Metabolic diversity of Saccharomyces cerevisiae strains from spontaneously fermented grape musts

One hundred and fifteen Saccharomyces cerevisiae strains from Aglianico del Vulture, a red wine produced in Southern Italy, were characterized for the production of some secondary compounds involved in the aroma and taste of alcoholic beverages. The strains exhibited a uniform behaviour in the production levels of n-propanol, active amyl alcohol and ethyl acetate, whereas isobutanol, isoamyl alcohol and acetaldehyde were formed with a wide variability. Only five strains produced wines close to the reference Aglianico del Vulture wine for the traits considered. Of these, two strains were selected, underwent to tetrad analysis and the single spore cultures were tested in grape must fermentation. The progeny of one strain showed a significant metabolic variability, confirming the necessity to test starter cultures for the segregation of traits of technological interest. Our findings suggest the selection of specific strains for specific fermentations as a function of the vine variety characteristics in order to take the major advantage from the combination grape must/S. cerevisiae strain.     

掘氏疫霉遗传学研究 Ⅰ.自交后代(S_1)生物学性状的遗传与变异

测定了掘氏疫霉Phytophthora drechsleri Tucker生长速率、菌落形态、菌体形态、产孢能力、耐35℃高温生长、致病力等生物学性状在自交S_1代的遗传与变异。2个掘氏疫霉野生型菌株(A_1、A_2各1株)经聚碳膜间隔配对诱导自交产生的卵孢子(保存110天左右)在S+L培养基上萌发,分别获得30和35个单卵孢株(萌发率10%左右)。测定结果表明,上述生物学性状在2个亲本自交后代均发生变异,部分单卵孢株生长速度减慢、菌落形态明显改变、孢子囊和菌丝畸形、产孢能力增强、在35℃中弱生长或不生长,或致病力减弱直至无致病力。在亲本的单游动孢子无性系后代未观察到上述变异,说明上述生物学性状变异主要因亲本遗传因子的杂合性所致。从遗传学角度探讨了在自然条件下掘氏疫霉生物学性状的变异性以及某些分类依据不稳定的原因,指出有性生殖过程中的基因重组可能是引起掘氏疫霉生物学性状变异的主要原因之一。     

Inheritable nature of enological quantitative traits is demonstrated by meiotic segregation of industrial wine yeast strains

Wine yeast strains exhibit a wide variability in their technological properties. The large number of allelic variants and the high degree of heterozygosity explain this genetic variability found among the yeast flora. Furthermore, most enological traits are controlled by polygenic systems presenting complex interactions between the alleles. Taking this into account, we hypothesized that the meiotic segregation of such alleles from a given strain might generate a progeny population with very different technological properties. In this work, a population of 50 progeny clones derived from four industrial wine strains of Saccharomyces cerevisiae was characterized for three major enological traits: ethanol tolerance, volatile-acidity production and hydrogen sulphide production. For this purpose, reliable laboratory fermentation tests were developed in accordance with enological practice. A wide variability in the values of the various parameters was found among spore clones obtained after sporulation. Many clones presenting better aptitudes than the parental strains were obtained. Moreover, analysis of the progeny demonstrated that: (1) traits are in part inheritable; (2) traits are clearly polygenic; (3) broad relations of dominance/recessivity can be established. All these findings constitute an initial step for establishing breeding strategies for wine yeast improvement.     

Screening of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine strains for the production of acetic acid

In this study 80 wine strains of Saccharomyces cerevisiae were characterized for the production of acetic acid. A significant variability in the production levels was determined among the strains, which produced from a few mg/l to more than 1 g/l. Fifteen strains, differing in acetic acid production, were tested in fermentation of grape musts of different varieties (Aglianico Basilicata, Aglianico Apulia, Cannonau, Bombino nero, Nero d'Avola, Vermentino, Fiano). The results emphasized a great strain variability, but the best strain behaviour was strictly related to the must composition, which is a determinant factor on the expression of the best strain potentiality. Therefore, this study, confirming the high/low production of acetic acid as a strain characteristic, demonstrated also that the inoculated fermentation becomes more advantageous when the starter culture is chosen in relation to the interaction of yeast strain/vine variety.     

Cytological and genetic studies of the life cycle of Saccharomycopsis lipolytica

Summary In the alkane yeast Saccharomycopsis lipolytica (formerly: Candida lipolytica) the variability in the ascospore number is caused by the absence of a correlation between the meiotic divisions and spore wall formation. In four spored yeasts, after meiosis II, a spore wall is formed around each of the four nuclei produced by meiosis II. However, in the most frequently occurring two spored asci of S. lipolytica, the two nuclei are already enveloped by the spore wall after meiosis I due to a delay of meiosis II. This division takes place within the spore during the maturation of the ascus. In this case germination of the binucleate ascospore is not preceded by a mitosis. It follows that the cells of the new haploid clones are mononucleate. In the three spored asci, which occur rarely, only one nucleus is surrounded by a spore wall after meiosis I; the other nucleus undergoes meosis II before the onset of spore wall formation. The result is one binucleate and two mononucleate spores. In the one spored asci the two meiotic divisions occur within the young ascospore, i.e. spore wall formation starts immediately after development of the ascus. These cytological observations were substantiated by genetic data, which in addition confirmed the prediction that binucleate spores may be heterokaryotic. This occurs when there is a postreduction of at least one of the genes by which the parents of the cross differ. This also explains the high frequency of prototrophs in the progeny on non-allelic auxotrophs since random spore isolates are made without distinguishing between mono-and binucleate spores. The possibility of analysing offspring of binucleate spores by tetrad analysis is discussed. These findings enable us to understand the life cycle of S. lipolytica in detail and we are now in a position to start concerted breeding for strain improvement especially with respect to single cell protein production.     

Influence of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains on wine total antioxidant capacity evaluated by photochemiluminescence

A preliminary investigation on 20 Aglianico del Vulture commercial wines from the Basilicata region proved the existence of a significant variability in total antioxidant capacity which can exert a potential impact on wine quality. Nineteen Saccharomyces cerevisiae strains were tested in Aglianico del Vulture on pilot scale fermentation and the experimental wines obtained were evaluated for the antioxidant capacity, ethanol and total polyphenols. At the ninth day of fermentation the experimental wines had an antioxidant capacity, measured by photochemiluminescence, between 2.88 and 6.25 mM of ascorbic acid equivalent, ethanol concentration, measured by GC, between 5.49% and 10.99% and total polyphenols, determined by Folin Ciocalteau reagent, from 1153 to 1867 mg catechins/l. After 12 days the total antioxidant capacity was increased in most wines but decreased in some wines. These results, statistically analysed by principal component analysis, revealed a significant influence of S. cerevisiae strain on total antioxidant capacity and total polyphenols content of wine.     

Nonparental progeny resulting from protoplast fusion inTrichoderma in the absence of parasexuality

The purpose of this study was to characterize a number of progeny from intra- and interstrain protoplast fusion within the genusTrichoderma. We wished to determine whether parasexuality or other genetic mechanisms occur in these fungi. When two different auxotrophs of the same strain were fused, rapidly growing prototrophic progeny were obtained in high frequencies. When single spore isolates of these strains were prepared, equal numbers of strains indistinguishable from the two parental auxotrophic strains were obtained, even though 10⁹–10¹⁰ conidia were tested per strain. Thus, progeny from intrastrain fusions all appeared to be balanced heterokaryons, and no evidence of recombination between the two parental strains was obtained. When 16 separate interstrain fusions were conducted, very different results were obtained, regardless of whether fusions were within or between species. Following interstrain fusions, presumptive somatic hybrids developed very slowly and in low numbers as compared with hybrids from intrastrain fusions. Most were weakly prototrophic. These slow-growing progeny were unstable and sectors developed from them. Such sectors themselves were unstable and gave rise to other progeny. Usually sectors were more strongly prototrophic and more rapid growing than the original progeny strain. Sectoring gave rise to a very wide range of morphotypes. Most of these morphotype variants were stable through conidiation; thus, these types did not occur as a consequence of heterokaryosis. Isozyme analysis was conducted on over 1000 progeny strains. Nearly all progeny were identical to one or the other parental isozyme phenotypes. A few progeny, when tested as soon as possible after fusion, exhibited the isozyme phenotypes of both parents, but such biparental banding patterns were rapidly lost upon subsequent reculturing. Isozyme banding patterns of multimeric enzymes never gave band patterns indicative of heterokaryosis or heterozygosis. Banding patterns indicative of heterozygous diploids or recombinants were never detected. Despite the extreme variation in morphotype and nutritional requirements among progeny, isozyme banding patterns of derived progeny from any fusion were invariably identical to one or the other parental strains. From these results, we conclude that protoplast fusion in the genusTrichoderma gives rise to great variability, but that the classical parasexual cycle is not required for variation to occur.     

Spontaneous Conversion of Nontransformed Avian Sarcoma Virus-Infected Rat Cells to the Transformed Phenotype

Normal rat kidney (NRK) fibroblasts were infected with the Schmidt-Ruppin strain (SR-D) of avian sarcoma virus (ASV) and cloned 20 h after infection without selection for the transformed phenotype. Most infected clones initially exhibited the flat, nontransformed morphology that is characteristic of uninfected NRK cells. In long-term culture, however, the majority of the SR-D NRK clones began segregating typical ASV-transformed cells. Transforming ASV could be rescued by fusion with chicken embryo fibroblasts from most of the infected clones tested. Three predominantly flat, independently infected clones were further analyzed by subcloning 8 to 10 weeks after infection. Most flat progeny subclones derived at random from two of these "parental" SR-D NRK clonal lines did not yield virus upon fusion with chicken embryo fibroblasts, although a nondefective transforming ASV was repeatedly recovered from the parental clones. This observation suggested that most, but not all, daughter cells in these SR-D NRK clones lost the ASV provirus after cloning. The progeny of the third independent parental cell clone, c17, gave rise to both flat and transformed subclones that carried ASV. In this case, ASV recovery by fusion and transfection from the progeny subclones was equally efficient regardless of the transformation phenotype of the cells. The 60,000-dalton phosphoprotein product of the ASV src gene was, however, expressed at high level only in the transformed variants. The results of a Luria-Delbruck fluctuation analysis and of Newcombe's respreading test indicated that the event leading to the spontaneous conversion to the transformed state occurred at random in dividing cultures of these flat ASV NRK cells at a rate predicted for somatic mutation.     

Analysis of yeasts derived from natural fermentation in a Tokaj winery

The diversity of yeast flora was investigated in a spontaneously fermenting sweet white wine in a Tokaj winery. The non-Saccharomyces yeasts dominating the first phase of fermentation were soon replaced by a heterogeneous Saccharomycespopulation, which then became dominated by Saccharomyces bayanus. Three Saccharomyces sensu stricto strains isolated from various phases of fermentation were tested for genetic stability, optimum growth temperature, tolerance to sulphur dioxide, copper and ethanol as well as for the ability to produce hydrogen sulphide and various secondary metabolites known to affect the organoleptic properties of wines. The analysis of the single-spore cultures derived from spores of dissected asci revealed high stability of electrophoretic karyotypes and various degrees of heterozygosity for mating-types, the fermentation of galactose and the production of metabolic by-products. The production levels of the by-products did not segregate in a 2:2 fashion, suggesting that the synthesis of these compounds is under polygenic control.     

Construction from a single parent of baker's yeast strains with high freeze tolerance and fermentative activity in both lean and sweet doughs.

From a freeze-tolerant baker's yeast (Saccharomyces cerevisiae), 2,333 spore clones were obtained. To improve the leavening ability in lean dough of the parent strain, we selected 555 of the high-maltose-fermentative spore clones by using a method in which a soft agar solution containing maltose and bromocresol purple was overlaid on yeast colonies. By measuring the gassing power in the dough, we selected 66 spore clones with a good leavening ability in lean dough and a total of 694 hybrids were constructed by crossing them. Among these hybrids, we obtained 50 novel freeze-tolerant strains with good leavening ability in all lean, regular, and sweet doughs comparable to that of commercial baker's yeast. Hybrids with improved leavening ability or freeze tolerance compared with the parent yeast and commercial baker's yeasts were also obtained. These results suggest that hybridization between spore clones derived from a single parent strain is effective for improving the properties of baker's yeasts.     

Isolation of SPO12–1 and SPO13–1 from a Natural Variant of Yeast That Undergoes a Single Meiotic Division

ATCC4117 is a strain of S. cerevisiae that undergoes a single nuclear division during sporulation to produce asci containing two diploid ascospores (Grewal and Miller 1972). All clones derived from these spores are sporulation-capable and, like the parental strain, form two-spored asci. In this paper, we describe the genetic analysis of ATCC4117. In tetraploid hybrids of vegetative cells of the ATCC4117 diploid and a/a or α/α diploids, the production of two-spored asci is recessive. From these tetraploids, we have isolated two recessive alleles, designated spo12–1 and spo13–1, each of which alone results in the production of asci with two diploid or near-diploid spores. These alleles are unlinked and segregate as single nuclear genes. spo12–1 is approximately 22 cM from its centromere; spo13–1 has been localized to within 1 cM of arg4 on chromosome VIII. This analysis also revealed that ATCC4117 carries a diploidization gene allelic to or closely linked to HO, modifiers that reduce the number of haploid spores per ascus and alleles affecting the total level of sporulation.     

Spore ultrastructure in Sporothrix schenckii

Pathogenic strains of Sporothrix schenkii may show triangular spores, whose angular shape is maintained by a tiebeam effect in the inner cell wall structure. This difference in wall structure lies adjacent to a folded and possibly more active part of the spore cytoplasm. The supposed generation of asci in old cultures was simulated by the death of hyphae which are reinvaded by intrahyphal growth with intrahyphal spore production, while true asci were not seen.     

Improvement of a Wine Saccharomyces cerevisiae Strain by a Breeding Program

Hybridization by spore conjugation was used to develop new and improved wine yeasts of Saccharomyces cerevisiae. The procedure was achieved with diploid, homothallic strains with high sporulation frequency and high spore viability. The method was verified by crossing flocculent and non-H₂S-forming strains. Single-spore descendants of the hybrids were studied by tetrad analysis with regard to the aforementioned characters and the other two winemaking traits, i.e., ethanol production and fermentation rate. A highly flocculent, non-H₂S-forming wine yeast strain with a high fermentation rate and high ethanol production was obtained.     

优良面包酵母菌株的杂交育种

本文对实验室保存的面包酵母进行筛选,以不加糖面团发酵力最高的菌株BY-14和高糖面团发酵力最高的菌株BY-6作为两杂交亲本.二倍体菌株经过单倍体制备、分离和筛选后,利用群体杂交方法,获得了一株兼备两亲本优良性能的面包酵母菌株,其不加糖面团发酵力达到菌株BY-14水平,且高糖面团发酵力比菌株BY-6提高了25%.     

优良面包酵母菌株的杂交育种

本文对实验室保存的面包酵母进行筛选, 以不加糖面团发酵力最高的菌株BY-14和高糖面团发酵力最高的菌株BY-6作为两杂交亲本。二倍体菌株经过单倍体制备、分离和筛选后, 利用群体杂交方法, 获得了一株兼备两亲本优良性能的面包酵母菌株, 其不加糖面团发酵力达到菌株BY-14水平, 且高糖面团发酵力比菌株BY-6提高了25％。     

Establishment of a quasi-field trial in <Emphasis Type="Italic">Abies nordmanniana</Emphasis>—test of a new approach to forest tree breeding

This study used DNA markers to establish a quasi-field trial within a production Christmas tree stand produced from seed collected in an open-pollinated clonal seed orchard (CSO). A total of 660 offspring from the CSO, which comprised 99 clones of Abies nordmanniana, were genotyped with 12 microsatellites. Parentage was assigned successfully to 93% and 98% of the progeny at 95% and 80% confidence, respectively. The assignment rate declined only to 90% when the number of markers was reduced to 10. The distribution of parentage to the offspring among the CSO clones was highly skewed. The most successful clone was assigned as parent in 7% of the cases, and only 92 of the 119 potential parental genotypes were assigned as parents. The obtained pedigree was used to estimate breeding values for the CSO clones for five characters relevant for Christmas tree breeding. For high-heritability traits, such as flushing, accurate breeding values could be estimated for a considerable proportion of the clones. To estimate breeding values for low-heritability traits, such as Christmas tree quality score, more genotyped offspring will be required. The largest drawback of the method is the highly skewed distribution of parentage among the parents in the seed orchards, making it difficult to calculate breeding values for all clones. The approach seems well suited for tree breeding that puts more emphasis on pure selection of parental genotypes and less on estimating quantitative genetic parameters.     

Regulation of ribosomal RNA cistron number in a strain of Neurospora crassa with a duplication of the nucleolus organizer region

Some progeny from a cross of the translocation mutant T(VL→IVL)AR33 with wild-type Neurospora crassa are double nucleolus organizer (DNO) strains, usually displaying two distinct nucleolus organizer regions. The DNO strain is sterile but displays the same growth response as normal laboratory strains of Neurospora. We used DNA-DNA hybridization techniques to quantify the number of rRNA cistrons in the DNO mutant and its vegetative progeny. Comparisons of the rate of hybridization of genomic DNA from the parental AR33 strain and from the DNO strain showed that hybridization was more rapid for the DNO strain than for the parental strain. Successive vegetative progeny of the DNO strain displayed hybridization rates intermediate to those of the original DNO strain and the parental single nucleolus strain, indicating that the number of rRNA cistrons had decreased during vegetative propagation. Estimates of rRNA cistron number obtained from comparisons of the amount of single copy DNA and rDNA hybridized to genomic DNO and AR33 DNA at saturation indicate that the parental AR33 strain contains 225 copies of the rRNA repeat unit, while the DNO strain has approx. 440 copies. The number of rRNA cistrons decreases gradually in the successive vegetative progeny, approximating the parental haploid value by the eleventh vegetative transfer.     

Genetic segregation of natural Saccharomyces cerevisiae strains derived from spontaneous fermentation of Aglianico wine

AIMS: Investigation of the meiotic segregation of karyotypes and physiological traits in indigenous Saccharomyces strains isolated from Aglianico (South Italy) red wine. METHODS AND RESULTS: Segregation was studied in F1 and F2 descendants. Tetrads were isolated from sporulating cultures by micromanipulation. The spore clones were subjected to karyotype analysis by pulse-field gel electrophoresis (Bio-Rad model CHEF-DR II) and to various physiological tests. Certain chromosomes of the isolates showed 2:2 segregation patterns in F1 but proved to be stable in F2. The ability of cells to utilize maltose also segregated in a 2 : 2 manner in F1 and did not segregate in F2. Resistance to CuSO4, SO2 tolerance, the fermentative power and the production of certain metabolites segregated in both F1 and F2 generations and showed patterns indicating the involvement of polygenic regulation. CONCLUSIONS: The analysis revealed a high degree of genetic instability and demonstrated that meiosis can improve chromosomal and genetic stability. SIGNIFICANCE AND IMPACT OF THE STUDY: Winemaking is critically dependent on the physiological properties and genetic stability of the fermenting Saccharomyces yeasts. Selection of clones from F2 or later generations can be a method of reduction of genetic instability.     

Interfertility and genetic variability among European and North American isolates of the basidiomycete fungus Chondrostereum purpureum

The conspecificity of Finnish and western Canadian isolates of the decay fungus Chondrostereum purpureum was investigated by several approaches, including the assessment of genetic variability, mating and progeny analysis, and the analysis of selected phenotypic traits. Eight second-generation single spore strains per fungal isolate pairing were investigated with specific genetic markers developed for both Finnish and Canadian parental isolates. Tests of linkage disequilibrium were used to analyze whether these markers assorted independently among single spore strains. This procedure was similarly applied to the third-generation spore progeny. Finally, global non-metric multidimensional scaling was used to analyze independent random amplified microsatellite marker data to assess the genetic variability of the parental Finnish and Canadian isolates, and their second- and third-generation progeny. Our results revealed that the parental isolates from Finland and western Canada were genetically divergent, but no interfertility barriers were identified between these geographically distant fungi. Furthermore, parental genetic markers used in mating studies demonstrated that second- and third-generation spore progenies underwent normal meiosis and genetic recombination without linkage disequilibrium. Based on this work, the studied C. purpureum isolates from Finland and Canada can be considered as belonging to a single biological species, although genetic and limited phenotypic differentiation was observed.     

Sexual reproduction and soil tillage effects on virulence of Pyrenophora teres in Finland

Knowledge of the virulence of a pathogen population and recognition of the risks of changes in the virulence spectrum are essential in breeding crops for disease resistance. Sexual recombination in a pathogen increases the level of genotypic diversity and can influence the virulence spectrum. This study aimed to determine how sexual recombination can change virulence of the barley pathogen Pyrenophora teres and whether the barley cultivation system, no‐tillage or normal tillage, influences P. teres virulence. The inheritance of avirulence/virulence in P. teres following sexual reproduction was studied in three artificially created pathogen populations. The first was a product of crossing two net forms of the pathogen, and the second and the third were products of crossing net and spot forms. None of the progeny generated resembled the parents exactly. The average similarity of the progeny isolates of the net by net cross with the parental type, based on avirulence/virulence tests, was 92%. That for net and spot form progenies was 58% in comparison with the net form parents and 73% with the spot form parents. The virulence reactions of the progeny isolates did not correlate with morphological traits of the isolates: growth rate on agar, spore production, spore width, spore length and numbers of septa per conidium. To study the effect of the barley cultivation method on P. teres virulence, 313 single‐spore cultures were obtained from barley fields. Two hundred and seventy‐six of the isolates represented the spot form and 37 represented the net form of P. teres. No association was established between the tillage method and virulence for either the net form or the spot form isolates.