killers to be capable of prevailing over sensitive strains to carry out the fermentation process (24). In selecting yeasts to be used as starters for the vinification of Brunello di Montalcino, the killer characteristic must be taken into consideration for the following reasons: (i) a high prevalence of killer yeasts is observed in the production area; (ii) there is a possibility of contamination with a wild killer strain, which could become prevalent if fermentation has begun with a sensitive strain; and (iii) since at the time when the starter culture is to be added the indigenous I population of the must is not known and since the occurrence of killer yeasts is widespread, a high probability of the presence of killer yeasts exists. Since the role of the killer phenotype as a competition factor is not known, it appears advisable to select a starter strain from killers which have prevailed at the end of a fermentation, since the probability that such a strain has the characteristics necessary for effective competition with the indigenous strains is likely to be enhanced. ACKNOWLEDGMENTS This work was supported by a 6% grant from the Ministero dell'universita e Ricerca Scientifica. We thank G. M. Rossolini and L. Marri for reading and correcting the manuscript, B. Bagnolesi and A. Vannocci for their technical assistance, and E. Sestini and F. Lissi for the preparation of the manuscript. We also thank the Consorzio del Vino Brunello di Montalcino for its cooperation. REFERENCES 1. Barnett, J. A., R. W. Payne, and D. Yarrow Yeasts: characteristics and identification. Cambridge University Press, Cambridge. 2. Barre, P Role du facteur "killer" dans la concurrence entre souches de levures. Bull. O.I.V. 53: Bevan, E. A., and M. Makower The physiological basis of the killer character in yeast. Proc. Int. Congr. Genet. 1: Bussey, H Physiology of killer factor in yeast, p In A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 2. Academic Press, London. 5. Bussey, H., T. Vernet, and A. M. Sdicu Mutual antagonism among killer yeasts: competition between Kl and K2 killers and a novel cdna-based K1-K2 killer strain of Saccharomyces cerevisiae. Can. J. Microbiol. 34: Ciolfi, G Distribuzione dei fenotipi killer, neutro, sensibile nel corso di fermentazioni spontanee. Riv. Vitic. Enol. 4: Cuinier, C., and C. Gros Enquete sur la r6partition des levures "killer" en France. Vigne Vines 318: Extremera, A. L., I. Martin, and E. Montoya A new killer toxin produced by Saccharomyces cerevisiae. Curr. Genet. 5: Heard, G. M., and G. H. Fleet Occurrence and growth of killer yeasts during wine fermentations. Appl. Environ. Microbiol. 51: Jacobs, C. J., I. Fourie, and H. J. J. Van Vuuren Occurrence and detection of killer yeasts on Chenin Blanc grapes and grape skins. S. Afr. J. Enol. Vitic. 9: Jacobs, C. J., and H. J. J. Van Vuuren Effects of different killer yeasts on wine fermentations. Am. J. Enol. Vitic. 42: Longo, E., J. B. Velazquez, J. Cansado, P. Calo, and T. G. 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7 VOL. 59, 1993 KILLER YEASTS IN SPONTANEOUS FERMENTATIONS Rosini, G., and M. Cantini Killer character in Kluyveromyces yeasts: activity on Kioeckera apiculata. FEMS Microbiol. Lett. 44: Schwartz, D. C., and G. R. Cantor Separation of yeast chromosome-sized pieces of DNA by pulsed field gradient gel electrophoresis. Cell 37: Starmer, W. T., P. F. Ganter, and V. Aberdeen Geographic distribution and genetics of killer phenotypes for the yeast Pichia kluyveri across the United States. Appl. Environ. Microbiol. 58: Tiourina, L. V., N. I. Bourjan, and T. K. Skarikova Emploi des cultures pures du phenotype "killer" dans la fermentation des mouts de raisin. Bull. O.I.V. 53: Tredoux, H. G., R. P. Tracey, and A. Tromp Killer factor in wine yeasts and its effect on fermentation. S. Afr. J. Enol. Vitic. 7: Van Vuuren, H. J. J., and C. J. Jacobs Killer yeasts in the wine industry: a review. Am. J. Enol. Vitic. 43: Van Vuuren, H. J. J., and B. D. Wingfield Killer yeastscause of stuck fermentations in a wine cellar. S. Afr. J. Enol. Vitic. 7: Wingfield, B. D., L. J. Van Der Meer, I. S. Pretorius, and H. J. J. Van Vuuren K3 killer yeast is a mutant K2 killer yeast. Mycol. Res. 94: Woods, D. R., and E. A. Bevan Studies on the nature of the killer factor produced by Saccharomyces cerevisiae. J. Gen. Microbiol. 51: Woods, D. R, I. W. Ross, and D. A. Hendry A new killer factor produced by a killer/sensitive yeast strain. J. Gen. Microbiol. 81: Young, T. W Killer yeasts, p In A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 2. Academic Press, London. 3. Young, T. W., and M. Yagiu A comparison of the killer character in different yeasts and its classification. Antonie Leeuwenhoek 44: Downloaded from on April 15, 219 by guest