Molecular Characterization and Genotypic ... - Science Direct

System. Appl. Microbiol. 19, 393-402 (1996) © Gustav Fischer Verlag· Stuttgart· Jena . New York

Molecular Characterization and Genotypic Identification of Metschnikowia Species KSENIJA LOPANDIC 1, HANSJORG PRILLINGER 1, ORSOLYA MOLNAR 1, and GABRIELLA GIMENEZ-JURAD0 2 1 2

Institute of Applied Microbiology, University of Agriculture, NuRdorfer Linde 11, A-1190 Vienna Portugese Yeast Culture Collection, Gulbenkian Institute of Science, Apartado 14,2781 Oeiras Codex, Portugal Received January 31, 1996

Summary Ten species currently described in the genus Metschnikowia (M. agaveae, M. australis, M. bicuspidata, M. gruessii, M. hawaiiensis, M. krissii, M. lunata, M. pulcherrima, M. reukaufii, M. zobellii) were examined for their cell wall carbohydrate composition and ubiquinone type. Glucose and mannose are the only carbohydrate components identified, and Q-9 is the main coenzyme-Q system. The RAPD-PCR fingerprinting supported the separation of the genus in the species. According to the molecular features a proper position for the genus Metschnikowia among ascomycetous yeasts is proposed.

Key words: Metschnikowia - Cell wall carbohydrate composition - RAPD-PCR - Taxonomy

Introduction According to Miller and Phaff (1984) the genus Metschnikowia Kamienski is characterized by spheroidal to ellipsoidal cells, sometimes pyriform, cylindrical or lunate which propagate by multilateral budding. Pseudomycelium with oval conidia at the terminals may appear in the majority of the strains, and the presence of "air plane" or cross-formations has been observed recently (GimenezJurado, 1992). Typical needle-shaped ascospores appear singly or in pairs in elongate or morphologically distinct asci which develope directly from vegetative cells or chlamydospores. Based on the distinct morphological features and selective physiological tests, ten species are recognized in the genus Metschnikowia presently: M. agaveae, M. australis, M. bicuspidata, M. gruessii, M. hawaiiensis, M. krissii, M. lunata, M. pulcherrima, M. reukaufii, M. zobellii. Out of the ten currently described Metschnikowia species six have been isolated from terrestrial environment, mainly from flowers, fruits, and insects, while the others are aquatic-associated free-living organisms or invertebrate parasites (Table 1). The extents of divergences among partial sequences of rRNAs suggested no close relationship between the aquatic and terrestrial species (Mendon~a-Hagler et al., 1985, 1993). A common deletion in the 25S-635-initiated region (position 434 to

483) differentiates the genus Metschnikowia from other ascomycetous yeasts (Kurtzman, 1992; Mendon~a-Hagler et al., 1993). M. hawaiiensis and M. lunata exhibited somewhat larger deletions in the specific region, attesting their unique position within the genus Metschnikowia. The reassignment of these species to a separate new genus has been proposed by some authors (Mendon~a-Hagleret al., 1993; Yamada et al., 1994). Chemotaxonomical characterization of the genus is limited to the ubiquinone identification for some species; Q-9 is the principal Co-Q system reported (Yamada et al., 1977; Yamada and Nagahama, 1991; Gimenez-Jurado, 1992). Dor{ler (1990) and Prillinger et al. (1990a, b, 1991a, b, 1993) demonstrated that carbohydrate composition of the purified cell walls have significance in the differentiation of higher ranking taxa. Seven clusters were identified among more than 200 ascomycetous and basidiomycetous yeasts and yeast stages examined. The purpose of this study was to determine a proper position of the Metschnikowia species within these seven groups and to demonstrate their potential molecular similarities with other ascomycetous yeasts. A second goal was to determine DNA similarities among the described Metschnikowia species using RAPD-PCR analysis that has been

394

K. Lopandic, H. Prillinger, O. Molnar, and G. Gimenez-Jurado

shown to be a highly sensitive method in species delimitation (Messner et al., 1994). In addition, genotypic identification of new Metschnikowia isolates was undertaken.

Materials and Methods Yeast strains

The ascomycetous strains examined are listed in Table 1. All strains are maintained in the Culture Collection of the Institute of Applied Microbiology, University of Agriculture, Vienna, Austria. Cell cultivation, disintegration, and purification of cell walls were prepared as described by Prillinger et al. (1993).

Preparation of cell walls for gas-liquid chromatography

Preparation of samples for gas-liquid chromatography was carried out according to the slightly modified procedure of Siiiimiinen and Tammi (1988) and Prillinger et al. (1993). Acid hydrolisis of polysaccharides. Approximately 2 mg of powdered cell walls were suspended in 0.5 ml of 2M trifluoroacetic acid, overlaid with gaseous nitrogen, and hydrolyzed for 2 hours at 120°C. The sediment was separated by membrane filtration (0.45 !tm, Millipore, U.S.A.). Reducing of monosaccharides. To remove trifluoroacetic acid, 30 !tl of the supernatant together with 9 !tg of myoinositol (internal standard) was evaporated in a water-bath at 36°C under a stream of gaseous nitrogen. After twofold addition of 200 !tl methanol, the blow-off procedure was repeated. The residue was alkalized with 70 !tl of 1M ammonia, and 70 !tl of 4% NaBH 4

Table 1. List of ascomycetous strains used in analyses Species

lAM collection number!

Other designation or source 2

Isolation source

Arxula terrestris (van der Walt et Johannsen) van der Walt Ashbya gossypii Candida albicans (Robin) Berkhout Candida albicans

HA 670T HA 88 HA 138 HA671 T

ATCC 60136T IFG 0101 ATCC 10231 CBS 562T

Candida ernobii (Loder et Kreger-van Rij) S. A. Meyer et Yarrow Candida haemulonii (van Uden et Kolipinski) S. A. Meyer et Yarrow Candida intermedia (Ciferri et Ashford) Langeron et Guerra Candida nemodendra (van der Walt et al.) S. A. Meyer et Yarrow Debaryomyces hansenii (Zopf) Lodder et Kreger-van Rij Eremothecium ashbyi Holleya sinecauda (Holley) Yamada Kluyveromyces aestuarii (Fell) van der Walt Kluyveromyces africanus (van der Walt) Mastigomyces philippovi Imshenetskij et Kriss Metschnikowia agaveae M. A. Lachance Metschnikowia agaveae Metschnikowia australis (Fell and Hunter) Mendon~a-Hagler et al. Metschnikowia bicuspidata Kamienski

HA 159T

CBS 1737T

HA 153 T

CBS 5149T

soil, South Africa unknown, authentic strain man with bronchomycosis man with interdigital mycosis, Uruguay intracellular symbiont of Ernobius mollis gut of Haemulon sciurus

HA 410T HA 158 T

CBS 572T CBS 6280 T

HA 413 T HA 89 HA 661 T HA 58 T HA59 T HA 664T HA 641 T HA 642 1 HA 635 T

CBS 767T IFG 0101 CBS 8199 T CBS 4438 T CBS 2517 T CBS 7047T

HA 672 T

CBS 5575 T

Metschnikowia gruessii G. Gimenez-Jurado Metschnikowia gruessii (Griiss) (Sydow et Sydow) G. Gimenez-Jurado Metschnikowia gruessi Metschnikowia gruessi

HA 638 T HA 639

CBS 7657T CBS 611

HA640 HA 901

CBS 7659 CBS 7658

Metschnikowia hawaiiensis Lachance et al.

HA 643 T

CBS 7432 T

Metschnikowia Metschnikowia van Uden Metschnikowia Metschnikowia Metschnikowia Metschnikowia Metschnikowia Metschnikowia Metschnikowia

hawaiiensis krissii (van Uden et Castelo-Branco)

HA644 HA 634 T

CBS 4823 T

sea-water, USA

lunata Golubev pulcherrima Pitt et Miller pulcherrima pulcherrima pulcherrima pulcherrima pulcherrima

HA HA HA HA HA HA HA

186T 665 T 187 879 880 881 882

CBS 5946T CBS 5833 T CBS 5534 IFG 0101 IFG 0104 IFG 0105 IFG 0114

flower of Lathyrus sp., USSR berries of Vitis labrusca, USA

CBS 5847T

faeces, Puerto Rico tunnel of Xyleborus aemulus, South Africa Carlsberg Lab. cotton boll? authentic strain seed of Brassica juncea, Canada mud of estuary, USA soil, South Africa litter of pine and spruce forest, USSR Agave tequilana, Mexico Agave tequilana, Mexico sea water, Antarctic ocean sporocysts of Diplostomum flexicaudum flower of Hebe salicifolia, Portugal flower of Linaria vulgaris, Germany flower of Salvia sp., Portugal flower of Citrus monospeliensis, Portugal flower of Ipomoea accuminata, Hawaii

Ribes rubrum Ribes uva-crispa Citrus limon

Genotypic Identification of Metschnikowia

395

Table 1. Continued Species

lAM collection number l

Other designation or source 2

Isolation source

* Metschnikowia pulcherrima

HA 883 HA 666 T

IFG 0115 CBS 5834T

Metschnikowia reukaufii Metschnikowia reukaufii * Metschnikowia reukaufii * Metschnikowia reukaufii Metschnikowia zobellii (van Uden et Castelo-Branco) van Uden Metschnikowia zobellii Nematospora coryli Pachytichospora transvaalensis (van der Walt) van der Walt Pichia minuta (Wickerham) Kurtzman Saccharomyces bayanus Saccardo Saccharomyces castellii Capriotti Saccharomyces cerevisiae Hansen Saccharomyces exiguus Reess Saccharomyces paradoxus Batschinskaya Saccharomyces pastorianus Reess ex E. e. Hansen Saccharomyces servazzii Capriotti Saccharomyces servazzii Saccharomycopsis fibuligera (Lindner) Klocker Saccharomycopsis fibuligera Stephanoascus ciferrii M. T. Smith er al.

HA HA HA HA HA

CBS 5554 IFG 0201 IFG 0211 IFG 0215 IGC 4600

sputum flower of Epilobium angustifolium, Canada fruit of Rubus strigosus, Canada

Metschnikowia reukaufii Pitt et Miller

184 884 885 886 636

HA 637 T HA 99 T HA 667 T

CBS 4821 T IFG 0101 CBS 2186 T

HA 86 HA 239 T HA 408 T HA 227 T HA 85 T HA 405 T HA 452 T HA55 T HA 83 HA 104 HA 105 HA 668 T

IFG 1901 CBS 380T CBS 4309 T CBS 1171 T CBS 379 T CBS 432 T CBS 1538 T CBS 4311 T IFG 1001 IFG 0112 CBS 6310 CBS 5295 T

Novak Ribes uva-crispa sea-water, Portugal sea-water, USA hazel nuts soil, South Africa turbid beer soil, Finland brewer's top yeast, Netherland tree exudares beer soil, Finland maca rom pig, Netherlands

Note: Superscript T indicates the type strain; superscript I indicates the isotype 1 lAM: Institut flir Angewandte Mikrobiologie, Universitat flir Bodenkultur, Wien, Austria 2 ATCC: American Type Culture Collection, Rockville, Maryland - CBS: Centraalbureau voor Schimmelcultures, Baarn-Delft, The Netherlands - IFG: Institut flir Garungsgewerbe und Biotechnologie zu Berlin, Berlin, Germany - IGC: Portugese Yeast Culture Collection, Gulbenkian Institut of Science, Portugal - JCM: Japan Collection of Microorganisms, Riken, Saitama, Japan " According to our results these strains are excluded from the genus Metschnikowia

was added. The reaction mixture was let to stand overnight at room temperature. Purification of alditols. Excess sodium borohydride was decomposed by twofold additions of 50 1-11 of 2M acetic acid, 20 1-11 of 1% acetic acid in methanol, and 200 1-11 methanol. The resulting mixture was evaporated to dryness under a stream of nitrogen. Acetylation. The residues left were acetylated with 100 1-11 of acetic acid anhydride for 1 hour at 100°e. The remaining anhydride was removed by evaporation under nitrogen-stream. Purification of alditol acetates. A 500 1-11 portion of dichlormethane was used to dissolve alditol acetate residues. Extraction of salts with approximately 2 ml of bidistilled water was repeated four times. The dichlormethane was evaporated to dryness. Prior to a GLC-analysis the dried residue was dissolved in 50 1-11 of dichlormethane. Gas-liquid chromatography

Gas-liquid chromatography was performed with a Hewlett Packard model 5890 Series II gas chromatograph (Hewlett Packard, U.S.A.) equipped with a hydrogen flame ionisation detector. 1 1-11 of samples were injected into a type Rtx-225 capillary column (30 m, 0.25 mm ID, 0.1 I-Im film thickness; Restek Corp., Bellefonte, U.S.A.). Nitrogen was used as the carrier gas at a pressure 1.3 X 105 Pa. The oven temperature was programmed to

increase from 140 to 190°C at a rate of 20°C min-I, and then to 225°C at a rate of 3°C min· l . Isolation of genomic DNA

Chromosomal DNA was extracted by the phenol method as described by De Graaff et al. (1988) with minor modifications. A 20 mg portion of wet yeast cells grown on solid growth medium was suspended in 0.4 mt of lysis buffer containing 0.1 M Tris (hydroxymethyl)-aminomethan, 1.4 M NaCl, and 50 mM ethylenedinitrilotetraacetate (EDTA), pH 8.0 in a 1.5 ml Eppendorf cup. The suspension was frozen at -20°C and thawed three times. An aliquot of 0.4 ml of phenol was added, and the freezing-thawing procedures were repeated. The mixture was heated in a water-bath at 55°C for 10 min., and supplemented with 0.4 ml of chloroform. After heating at 55°C for 10 min. the mixture was centrifuged at 15,500 x g for 30 min. A 0.3 ml volume from the upper aqueous layer containing the nucleic acids was transfered into a new Eppendorf cup and precipitated with 0.45 ml of 2-propanol. The sediment containing DNA was separated from the protein-solution by centrifugation at 15,500 x g for 10 min. The pellet was washed with 0.8 ml of 70% (vol/vol) ethanol and subsequently dried under vacuum. The precipitated DNA was dissolved in 50 1-11 of 1 xTE buffer (10 mM Tris.HCl, 1 mM EDTA, pH 8.0). To control purity and concentration, 15 1-11 of the DNA preparation mixed with 5 1-11 of loading buffer (40% Suc-

396


rose, 0.1 % Bromphenol blue) was analysed by electrophoresis in 1% agarose gel run in 0.5 xTBE buffer (SAg 1- 1 Tris.HCl, 2.75 g 1- 1 Boric acid, 2 ml 0.5 M EDTA) at 6 V cm- l for 45 min. DNA preparation was diluted to a concentration of approximately 5 ng 1t1- 1 and stored at -20°C.

Random amplified polymorphic DNA (RAPD) assay Polymerase chain reaction (PCR) was performed with 2 Itl aliquot of a diluted DNA preparation (10 to 20ng DNA) and 28 Itl of PCR-mixture of the following composition: Stock solutions

End concentration in 30 Itl of PCR-mixture

sterile bidistilled water 10 x buffer pH 8.8 (100 mM KCl, 100 mM (NH4hS04' 200 mM Tris.HCl, 1% (wtlvol) Triton X-lOa) 0.1 M MgS0 4 10 mg ml- 1 Bovine serum albumin (BSA) 5 mM dNTPs (Promega, Madison, Wisconsin, U.S.A.) 0.1 g 1-1 Primer (decamers) (Codon Genetic System, Vienna, Austria) 5 U Itl- l Taq-DNA Polymerase (Biomedica, Vienna, Austria)

30 Itl 1 x buffer 4.5 mM 4.5 Itg 0.1 mM 30 ng 0.5 U

Template DNA and PCR-mixture were mixed in a 0.5 ml Eppendorf cup, overlaid with 50 Itl of mineral oil (Sigma Chemie GmbH, Gisenhofen, Germany) and processed with a Trio-Termoblock TB1 thermocycler (Biometra, G6ttingen, Germany). Amplification was performed under following cycling conditions: DNA-denaturation 98°C for 15 s annealing 32°C for 90 s extension 72 °C for 100 s A total of 40 cycles were performed in the analysis with three decamer primers synthesized by Codon Genetic Systems (Vienna, Austria) with a model 392 DNA synthesizer (Applied Biosystems, Foster City, California): ACGGTCTTGG (Primer 1), TGCAGCGTGG (Primer 2), and GGGTAACGCC (Primer 3), respectively. A 20 Itl aliquot of the reaction mixture was mixed with PCRstop solution (30% sucrose, 0.2% Bromphenol blue, 0.1 M EDTA), loaded, and DNA fragments were resolved by electrophoresis (5 Vcm- l ) in 1.3% (wt/vol) agarose gel run in 0.5 x TBE buffer supplemented with 0.5 Itg ml- l of ethidium bromide, for 3 hours. The gel image was photographed under UV light with polaroid camera. The percentages of similarity berween investigated strains were calculated according to the formula of Nei and Li (1979): similarity = 2 x bands in common/total of bands.

Results and Discussion The carbohydrate composition of purified yeast cell walls has been proven to be a valuable criterion in the delimitation of higher taxa (Darfier, 1990; Prillinger et al., 1990a, b, 1991a, b, 1993). It is generally accepted that all budding yeasts with characteristic glucose-mannose

profile cluster together within the Saccharomyces-type. Table 2. demonstrates results of the cell wall carbohydrate analysis and ubiquinone determination of fifteen ascomycetous genera: Arxula, Ashbya, Candida, Debaryomyces,

Eremothecium, Holleya, Kluyveromyces, Mastigomyces, Metschnikowia, Nematosp 0 ra, Pachytichospora, Pichia, Saccharomyces, Saccharomycopsis, and Stephanoascus.

Genera exhibiting Co-Q5, Co-Q6, Co-Q7, Co-Q8 and Co-Q9 cluster within one group and have glucose and mannose as the only sugar components. It is obvious that despite different morphological traits, distinct shapes of vegetative cells, formation of mycelium in some genera, presence or absence of asci, different ascospore-shape, and distinct physiological properties, all strains examined have cell wall carbohydrate composition in common. Despite qualitatively the same carbohydrate pattern, differences in relative amounts and relationships of glucose to mannose between some genera, and even within one genus are noted. Within the investigated strains some species show predominance of glucose (> 60%) in their cell walls, while the others exhibit preponderance of mannose or equilibrated amounts of both sugars. Presumptions that different hosts or natural surrounding media may influence to a certain extent both qualitative and quantitative organisation of the carbohydrate part of the cell wall within one yeast-type or even one genus is only a speculation. Price (1987) suggested that proton magnetic resonance-spectra of extracted mannans were not highly stable because a single-gene mutation might provoke alteration in the structure of the macromolecule. These assumptions should be taken in consideration by any estimation of the cell wall Table 2. Carbohydrate composition of purified yeast cell walls and major ubiquinone component of different species belonging to the Saccharomyces-type Species

Strain

Cell wall carbohydrate composition (mol%)

Co-Q system 1

Glucose

Mannose

HA 670 T

68

32

9

HA 88

60

40

6

HA HA HA HA HA HA

138 671 T 159 T 153 T 410T 158 T

53 55 57 50 34 41

47 45 43 50 66 59

9 9 8 9 9 7

HA 413 T

66

34

9

HA 89

78

22

6

Arxula

A. terrestris Ashbya

A.gossypii Candida C. albicans C. albicans

C.ernobii C. haemuloni C. intermedia C. nemodendra Debaryomyces

D.hansenii Eremothecium

E.ashbyii

Genotypic Identification of Metschnikowia Table 2. Continued Species

Strain

Cell wall carbohydrate composition (mol%)

Co-Q system 1

Glucose

Mannose

37

63

9

37 37

63 63

6

41

59

9

46 51 70 69 69 57 59 61 38 42 74 49 50 47 74 77

54 49 30 31 31 43 41 39 62 58 26 51 50 53 26 23

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

N. coryli

47

53

5

Pachytichospora P. transvaalensis

47

53

6

Holleya H. sinecauda

HA 661 T

Kluyveromyces

K. aestuarii K. africanus

6

Mastigomyces M. philippovi

Metschnikowia

M.agaveae M.agaveae M. australis M. bicuspidata M.gruessii M.gruessii M.gruessii M.gruessii M. hawaiiensis M. hawaiiensis M. krissii M.lunata M. pulcherrima M. reukaufii M.zobellii M. zobellii

HA 641 T HA64i HA 635 T HA 672 T HA 638 T HA 639 HA 640 HA 901 HA 643 HA644 HA 634T HA 186T HA 665 T HA 666 T HA 636 HA 637T

Nematospora

Pichia

P.minuta

HA 86

44

56

7

Saccharomyces S. bayanus S. castellii S. cerevisiae S. exiguus S. paradoxus S. pastorianus S. servazzii S. servazzii

HA 239 T HA 408 T HA 227 T HA 85 T HA 405 T HA 452 T HA SST HA 83

33 48 42 40 27 36 50 48

67 52 58 60 73 64 50 52

6 6 6 6 6 6 6 6

Saccharomycopsis S. fibuligera S. fibuligera

HA 104 HA 105

55 51

45 49

8

Stephanoascus S. ciferrii

HA 668 T

68

32

9

1

Data from references: Yamada and Kondo, 1972; Yamada et aI., 1973, 1976, 1977, 1981; Lee and Komagata, 1980; Yamada, 1986; Gimenez-Jurado, 1992; and personal results

27 System. Appl. Microbiol. Vol. 19/3

397

monosaccharide composition, especially when a relationship of the individual sugars has importance in delimitation of the yeast groups (e.g. Ustilago- and Microbotryumtype; Prillinger et aI., 1993; Messner et aI., 1994). In any case, the defining of specific assemblage based on the determination of the cell wall carbohydrate composition has been shown to be valuable approach to the estimation of the different evolutionary events within the yeasts, and therefore such an approach is increasingly being used in improving yeast taxonomy. Results of our investigation with yeasts and yeast stages of 15 different ascomycetous yeasts and yeast-like fungi demonstrate that glc-man group comprises beside budding yeasts, two filamentous plant pathogens Ashbya gossypii, and Eremothecium ashbyi, and dimorphic yeasts Holleya sinecauda as well as Nematospora coryli. Recently Messner et al. (1995), based their analysis on partial nucleotide sequences of 18S and 25S rDNA, and ITS-regions, supported the inclusion of the genera Ashbya, Eremothecium and Nematospora within the family Saccharomycetaceae. These results are consistent with exhaustive work of Kurtzman and Robnett (1994) who have indicated that yeasts and yeast-like fungi constitute a monophyletic group separate from the filamentous Ascomycetes. Our studies with yeast cell walls corroborate the investigations relied on the determination of the genetic relationships among yeasts. Traditionally, phenotypic features represented the main criteria used to separate taxa and discern relationships among organisms. More recently, powerful biochemical techniques permit us to examine structural features of more reliable cell characters, such as cell wall carbohydrates, ubiquinones, DNA and RNA. Numerous advances in this direction contributed to an improved approach to evolutionary divergence in yeasts. The outcome of these studies on phylogenetic inferences has been widely discussed (e.g. Prillinger et aI., 1993; Kurtzman and Robnett, 1994). All strains examined belonging to the 10 species currently accepted in the genus Metschnikowia cluster within the Saccharomyces-type. The species could be separated in two groups with respect to their cell wall carbohydrate composition (Table 2.). The first group characterized by a predominance of glucose over mannose is comprised mainly of aquatic species with the exception of M. gruessii isolated from flowers. Terrestrial species, which make the second group, have equilibrated amount of mannose and glucose, whereas by M. hawaiiensis a light preponderance of mannose was noted. Similar observations were reported by Mendon~a-Hagler et al. (1993) who studying Metschnikowia species of different habit-origin, recognized two distinct phylogenetic groups within the genus from ribosomal RNA sequence data (M. agavae and M. gruessii were not examined). M.lunata and M. hawaiiensis were shown to be separated from other Metschnikowia species and their reassigment to a new genus was discussed (Mendon~a-Hagler et aI., 1993; Yamada et aI., 1994). Analysing cell wall monosaccharide composition we expected different carbohydrate pattern in M. lunata and M. hawaiiensis in comparison with other Metschnikowia species. However, the obtained values are not significantly different to show distinct lineage from other species indi-

398


of M. hawaiiensis, are the only characters, that define phylogenetic distances of these organisms at present. Recently, Molnar et al. (1995) using RAPD-PCR analysis, were aple to distinguish 10 genetically distinct species within 66 investigated Saccharomyces strains. The same approach was applied in the present study to evaluate the genomic similarities among Metschnikowia species. Three randomly selected primers (decamers) were used in the amplification reaction of genomic DNA isolated from 16

eating that estimation of cell wall carbohydrate composition is the method of limited value, very usefull in discriminating higher taxa, but in species characterization genome comparison is the method of ,choice. Neither monosaccharide composition nor coenzyme-Q analysis were able to distinguish M. lunata and M. hawaiiensis from the other species in the genus. It seems that genomic differences which coincide with morphological appearance, lunate shape of M. lunata, and large ascospore size 1

2 3 4

5 6 7 8 9 10 St 111213 14 1516 1718 19 20 St 21 22 2324252627282930 St

- 5.080 - 2.840 1.700 -

1.159

-

0.805

-

0.516

KB Fig. 1. PCR amplified genomic DNA of the Metschnikowia type strains primed with Primer 1 (lane 1, M. australis HA 63S T ; lane 2, M. bicuspidata HA 672T j lane 3, M. krissii HA 634 T ; lane 4, M. zobellii HA 637 T ; lane 5, M. agaveae HA 641 T j lane 6, M. gruessii HA 638 T j lane 7, M. hawaiiensis HA 643 T j lane 8, M.lunata HA 186 T j lane 9, M. pulcherrima HA 665 T ; lane 10, M. reukaufii HA 666 T ), Primer 2 (lane 11, M. australis HA 635 T j lane 12, M. bicuspidata HA 672 T j lane 13, M. krissii HA 634 T ; lane 14, M. zobellii HA 637T ; lane 15, M. agaveae HA 641 T ; lane 16, M. gruessii HA 638\ lane 17, M. hawaiiensis HA 643 T j lane 18, M.lunata HA 186T j lane 19, M. pulcherrima HA 665\ lane 20, M. reukaufii HA 666 T ), and Primer 3 (lane 21, M. australis HA 635 T ; lane 22, M. bicus~idata HA 672 T j lane 23, M. krissii HA 634 T ; lane 24, M. zobellii HA 637 T ; lane 25, M. agaveae HA 641\ lane 26, M. gruessii HA 638 j lane 27, M. hawaiiensis HA 643 T ; lane 28, M. lunata HA 186T ; lane 29, M. pulcherrima HA 665 T ; lane 30, M. reukaufii HA 666 T ). Lane St represents ADNA digested with Pst!'

1

2 3 4

5 6 7 8 9 10 11 12 St 13 14 1516 1718

- 5.080 - 2.840 1.700 1.159 - 0.805 -

0.516

KB

Fig. 2. RAPD-PCR-fingerprints of M. zobellii, M. agaveae, and M. hawaiiensis generated by Primer 1 (lane 1, M. zobellii HA 637 T j lane 2, HA 636; lane 3, M. agaveae HA 641 T , lane 4, HA 642 1j lane 5, M. hawaiiensis HA 643 T j lane 6, HA 644), Primer 2 (lane 7, M. zobellii HA 637 T j lane 8, HA 636j lane 9, M. agaveae HA 641 T j lane 10, HA 642 1j lane 11, M. hawaiiensis HA 643 T j lane 12, HA 644), and Primer 3 (lane 13, M. zobellii HA 637T j lane 14, HA 636; lane 15, M. agaveae HA 641 T j lane 16, HA 642 1j lane 17, M. hawaiiensis HA 643 T ; lane 18, HA 644). Lane St represents ADNA digested with PstI.

Genotypic Identification of Metschnikowia 1 2 3

4

399

Metschnikowia strains as described under Materials and Methods. Figures 1, 2, and 3 display DNA patterns of different Metschnikowia type strains, and additional isolates of different origin generated by the RAPD-PCR analysis. Genomic similarity values were calculated individually for each primer and mean values are presented in Tables 3, 4, and 5. It is evident that genetically distinct species are separated by the level of similarity lower than 30%, and most species exhibit the values below 20%. According to these results the genus Metschnikowia is comprised of ten species separated by their genomic features. Conspecific strains, like strains of M. zobellii (Fig. 2, lanes 1,2,7,8,13,14), M. agaveae (Fig. 2, lanes 3, 4,9, 10, 15, 16), M. hawaiiensis (Fig. 2, lanes 5,6, 11, 12, 17, 18), and M. gruessii (Fig. 3) show levels of DNA similarity higher than 70% (Table 4, 5). These results are supported by the recently published reports with Mrakia and Sterigmatomyces strains (Messner et al., 1994) and Saccharomyces strains (Molnar et al., 1995), respectively. The results of Messner et al. (1994) using a broader spectrum of ascomycetous yeasts, together with the present study, indicate that similarity values in the range of 30-50% might be considered characteristic of closely related species. Strains exhibiting similarity values of 50% or higher are considered conspecific. Two strains of M. gruessii, HA 640 and HA 901, with the similarity values of

5 6 7 8 9 10 11 12 St

- 5.080 - 2.840 1.700 - 1.159 - 0.805 - 0.516

KB Fig. 3. RAPD-PCR-fingerprints of M. gruessii strains generated by Primer 1 (lane 1, HA 638 T ; lane 2, HA 640; lane 3, HA 901; lane 4, HA 639), Primer 2 (lane 5, HA 638 T ; lane 6, HA 640; lane 7; HA 901; lane 8, HA 639), and Primer 3 (lane 9, HA 638 T ; lane 10, HA 640; lane 11, HA 901; lane 12, HA 639). Lane St represents ADNA digested with Pst!'

Table 3. Levels of similarity among Metschnikowia type strains determined by RAPD-PCR fingerprinting % similarity with strain:

Strains

M. australis HA 63S T M. bicuspidata HA 672 T M. krissii HA 634T M. zobel/ii HA 637T

HA 63S T

HA 672 T

HA 634 T

HA 637T

HA 641 T

HA 638 T

HA 643 T

HA 186T

HA 66S T

HA 666 T

100

18 100

18 3 100

16 16 6 100

20 14 23 19 100

23 8 27 21 28 100

19 13 21 20 25 18 100

14 20 9 28 13 27 21 100

6 23 29 29 29 18 27

10 23 14 13 18 14 11

100

19 100

M. agaveae HA 641 T M.gruessii HA 638 T M. hawaiiensis HA 643 T M.lunata HA 186T M. pulcherrima HA 66S T M. reukaufii HA 666 T

Table 4. Levels of similarity among strains of M. zobel/ii, ,\-/. agaveae, and M. hawaiiensis determined by RAPD-PCR fingerprinting % similarity with strain:

Strains

M. zobellii HA 637T HA 636 M. agaveae HA 641 T HA64i M. hawaiiensis HA 643 T HA 644

HA 637T

HA 636

HA 641 T

HA 642 1

HA 643 T

HA 644

100

93 100

19 14 100

18 16 73 100

20 14

20 12 24 20 96 100

25

22 100

25

15

400


Table 5. Levels of similarity among M. gruessii strains determined by RAPD-PCR fingerprinting % similarity with strain:

Strains

M.gruessii HA HA HA HA

638 T 640 901 639

HA 638 T

HA 640

HA 901

HA 639

100

71 100

72 95 100

56 48 46 100

71 and 72%, respectively with the type strain (HA 638 T ) clearly demonstrate conspecificity (Fig. 3, lanes 1, 2, 3, 5, 6, 7, 9,10,11; Table 5). The third strain, HA 639, showed an unexpectedly low DNA similarity value of 56% with the type strain (Fig. 3, lanes 1,4,5, 8,9, 12; Table 5), and somewhat lower values with strains HA 640 and HA 901, 46 and 48%, respectively (Fig. 3, lanes 2, 3, 4, 6, 7, 8, 10, 11, 12; Table 5). The greatest difference among HA 639 strain and other M. gruessii strains is noted in the DNAfingerprints raised with the Primer 2 (Fig. 3, line 5, 6, 7, 8). Fragments of higher molecular size were amplified in the HA 639 strain, while the main PCR products in the other M. gruessii strains were of lower molecular weights «1 1 2 3 4

kb). It could be that differences in the primary structure of the DNA from the type strains HA 638 T and strain HA 639 are more accentuated when Primer 2 is used in PCR. Strain HA 639 originally described as Nectaromyces reukaufii (Griiss) Sydow et Sydow (Sydow and Sydow, 1918) gave a very high reassociation value (97%) with the type strain of M. gruessii (Gimenez-Jurado, 1992). This strain represents the anamorphic stage of a perfect yeast. Comparison of nuclear DNA relatedness with RAPD-PCR analysis (Messner et al., 1994) attests to the inherent differences in resolution detected by diverse methologies; RAPD analysis is most powerful in differentiating strains. The differences in similarity values for M. gruessii strains, HA 638 T and HA 639, obtained with two methods, are somewhat high, even though a similarity value above 50% could be considered as confirmation of conspecificity. The reason for this discrepancy probably lies in the specificity of the methods and inherent strain variance. Of the four strains tested, HA 639 was the only anamorphic strain investigated, and is the only strain of M. gruessii isolated from a different geographic region. Additional investigations combined with sequencing data should resolve any dubious results. Six strains phenotypically identified as M. pulcherrima (HA 187, HA 879, HA 880, HA 881, HA 882, HA 883), and four strains originally specified as M. reukaufii (HA

5 6 7 8 91011 12St13 14 15161718 St

- 5.080 - 2.840 1.700 1.159 - 0.805 - 0.516

KB

Fig. 4. Identification of M. pulcherrima strains using RAPD patterns generated by Primer 1 (lane 1, HA 665 T ; lane 2, HA 187; lane 3, HA 880; lane 4, HA 881; lane 5, HA 882; lane 6, HA 879), Primer 2 (lane 7, HA 665 T ; lane 8, HA 187; lane 9, HA 880; lane 10, HA 881; lane 11, HA 882; lane 12, HA 879), and Primer 3 (lane 13, HA 665 T ; lane 14, HA 187; lane 15, HA 880; lane 16, HA 881; lane 17, HA 882; lane 18, HA 879). Lane St represents "DNA digested with Pst!'

Table 6. Percentages of similarity among M. pulcherrima type strain and its nature isolates Strains

% similarity with strain:

M. pulcherrima HA 665 T HA 187 HA 880

HA 881 HA 882 HA 879

HA 665 T

HA 187

HA 880

HA 881

HA 882

HA 879

100

65 100

69 72

69 72

68 66

53 54

100

66 100

52 59 100

100

100

66

52

Genotypic Identification of Metschnikowia 1

2 3

4

5 6

7 8

401

cherrima or M. reukaufii. Further investigations are required to elucidate their position.

9 St

- 5.080 - 2.840 1.700 1.159

Acknowledgements. The authors wish to thank Dr. R. Messner for his help and fruitful suggestions in the initial stage of this study. We thank also Prof. Dr. I. Spencer-Martins, Prof. Dr. U. Stahl and K. Scheide for providing strains. The research was supported by a grant from the ]ubilaumsfonds der Osterreichischen Nationalbank.

- 0.805 - 0.516

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KB Fig. 5. Identification of M. reukaufii strains using RAPD patterns generated by Primer 1 (lane 1, HA 666 T ; lane 2, HA 184; lane 3, HA 884), Primer 2 (lane 4, HA 666 T ; lane 5, HA 184; lane 6, HA 884), and Primer 3 (lane 7, HA 666 T ; lane 8, HA 184; lane 9, HA 884). Lane St represents ADNA digested with Pst!'

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Strains

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HA 666 T

HA 184

HA 884

100

51 100

56 63 100

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Prof. Dr. Hansjorg Prillinger, University of Agriculture, Institute of Applied Microbiology, NuBdorfer Linde 11, A-1190 Vienna