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Molecular and Physiological Investigations of Exophiala Species Described from Fish
System. Appl. Microbiol. 20, 585-594 (1997) © Gustav Fischer Verlag
Molecular and Physiological Investigations of ExophiaJa Species Described from Fish J. M. J. UIJTHOF, M. J. FIGGE, and G. S.
DE HOOG
Centraalbureau voor Schimmelcultures, Baarn, The Netherlands Received.
Summary Eighteen strains of black yeasts, phenetically identified as Exophiala pisciphila, E. psychrophila or E. salmonis, were compared using ITS1 sequencing and RFLP of PCR-amplified ribosomal genes; in addi-
tion, their physiological profiles were determined. One strain contained an insert in its SSU ribosomal gene. The strains were found to have very similar SSU rDNA RFLP patterns, but to be heterogeneous in ITS RFLP patterns and ITS1 sequences. Physiological data corresponded with these findings. A distinction of species in psychrophilic Exophiala species remains difficult.
Key words: Black yeasts - Taxonomy - ITS1 sequences - PCR RFLP - Nutritional physiology - Fish pathology - Soil fungi
Introduction The black yeast genus Exophiala was erected by CARMICHAEL (1966) on the basis of E. salmonis Carmichael, a species causing a systemic infection in trout. Subsequently, two further fish pathogens were added, E. pisciphila McGinnis & Ajello (MCGINNIS and AJELLO, 1974) and E. psychrophila Pedersen & Langvad (PEDERSEN and LANGVAD, 1989), as well as species from other sources (for reviews see DE HOOG and MCGINNIS, 1987; UIJTHOF and DE HOOG, 1995). Species delineation in the genus Exophiala is problematic because of the limited number of reliable morphological characteristics. Hence a wide array of nonmorphological techniques have been introduced, among which are karyology (TAKEO and DE HOOG, 1991), coenzyme Q analysis (SUGIYAMA et al., 1987; YAMADA et al., 1989; GOTO et al., 1981), RFLP of mtDNA (KAWASAKI et al., 1990; 1993), DNA-DNA hybridization (MASUDA et al., 1989), SSU rDNA sequencing (HAASE et al., 1995; SPATAFORA et al., 1995) and LSU rRNA sequencing (MASCLAUX et al., 1995). For the distinction of type isolates of recognized and synonymized species in the genus Exophiala and related taxa we tested nutritional physiology (DE HOOG et al., 1995) and applied molecular techniques like PCR-RFLP (UIJTHOF and DE HOOG, 1995) and ITS1 sequencing (UIJTHOF, 1996). Nearly all described species were proven to represent separate entities. The three species from fish
clustered in a monophyletic branch at some distance from the remaining Exophiala species (UIJTHOF, 1996). The separate position of these species was suggested earlier on the basis of coenzyme Q hydrogenation (YAMADA et al., 1989). Their phylogenetic relationship might result from a shared ecological niche in cold environments. To elucidate their taxonomic structure, morphology, physiology and molecular characteristics of 18 strains previously identified as Exophiala salmonis, E. pisciphila or E. psychrophila were studied.
Materials and Methods Strains and culture conditions: The strains studied are listed in Table 1. Stock cultures were maintained on oatmeal agar at 4°C. Strains were grown for 7 days in 100 mlliquid 2% malt extract / 0.5% yeast extract medium on a rotary shaker at 300 rpm at 28°C. Nutritional physiology: Growth and fermentative abilities were tested in duplicate in liquid medium according to V AN DER WALT and YARROW (1984) with modifications according to DE HOOG et al. (1995). Assimilation tests were performed in liquid medium in test tubes at 25°C, rocked in a nearly horizontal position at 50 rev. min-I. Biomass production was verified weekly, with final reading 2 wk after the exhaustion of the C-source in the glucose assimilation test tube. Fermentation was tested in glucose over a 2 wk period in vertical test tubes with Durham
J. M. J. UIJTHOF et al.
586
Table 1. List of strains studied. Strains 100.68 537.73 515.76 660.76 661.76 217.79 260.80 404.81 159.89 160.89 256.92 150.93 191.87 587.66 157.67 665.76 510.81 dH9733
T
T T T
Initial identification
Source
Geographic origin
E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila aff. E. pisciphila E. psychrophila E. brunnea E.salmonis E. salmonis E. species E. species
Plant, Pisum sativum Fish, kidney Soil Rhizosphere of Triticum aestivum Cyst nematode Soil Fish, kidney Soil Root, Hordeum vulgare Root, Hordeum vulgare Fish, kidney Washed root, Tilia platyphylla Fish, kidney Leaf litter Fish, brain Wood Compost Plant, Triticum aestivum
The Netherlands USA Canada Australia Germany Chile Norway The Netherlands The Netherlands The Netherlands Ireland Germany Norway South Africa Canada Chile Austria Italy
inserts kept stationary apart from brief manual shaking every 2 days prior to reading. Urease activity (V AN DER WALT and Y ARROW, 1984) was tested on Christensen's agar. Gelatin (12%; GistBrocades, Delft, The Netherlands) liquefaction was tested on agar plates, using HgCIz in 7.6% HCl as a reagent. Thermotolerance was tested with 4% malt extract agar (MEA) slants inoculated with fresh cell suspensions. Cycloheximide tolerance was tested both in liquid medium at 0.01, 0.05 and 0.1 % (VAN DER WALT and YARROW, 1984) and on commercial Mycosel agar (0.04%; Becton Dickinson, Cockeysville, USA). Extracellular DNAse medium was provided by Difco (Detroit, USA). Halotolerance was tested in rocked liquid malt medium containing 2.5, 5 and 10% (w:v) NaCl or MgCI 2 • DNA-Isolation: Two ml of cell suspension were centrifuged at 14000 rpm and the cells were washed twice with sterile water. The resulting pellets (approximately 100 Ill) were stored at -70°C. Total DNA was extracted by using a miniprep protocol (UIJTHOF and DE HOOG, 1995). The pellet was dissolved in 0.5 ml TES buffer (0.1 mM Tris-HCl, pH 8.0; 10 mM EDTA; 2% SDS), and approximately 400 III of glass beads (<1> 0.45-0.50 mm) were added. After homogenization, the mixture was vortexed for at least 10 min. Proteinase K was added and the mixture was incubated for 30 min at 55-60 °C. After centrifugation, the supernatant was transferred to a new tube. Subsequently the procedure of MOLLER et al. (1992) was followed, starting with the addition of hexadecyltrimethylammoniumbromide (CTAB; Sigma) in high salt concentration. The resulting DNA pellet was dissolved in water and the DNA concentration was determined spectrophotometrically. DNA Amplification: PCR was performed in 50 III volumes of a " Tris-based (10 mM, pH 8.3) reaction mixture (50 mM KCI, 1.5 mM MgCh, 0.01 % gelatin) containing 200 IlM of "each deoxynucleotide triphosphate, 50 pmol of each primer, 10-100 ng nuclear DNA and 0.25 U of Taq DNA polymerase (Super Taq; Sphaero Q, Leiden, The Netherlands). Restriction analysis: The amplicons were digested for at least 2 h with one of the following restriction enzymes (1 U): DdeI, HaeIII, HhaI, HinfI, NdeII, MspI, RsaI and Taqi. Digests were subjected to electrophoresis on 2% agarose gels (TAE buffer; SAMBROOK et al., 1989), after which the gels were stained with ethidium bromide and photographed.
Sequencing: Primers v9 and its4 (WHITE et al., 1990) were emplqyed. Forty amplification cycles were performed: 94°C, 1 min; 48 °C, 2 min; 74°C, 3 min with an initial delay of 1 min at 94°C in a Biomed thermocycler (type 60). The remaining primers and dNTPs were removed by isolating the amplicons with Geneclean according to instructions given by the manufacturer. The amplicon was sequenced with a primer terminating protocol on an ABI automatic sequencer with primers its1 and its2 (WHITE et al.,1990). Alignment and phylogenetic analysis: The sequences obtained with both primers were aligned with Clustal V and adjusted by using the DCSE program (DE RIJK and DE WACHTER, 1993). The phylogenetic tree was calculated with several distance methods. The final tree was constructed using the Neighbor Joining method of the Treecon package (VAN DE PEER and DE WACHTER, 1993). Bootstrap values of over 75 are indicated near the branches.
Results PCR amplification of the relatively conserved SSU rRNA genes, using primers ns1 and ns24, resulted in amplicons of 1800 bp, except for CBS 157.67 (E. salmonis) which had an amplicon of about 2100 nucleotides (Table 2). The RFLP patterns obtained after Hinfl, N dell and DdeI digestion of amplicon NS1-NS24 were identical for all isolates with an amplicon length of 1800 nucleotides. With enzyme HaeIII, isolates CBS 587.66 and 660.76 each had a unique pattern. PCR amplification of the more variable ITS region, covering the internal transcribed spacers and the 5.8S rRNA gene, using primers its1 and its4, resulted in amplicons of 600 nucleotides for all isolates. The restriction patterns of these amplicons are listed in Table 3 and displayed graphically in Fig. 1. The type strains of the species investigated in the present study are all in the center. A phylogenetic tree based on ITS1 sequences is presented in Fig. 3. Close similarity in RFLP patterns corre-
Molecular and Physiological Investigations Table 2. RFLP patterns of amplicon NSI-NS24. Strains 100.68 537.73 515.76 660.76 661.76 217.79 260.80 404.81 159.89 160.89 256.92 150.93 191.87 587.66 157.67 665.76 510.81 dH9733
T
T T T
Amplicon length
HinfI
HaeIII
DdeI
NdeIl
1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 2100 1800 1800
A A A A A A A A A A A A A A B A
A A A B A A A A A A A A A C D A A
A A A A A A A A A A A A A A B A A
A A A A A A A A A A A A A A B A A
587
Results of physiological tests are shown in Table 4. Most strains assimilate D-glucosamine relatively well; soluble starch is not or poorly utilized except by dH9733. Growth responses to lactose and inulin are variable. Erythritol, nitrate, creatine and creatinine are assimilated, though sometimes weakly. All strains were tolerant to cycloheximide but relatively intolerant to salt, mostly growing only weakly with 5% NaCl. Only CBS 510.81 grew weakly at 3rC; growth at 30°C was observed in CBS 587.66, 150.93, 217.79 and 537.73. A numerical comparison of the physiological dataset (Fig. 4) indicates a close similarity between CBS 256.92 and 191.87, and between CBS 159.89, 160.89,515.76 and 661.76, with CBS 150.93 at some distance. The type strain of E. pisciphila, CBS 537.73 was physiologically similar to CBS 510.81, while that of E. salmonis CBS 157.67, was similar to CBS 665.76. Because of their long terminal branches, the cluster of isolates CBS 660.76, 217.79 and 100.68 is not regarded as being homogeneous.
Discussion sponded with short distances in the phylogenetic tree based on ITS1 sequencing (Fig. 3). The ITS1 sequences of the strains were variable in 70 positions. Strains CBS 159.89,160.89,515.76 and 661.76 were identical to each other. Strains dH9733 and CBS 150.93 were closely related to this group, but differed in 1 and 5 positions, respectively. The ITS1 sequence of CBS 256.92 was identical to that of CBS 191.87, the type strain of E. psychrophila. A third group, composed of strains CBS 510.81 and 217.79, was interconnected by rather long branches. Strains CBS 157.67, 660.76, 587.66 and 537.73 were found at relatively long distances to all other isolates studied.
YAMADA et al. (1989) and SUGIYAMA et al. (1987) analyzed coenzyme Q systems of numerous black yeasts. Species originating from fish were found to differ from the remaining Exophiala species in having Co-QlO(H 2 ) rather than QlO systems. With ITS1 phylogeny the fish-associated strains clustered together in a monophyletic branch at some distance from the core of the genus (UUTHOF, 1996). In the present study we analyzed the strains composing this branch, also comprising a number of similar strains from soil and leaf litter. In their original descriptions, the species Exophiala salmonis, E. pisciphila and E. psychrophila were characterized by 1-3 septate conidia, 0-1 septate conidia and a maximum growth temperature of 23°C, respectively. The
Table 3. RFLP patterns of amplicon ITSI-ITS4. Strains 100.68 537.73 515.76 660.76 661.76 217.79 260.80 404.81 159.89 160.89 256.92 150.93 191.87 587.66 157.67 665.76 510.81 dH9733
T
T T T
Amplicon length
RsaI
NdeIl
TaqI
MspI
HaeIII
HinfI
HhaI
DdeI
600 600 600 600 600 600 600 600 600 600 606 600 600 600 600 600 600 600
A B A B A
A B B
A B A B A B
A B A
A B B
C
C
A A A B A B B A A B A
A A A
A A A A A B B A A A B A B A A D B A
A A A A A B A A A A A A A B A A B A
A A A A A B A A A A A A A A A A B A
C
A A A C
A C
D A A C
A
C
B A B A B B B A B A A A A B
A C
C
A C
B B B C
A
B B B A B B B B B A A A B B
Galactitol myo-Inositol Glucono-o-lactone keto-2-D-GI ucona te keto-5-D-Gluconate D-Gluconate D-Glucuronate D-Galacturona te DL-Lactate Succinate
Melezitose Inulin Sol. starch Glycerol meso-Erythritol Ribitol Xylitol L-Arabinitol D-Glucitol D-Mannitol
Sucrose Maltose a,a-Trehalose methyl-a-D-Glucoside Cellobiose Salicin Arbutin Melibiose Lactose Raffinose
D-Glucose D-Galactose L-Sorbose D-Glucosamine D-Ribose D-Xylose L-Arabinose D-Arabinose L-Rhamnose
+ +
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w
w
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?
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w
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+ + + + +
+
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+
w
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160.89
+ + + + + +
+ + + + + + + + +
150.93
+ + + + + +
w
+ + +
217.79
+ + + + + +
w
+ + +
510.81
+ + +
w
+ + +
660.76
+ + + + + +
w
+ +
+ + +
w
256.92
191.87
Table 4. Growth reactions and other tests carried out with the isolates.
+ + + + + + + + + +
+ +
w
+ + + +
w
+
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w
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159.89
+ + + + + + + + + + +
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661.76
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515.76
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+ + + + + + +
w
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w
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dH9733 157.67
w
+
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+ +
w
+ + + +
+ +
+
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w
+ + + + +
+ + + + +
w
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100.68
+
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w
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+ + + + + +
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537.73
+ + + + + + +
+ +
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w
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665.76
+
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587.66
v.
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~
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~
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40C Fermen ta tion Gelatine Source Status
5 % MgCh 10% MgCh 5% NaCI 10% NaCI 0.01 % Cycloheximide 0.1 % Cycloheximide Mycosel Urease 30C 37C
Citrate Methanol Ethanol Nitrate Nitrite Ethylamine L-Lysine Cadaverine Creatine Creatinine
Table 4. Continued
+
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T
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Fish
w
w w
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256.92
w
191.87
w
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Soil
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Compost Soil
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w
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217.79
510.81
660.76
+
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w
Soil
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160.89
150.93
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661.76
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dH9733 157.67
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100.68
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J.M. J.UIJTHOFetal.
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100.68 404.81
1587.661 157.67 T
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1 510.8~ I 217.79
150.93 661 .76
537.73 T
515.76
191.87 T
159.89
256.92
160.89
260.80
dH9733
11660.761 1
Fig. 1. Graphical representation of ITSI-ITS4 restriction profiles (see Tab. 3). For each restriction enzyme used, the isolates showing identical patterns are surrounded with a line.
preliminary identification of the studied isolates was based on these key features. However, these criteria are insufficient for a reliable species identification. In the course of this study some of the CBS strains listed under these names proved to be degenerate cultures of other Exaphiala species, and were therefore excluded. Length variations in the NS1-NS24 amplicon containing the 18S rRNA gene sequence are a common feature in Exaphiala species. In E. dermatitidis these variations were supposed to be due to Intron Group I elements (HAASE et al., 1995) which have been found in many other fungi (DEPRIEST, 1993; GARGAS et al., 1995). They occur in isolates of E. dermatitidis sensu stricta, which had nearly identical ITS1 sequences (UIJTHOF et al., 1996). Their taxonomic significance is as yet unclear. The RFLP patterns of the SSU rDNA of the isolates, with an amplicon length of 1800 bp, were similar. In fact these were patterns observed in most other Exaphiala species as well (Patterns A, Table 2; UUTHOF and DE HOOG, 1995). Strains CBS 660.76 and 587.66 showed unique patterns with HaeIII. The ITS1-ITS4 amplicons, containing the relatively variable ITS regions, had a constant length, but gave more heterogeneous RFLP patterns. The type strains of the three species analyzed yielded clearly different patterns with all enzymes (Fig. 1). Identical patterns were observed with all enzymes in isolates CBS 776.76, 515.76, 159.89, 160.89 and dH9733; the type strain of E. salman is was relatively close to this group (Fig. 1, Table3). Phylogenetic analysis of ITS 1 sequences confirmed the close affinity of E. salmanis to the group of five identical isolates, which also includes CBS 150.93 at 5 bp distance (Fig. 1). All strains of
this group, except CBS 157.67, ongmate from soil or rhizospheres. Given the relatively large distance of CBS 157.67 to these soil isolates, it is doubtful whether these strains all belong to a single species. CBS 256.92 and 260.80 proved to be identical to the type strain of E. psychraphila, CBS 191.87. As this small group clearly differs from the remaining strains, E. psychraphila is considered to be a separate species. CBS 537.73, the type strain of E. pisciphila, took a rather isolated position. In this representation the isolate CBS 665.76 was found in the same branch as E. salmanis, CBS 157.67, but the terminal branch lengths were quite long. DE HOOG (1977) synonymized E. brunnea, CBS 587.66 (PAPENDORF, 1969) with E. salmanis, CBS 157.67, but this is not supported by the results obtained in the present study. ITS1 sequencing data suggest that this species should be maintained as a separate taxon. Physiological data in main traits corresponded well with phylogenetic analyses on the basis of ITS1 sequencing and ITS1-ITS4 RFLP. The five isolates that were closely related (Fig. 3) were phenetically similar to each other, with strain CBS 150.93 at some distance (Fig. 4) . Isolates CBS 256.92 and 191.87, which were found to be identical on the basis of ITS1 sequencing data were also the nearest neighbours in the physiological comparison (Fig.4). The strains CBS 256.92 and 191.87 (E. psychraphila) do not grow with lactose, D-glucuronate and D-galacturonate while E. salmanis, CBS 157.67 and E. pisciphila, CBS 537.73 are positive for all these compounds. E. pisciphila contrasts from the others by its ability to grow on L-arabinitol. DE HOOG and GUARRO (1995) listed E. pisciphila as negative for ethanol, while in the present study it was found to be
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salmonis, T species pisciphila pisciphila pisciphila pisciphila pisciphila psychrophila, T pisciphila pisciphila species pisciphila brunnea, T pisciphila, T lecanii-corni, T
I
. . . . . . . . . . . . . . A.... .. .. . A . . ..
CC .. - T . .. CC .. - T ... CC .. A . C - .. CT .. AA ... CTT. AA .. C - A . . . ... . -A ... T ... -
157 .67 dH97 33 661.76 515.76 1 59.89 160. 89 150.93 191.87 256.92 660.76 510.81 217.79 587.66 537.73 123.33
I
. . . . . . . . . . . . . . A....
· .. A . A. . . . · .. A . A. . .. · .. A. A. . . . · .. ACA . T . . · .. ACA ... T · . . A. . . . .. ... A.A ... -
salmonis, T species pisciphila pisciphila pisciphila pisc iphil a pisciphila psychrophila, T pisciphila pisciphila specie s pisciphila brunnea, T pisci phil a , T lecanii-corni, T
210 ••••
CCGCGCTCG TCGGTGGCCC - -AACCTTTA TAAAATCTTT AACCAAACGT GCCTC-A-AT - -CTAAGTAC . . ..... . . . . . . . A.... . . ..... . . " .. T ...... . ... T-. -. . . . . . . .. . .. ...... . . . .. . A.... .. . . . . . . . . . . T ...... .... T-. -.. .. .... ..
. . . . . A ....
157.67 dH9733 661.76 515.76 159.89 16 0 .89 150.9 3 191.87 256.92 660.76 510.81 217.79 587.66 537.73 123 . 33
I
... . G .. . - - T ... T- - - -G TA ... CT .. .
.. . A .--- --
salmo nis,T species pisciphila pisciphila pisciphila pisciphila pisciphila psychrophila, T pisciphila pisciphila species pisciphila brunnea,T pisciphila,T lecanii-co rni, T
I
.... - .. CCT ... - .. CC T ... - .. CC -
157.6 7 dh9 733 661.76 515.76 159.89 160 .89 150.93 191.87 256.92 660.76 510.81 217.79 587.66 537.73 123.33
140 ••••
- - ACGGACCG CCGGAGGG -A CCTT-CATT- GGCCCTCTGG .. . . . . . . . . . . . . . . . T ... -T.C . - ........ .. T . . . - T.C. T . .. - T.C......... - . T ... -T .C. -. T ... -T . C.-
..... ... - .
591
....... A .. . . . . . . . A .. ..... . . A .. . . . . . . . A ..
.. .... . A ..
C . A-.. T . . . . . . . . . . . . . . . . . G . . . . . T . . . . .. ..
..... T . . . . T ...... ..
1 57 .67 dH9733 661 .7 6 515 . 76 159.89 160.89 150.93 191.87 256 .92 660.76 510.81 2 17 . 79 587.66 537 .7 3 123.33
salmonis, T species pisciphila pisciphila pisciphi la pisciphila pisciphila psychrophila, T pisciphila pisciphila species pisciphila b runnea , T pisciphila, T lecanii-c orni, T
Fig.2. Alignment of ITS1 sequences from the isolates studied. Strain CBS123.33 Exophiala jeanselmei var. lecanii-corni (type) is introduced as outgroup. The first six nucleotides represent the 3' end of the 185 small subunit ribosomal DNA and the last four nucleotides represent the 5' end of the 5.85 ribosomal RNA gene. A dot indicates identity to the leading strand, a "-" a gap introduced for optimal alignment.
592
J. M. J. UIJTHOF et
al.
Distance 0.1
r--
dH9733 spec
515.76 pisciphila 160.89 pisciphila
~ 661.76 ¢"iphil. 84
159.89 pisciphila 150.93 pisciphila
87
157.67 salm onis T
-
100 87
100 100
217.79 pisciphila 510.81 Spec 660.76 pisciphila
Y
1001 256.92 pisciphila
I 191.87 psychrophila T 537.73 pis ciphila T
587.66 brunnea T 123.331ecanii-comi Fig. 3. Neighbor joining tree constructed with Treecon, using the ITSI sequence alignment in Fig. 1. The tree was bootstrapped 100 times. Bootstrap values over 75 are indicated.
r - - - - - CBS 191.87 L..-_ _ _ _ CBS 256.92 r----CBS 510.81
' - - - - - CBS 537.73 CBS 160.89 CBS 159.89 ~-CBS
661.76
"--CBS 515.76 '------CBS 150.93 .......- - - - - - C B S 587.66 ~----CBS
157.67
L..-_ _ _ _ CBS 665.76 ~----CBS
217.79
' - - - - - - C B S 660.76 L..-------CBS 100.68 Fig. 4. Phenogram based on similarity of physiological properties (UPGMA) of isolates studied.
positive. This discrepancy is explained by a late growth reaction with some compounds. The remaining strains can be discriminated on the basis of assimilation of lactose, inulin, L-arabinitol, galactitol, D-glucuronate and Dgalacturonate, their tolerance of 5% NaCl and elevated temperatures. These characters are among the most important physiological features in diagnostics of Exophiala (DE HOOG et aI., 1995). This confirms the conclusion, based on molecular differences, that the initially distinguished groups represent individual taxa. Thus, in contrast to the situation in E. dermatitidis (UIJTHOF et aI., 1996), the species from fish seem to form a heterogeneous assemblage. Black yeasts in fish mainly affect salmon and trout held in hatcheries (RICHARDS et aI., 1978; CARMICHAEL, 1966; OTIS et aI., 1985; LANGVAD et aI., 1985; LANGDON and McDONALD, 1987), fish in pond water (FIJAN, 1969) or aquaria (BLAZER and WOLKE, 1979). All infections reported thus concerned fish raised under artificial conditions which may predispose them for infection by opportunistic pathogens (OTIS et aI., 1985). This is further underlined by histopathological data, the prevalent form of growth of the fungus in internal organs being loose hyphae. A possible source of infection are biological filter systems with garden soil which are used for recirculation of water for the containers in which the young fish are maintained in anticipation of their transfer to seawater (LANGVAD et aI., 1985). In the present study the strains from soil indeed were found in the same ITS 1 branch as strains from fish. Some of the soil strains were isolated from nematode cysts. MORGAN-JONES et al. (1984) sup-
Molecular and Physiological Investigations posed that E. pisciphila may be a regular invader of nematode eggs. IWATSU and UDAGAWA (1984), however reported several strains matching E. pisciphila from rotten plant material and food. A moderate degree of psychrophily seems to be a common ecological factor, since none of the strains were able to grow at temperatures above 30°C, except for a strain from compost (T able3). The fish inhabiting strains comprise two distinct types of pathogenicity. All are systemic, but may be either cranial and neurotropic (e. g. CARMICHAEL, 1966; represented by strain CBS 157.67) or visceral, particularly in the kidney (e. g. FIJAN, 1969, represented by strain CBS 537.73; LANGVAD et ai., 1985, represented by strain CBS 191.87). Symptoms are consistent within each epidemic. These three strains turned out to be phylogenetically distinct (Fig. 3). Thus, separate species with different pathology might be concerned. In conclusion, the possibility is not excluded that the fish branch comprises a number of species different from those found in other ecological niches, in addition to primary fish pathogens. This might explain the apparent heterogeneity of the group under study. In contrast, the mainly human pathogenic E. dermatitidis was remarkably homogeneous (UIJTHOF et ai., 1996). In order to establish whether there are well-adapted fish pathogens among the strains studied in this paper, or whether just a wide array of moderately psychrophilic opportunists from soil are concerned, a larger set of strains will have to be studied.
References BLAZER, V. S., WOLKE, R. E.: An Exophiala like fungus as the cause of a systemic mycosis of marine fish. J. Fish Dis. 2,
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CARMICHAEL, J. W.: Cerebral mycetoma of trout due to a Phialophora like fungus. Sabouraudia 5, 120-123 (1966) DE HaaG, G. S.: Rhinocladiella and allied genera. Stud. Mycol.
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GARGAS, A. , DEPRIEST, P. T., TAYLOR, 1. W.: Positions of multiple insertions in SSU rDNA of lichen-forming fungi. Mol. BioI. Evol. 12,208-218 (1995) GOTO, S., AONO, R., SUGIYAMA, J., HORIKOSHI, K.: Exophiala alcalophila, a new black yeast-like hyphomycete with an accompanying Phaeococcomyces alcalophilus morph, and its physiological characteristics. Trans. Mycol. Soc. Japan. 22,
429-439 (1981)
HAASE, G ., SONNTAG, L., VAN DE PEER, Y., UIJTHOF, J. M. J., PODBIELSKI, A., MELZER-KRICK, B.: Phylogenetic analysis of ten black yeast species using small subunit rRNA gene sequences. Antonie van Leeuwenhoek 68, 19-33 (1995) IWATSU, T., UDAGAWA, S.: Materials for the fungus flora of Japan. Trans. Mycol. Soc. Japan 25, 389-394 (1984) KAWASAKI, M. , ISHIZAKl , H. , NISHIMURA, K., MIYAJI, M.: Mitochondrial DNA analysis of Exophiala jeanselmei and Exophiala dermatitidis. Mycopathologia 110, 107-112 (1990) KAWASAKI , M., ISHIZAKI, H., NISHIMURA, K., MIYAJI, M.: Mitochondrial DNA analysis of Exophiala moniliae. Mycopathologia 121, 7-10 (1993) LANGDON, J . S., McDONALD, W. L.: Cranial Exophiala pisciphila infection in Salmo solar in Australia. Bull. Eur. Assoc. Fish Pathol. 7, 35-37 (1987) LANGVAD, F., PEDERSEN, 0., ENGJOM, K.: A fungal disease caused by Exophiala sp. nova in farmed Atlantic salmon in Western Norway, pp. 323-328. In: Fish and Shellfish Pathology; First Int. Conf. Eur. Assoc. Fish Pathol. (A. E. ELLIS, ed.) London, Academic Press 1985 MASCLAUX, F., GUEHO, E., DE HOOG, G. S.: Phylogenetic relationships of human pathogenic Cladosporium (Xylohypha) species inferred from partial LSU rRNA sequences. J. Med. Vet. Mycol. 33, 327-338 (1995) MASUDA, M., NAKA, W., TAJIMA, S., HARADA, T ., NISHIKAWA, T., KAUFMAN, L., STANDARD, P.: Deoxyribonucleic acid hybridization studies of Exophiala dermatitidis and Exophiala jeanselmei. Microbiol. Immunol. 33, 631-639 (1989) MCGINNIS, M. R., AJELLO, L.: A new species of Exophiala isolated from channel catfish. Mycologia 66, 518-520 (1974) MOLLER, E. M., BAHNWEG, G., SANDERMANN , H ., GEIGER, H. H.: A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissue. Nucleic Acids Res. 20, 6115-6116
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DE HOOG, G. S., GERRITS VAN DEN ENDE, A. H. G., UlJTHOF, J. M. J ., UNTEREINER, W . A.: Nutritional physiology of type isolates of currently accepted species of Exophiala and Phaeococcomyces. Antonie van Leeuwenhoek 68, 43-49
MORGAN-JONES, G., CULBREATH , A. K., RODRIGUEz-KABANA, R. : Notes on hyphomycetes. XLIX. Xenokylindria obovata, a new species isolated from diseased eggs of the nematode Meloidogyne arenaria, and X. prolifera. Mycotaxon 20,
DE HOOG, G. S., GUARRO, J. (eds.): Atlas of Clinical Fungi. Baarn / Reus, Centraalbureau voor Schimmelcultures / Universitat Rovira i Virgili 1995 DE HOOG, G. S., GUEHO, E. MASCLAUX, F., GERRITS VAN DEN ENDE, A. H. G., KWON-CHUNG, K. J., MCGINNIS, M. R.: Nutritional physiology and taxonomy of human-pathogenic Cladosporium-Xylohypha species. J. Med. Vet. Mycol. 33,
OTIS, E. J., WOLKE, R. E., BLAZER, V. S.: Infection of Exophiala salmon is in Atlantic salmon (Salmo sa/ar L.). J. Wildlife Dis.
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DE HOOG, G. S., MCGINNIS, M. R.: Ascomycetous black yeasts. Stud. Mycol. 30, 187-:199 (1987) DEPRIEST, P. T.: Small subunit rDNA variation in a population of lichen fungi due to optional group I introns. Gene 134, 67-
71 (1993) DE RlJK, P., DE WACHTER, R .: DCSE, an interactive tool for sequence alignment and secondary structure research. Compo Appl. Biosci. 9, 315-322 (1993) FIJAN, N.: Systematic mycosis in channel fish. Bull. Wildlife Dis. Assoc. 5, 109 (1969)
599-606 (1984)
21,61-64 (1985)
PAPENDORF, M. c.: New South African soil fungi. Trans. Br. Mycol. Soc. 52, 483-489 (1969) PEDERSEN, O. A., LANGVAD, F.: Exopmala psychrophila sp. nov., a pathogenic species of the black yeasts isolated from farmed Atlantic salmon. Mycol. Res. 92, 153-156 (1989) RICHARDS, R. H. , HOLLIMAN, A., HELGASON, S.: Exophiala salmonis infection in Atlantic salmon Salmo salar L. ]. Fish Dis.
1, 357-368 (1978)
SAMBROOK, J., FRITSCH, E. F., MANIATIS, T.: Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York 1989 SPATAFORA, J. W. , MITCHELL, T. G., VILGALYS , R.: Analysis of genes coding for small subunit rRNA sequences in studying phylogenetics of dematiaceous fungal pathogens J. Clin. Microbiol. 33, 1322-1326 (1995)
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SUGIYAMA, J., NAGAI, K., KOMAGATA, K.: Ubiquinone systems in strains of species in the black yeast genera Phaeococcomyces, Exophiala, Hortaea and Rhinocladiella. J. Gen. Appl. MicrobioI. Tokyo 33, 197-204 (1987) TAKEO, K., DE HOOG, G. S.: Karyology and hyphal characters as taxonomic criteria in Ascomycetous black yeasts and related fungi. Antonie v. Leeuwenhoek 60, 35-42 (1991) UUTHOF, J. M. J.: Relationships within the black yeast genus Exophiala based on ITS1 sequences. Mycol. Res. 100, 1265-1271 (1996) . tJIJTHOF, J. M. J., DE HOOG, G. S.: PCR-Ribotyping of isolates of currently accepted Exophiala and Phaeococcomyces species. Antonie v. Leeuwenhoek 68, 35-42 (1995) UUTHOF, J. M. J., VAN BELKUM, A., DE HOOG, G . S., HAASE, G.: Exophiala dermatitidis and Sarcinomyces phaeomuriformis: ITS1 sequencing and development of a molecular probe. ]. Med. Vet. Mycol. 1996 (submitted) VAN DE PEER, Y., DE WACHTER, R.: Treecon: a software package for the construction and drawing of evolutionary trees. Compo Appl. Biosci. 9,177-182 (1993)
VAN DER WALT, J. P., YARROW, D.: Methods for the isolation, maintenance, classification and identification of yeasts, pp.45-104. In: The Yeasts, a Taxonomic Study (N.J. W. KREGER-VAN Ru, ed.) 4th edn, Amsterdam, Elsevier Science· Publishers 1984 WHITE, T. J., BRUNS, T., LEE, S., TAYLOR, J.: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, pp.315-322. In: PCR Protocols: A guide to methods and applications (N. INNIS, J. GELFLAND, T. WHITE, eds.) New York, Academic Press 1990 YAMADA, Y., SUGIHARA, K., VAN EUK, G. W., ROEIJMANS, H. J., DE HOOG, G. S.: Coenzyme Q systems in ascomycetous black yeasts. Antonie V. Leeuwenhoek 56,349-356 (1989) Corresponding author: G. S. DE HOOG, Centraalbureau voor Schimmelcultures, P.O. Box 273, NL-3740 AG Baarn, The Netherlands Tel.: 31-35-5481253; Fax: 31-35-5416142; e-mail: de.hoog@ cbs.knaw.nl
Molecular and Physiological Investigations of ExophiaJa Species Described from Fish J. M. J. UIJTHOF, M. J. FIGGE, and G. S.
DE HOOG
Centraalbureau voor Schimmelcultures, Baarn, The Netherlands Received.
Summary Eighteen strains of black yeasts, phenetically identified as Exophiala pisciphila, E. psychrophila or E. salmonis, were compared using ITS1 sequencing and RFLP of PCR-amplified ribosomal genes; in addi-
tion, their physiological profiles were determined. One strain contained an insert in its SSU ribosomal gene. The strains were found to have very similar SSU rDNA RFLP patterns, but to be heterogeneous in ITS RFLP patterns and ITS1 sequences. Physiological data corresponded with these findings. A distinction of species in psychrophilic Exophiala species remains difficult.
Key words: Black yeasts - Taxonomy - ITS1 sequences - PCR RFLP - Nutritional physiology - Fish pathology - Soil fungi
Introduction The black yeast genus Exophiala was erected by CARMICHAEL (1966) on the basis of E. salmonis Carmichael, a species causing a systemic infection in trout. Subsequently, two further fish pathogens were added, E. pisciphila McGinnis & Ajello (MCGINNIS and AJELLO, 1974) and E. psychrophila Pedersen & Langvad (PEDERSEN and LANGVAD, 1989), as well as species from other sources (for reviews see DE HOOG and MCGINNIS, 1987; UIJTHOF and DE HOOG, 1995). Species delineation in the genus Exophiala is problematic because of the limited number of reliable morphological characteristics. Hence a wide array of nonmorphological techniques have been introduced, among which are karyology (TAKEO and DE HOOG, 1991), coenzyme Q analysis (SUGIYAMA et al., 1987; YAMADA et al., 1989; GOTO et al., 1981), RFLP of mtDNA (KAWASAKI et al., 1990; 1993), DNA-DNA hybridization (MASUDA et al., 1989), SSU rDNA sequencing (HAASE et al., 1995; SPATAFORA et al., 1995) and LSU rRNA sequencing (MASCLAUX et al., 1995). For the distinction of type isolates of recognized and synonymized species in the genus Exophiala and related taxa we tested nutritional physiology (DE HOOG et al., 1995) and applied molecular techniques like PCR-RFLP (UIJTHOF and DE HOOG, 1995) and ITS1 sequencing (UIJTHOF, 1996). Nearly all described species were proven to represent separate entities. The three species from fish
clustered in a monophyletic branch at some distance from the remaining Exophiala species (UIJTHOF, 1996). The separate position of these species was suggested earlier on the basis of coenzyme Q hydrogenation (YAMADA et al., 1989). Their phylogenetic relationship might result from a shared ecological niche in cold environments. To elucidate their taxonomic structure, morphology, physiology and molecular characteristics of 18 strains previously identified as Exophiala salmonis, E. pisciphila or E. psychrophila were studied.
Materials and Methods Strains and culture conditions: The strains studied are listed in Table 1. Stock cultures were maintained on oatmeal agar at 4°C. Strains were grown for 7 days in 100 mlliquid 2% malt extract / 0.5% yeast extract medium on a rotary shaker at 300 rpm at 28°C. Nutritional physiology: Growth and fermentative abilities were tested in duplicate in liquid medium according to V AN DER WALT and YARROW (1984) with modifications according to DE HOOG et al. (1995). Assimilation tests were performed in liquid medium in test tubes at 25°C, rocked in a nearly horizontal position at 50 rev. min-I. Biomass production was verified weekly, with final reading 2 wk after the exhaustion of the C-source in the glucose assimilation test tube. Fermentation was tested in glucose over a 2 wk period in vertical test tubes with Durham
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Table 1. List of strains studied. Strains 100.68 537.73 515.76 660.76 661.76 217.79 260.80 404.81 159.89 160.89 256.92 150.93 191.87 587.66 157.67 665.76 510.81 dH9733
T
T T T
Initial identification
Source
Geographic origin
E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila E. pisciphila aff. E. pisciphila E. psychrophila E. brunnea E.salmonis E. salmonis E. species E. species
Plant, Pisum sativum Fish, kidney Soil Rhizosphere of Triticum aestivum Cyst nematode Soil Fish, kidney Soil Root, Hordeum vulgare Root, Hordeum vulgare Fish, kidney Washed root, Tilia platyphylla Fish, kidney Leaf litter Fish, brain Wood Compost Plant, Triticum aestivum
The Netherlands USA Canada Australia Germany Chile Norway The Netherlands The Netherlands The Netherlands Ireland Germany Norway South Africa Canada Chile Austria Italy
inserts kept stationary apart from brief manual shaking every 2 days prior to reading. Urease activity (V AN DER WALT and Y ARROW, 1984) was tested on Christensen's agar. Gelatin (12%; GistBrocades, Delft, The Netherlands) liquefaction was tested on agar plates, using HgCIz in 7.6% HCl as a reagent. Thermotolerance was tested with 4% malt extract agar (MEA) slants inoculated with fresh cell suspensions. Cycloheximide tolerance was tested both in liquid medium at 0.01, 0.05 and 0.1 % (VAN DER WALT and YARROW, 1984) and on commercial Mycosel agar (0.04%; Becton Dickinson, Cockeysville, USA). Extracellular DNAse medium was provided by Difco (Detroit, USA). Halotolerance was tested in rocked liquid malt medium containing 2.5, 5 and 10% (w:v) NaCl or MgCI 2 • DNA-Isolation: Two ml of cell suspension were centrifuged at 14000 rpm and the cells were washed twice with sterile water. The resulting pellets (approximately 100 Ill) were stored at -70°C. Total DNA was extracted by using a miniprep protocol (UIJTHOF and DE HOOG, 1995). The pellet was dissolved in 0.5 ml TES buffer (0.1 mM Tris-HCl, pH 8.0; 10 mM EDTA; 2% SDS), and approximately 400 III of glass beads (<1> 0.45-0.50 mm) were added. After homogenization, the mixture was vortexed for at least 10 min. Proteinase K was added and the mixture was incubated for 30 min at 55-60 °C. After centrifugation, the supernatant was transferred to a new tube. Subsequently the procedure of MOLLER et al. (1992) was followed, starting with the addition of hexadecyltrimethylammoniumbromide (CTAB; Sigma) in high salt concentration. The resulting DNA pellet was dissolved in water and the DNA concentration was determined spectrophotometrically. DNA Amplification: PCR was performed in 50 III volumes of a " Tris-based (10 mM, pH 8.3) reaction mixture (50 mM KCI, 1.5 mM MgCh, 0.01 % gelatin) containing 200 IlM of "each deoxynucleotide triphosphate, 50 pmol of each primer, 10-100 ng nuclear DNA and 0.25 U of Taq DNA polymerase (Super Taq; Sphaero Q, Leiden, The Netherlands). Restriction analysis: The amplicons were digested for at least 2 h with one of the following restriction enzymes (1 U): DdeI, HaeIII, HhaI, HinfI, NdeII, MspI, RsaI and Taqi. Digests were subjected to electrophoresis on 2% agarose gels (TAE buffer; SAMBROOK et al., 1989), after which the gels were stained with ethidium bromide and photographed.
Sequencing: Primers v9 and its4 (WHITE et al., 1990) were emplqyed. Forty amplification cycles were performed: 94°C, 1 min; 48 °C, 2 min; 74°C, 3 min with an initial delay of 1 min at 94°C in a Biomed thermocycler (type 60). The remaining primers and dNTPs were removed by isolating the amplicons with Geneclean according to instructions given by the manufacturer. The amplicon was sequenced with a primer terminating protocol on an ABI automatic sequencer with primers its1 and its2 (WHITE et al.,1990). Alignment and phylogenetic analysis: The sequences obtained with both primers were aligned with Clustal V and adjusted by using the DCSE program (DE RIJK and DE WACHTER, 1993). The phylogenetic tree was calculated with several distance methods. The final tree was constructed using the Neighbor Joining method of the Treecon package (VAN DE PEER and DE WACHTER, 1993). Bootstrap values of over 75 are indicated near the branches.
Results PCR amplification of the relatively conserved SSU rRNA genes, using primers ns1 and ns24, resulted in amplicons of 1800 bp, except for CBS 157.67 (E. salmonis) which had an amplicon of about 2100 nucleotides (Table 2). The RFLP patterns obtained after Hinfl, N dell and DdeI digestion of amplicon NS1-NS24 were identical for all isolates with an amplicon length of 1800 nucleotides. With enzyme HaeIII, isolates CBS 587.66 and 660.76 each had a unique pattern. PCR amplification of the more variable ITS region, covering the internal transcribed spacers and the 5.8S rRNA gene, using primers its1 and its4, resulted in amplicons of 600 nucleotides for all isolates. The restriction patterns of these amplicons are listed in Table 3 and displayed graphically in Fig. 1. The type strains of the species investigated in the present study are all in the center. A phylogenetic tree based on ITS1 sequences is presented in Fig. 3. Close similarity in RFLP patterns corre-
Molecular and Physiological Investigations Table 2. RFLP patterns of amplicon NSI-NS24. Strains 100.68 537.73 515.76 660.76 661.76 217.79 260.80 404.81 159.89 160.89 256.92 150.93 191.87 587.66 157.67 665.76 510.81 dH9733
T
T T T
Amplicon length
HinfI
HaeIII
DdeI
NdeIl
1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 1800 2100 1800 1800
A A A A A A A A A A A A A A B A
A A A B A A A A A A A A A C D A A
A A A A A A A A A A A A A A B A A
A A A A A A A A A A A A A A B A A
587
Results of physiological tests are shown in Table 4. Most strains assimilate D-glucosamine relatively well; soluble starch is not or poorly utilized except by dH9733. Growth responses to lactose and inulin are variable. Erythritol, nitrate, creatine and creatinine are assimilated, though sometimes weakly. All strains were tolerant to cycloheximide but relatively intolerant to salt, mostly growing only weakly with 5% NaCl. Only CBS 510.81 grew weakly at 3rC; growth at 30°C was observed in CBS 587.66, 150.93, 217.79 and 537.73. A numerical comparison of the physiological dataset (Fig. 4) indicates a close similarity between CBS 256.92 and 191.87, and between CBS 159.89, 160.89,515.76 and 661.76, with CBS 150.93 at some distance. The type strain of E. pisciphila, CBS 537.73 was physiologically similar to CBS 510.81, while that of E. salmonis CBS 157.67, was similar to CBS 665.76. Because of their long terminal branches, the cluster of isolates CBS 660.76, 217.79 and 100.68 is not regarded as being homogeneous.
Discussion sponded with short distances in the phylogenetic tree based on ITS1 sequencing (Fig. 3). The ITS1 sequences of the strains were variable in 70 positions. Strains CBS 159.89,160.89,515.76 and 661.76 were identical to each other. Strains dH9733 and CBS 150.93 were closely related to this group, but differed in 1 and 5 positions, respectively. The ITS1 sequence of CBS 256.92 was identical to that of CBS 191.87, the type strain of E. psychrophila. A third group, composed of strains CBS 510.81 and 217.79, was interconnected by rather long branches. Strains CBS 157.67, 660.76, 587.66 and 537.73 were found at relatively long distances to all other isolates studied.
YAMADA et al. (1989) and SUGIYAMA et al. (1987) analyzed coenzyme Q systems of numerous black yeasts. Species originating from fish were found to differ from the remaining Exophiala species in having Co-QlO(H 2 ) rather than QlO systems. With ITS1 phylogeny the fish-associated strains clustered together in a monophyletic branch at some distance from the core of the genus (UUTHOF, 1996). In the present study we analyzed the strains composing this branch, also comprising a number of similar strains from soil and leaf litter. In their original descriptions, the species Exophiala salmonis, E. pisciphila and E. psychrophila were characterized by 1-3 septate conidia, 0-1 septate conidia and a maximum growth temperature of 23°C, respectively. The
Table 3. RFLP patterns of amplicon ITSI-ITS4. Strains 100.68 537.73 515.76 660.76 661.76 217.79 260.80 404.81 159.89 160.89 256.92 150.93 191.87 587.66 157.67 665.76 510.81 dH9733
T
T T T
Amplicon length
RsaI
NdeIl
TaqI
MspI
HaeIII
HinfI
HhaI
DdeI
600 600 600 600 600 600 600 600 600 600 606 600 600 600 600 600 600 600
A B A B A
A B B
A B A B A B
A B A
A B B
C
C
A A A B A B B A A B A
A A A
A A A A A B B A A A B A B A A D B A
A A A A A B A A A A A A A B A A B A
A A A A A B A A A A A A A A A A B A
C
A A A C
A C
D A A C
A
C
B A B A B B B A B A A A A B
A C
C
A C
B B B C
A
B B B A B B B B B A A A B B
Galactitol myo-Inositol Glucono-o-lactone keto-2-D-GI ucona te keto-5-D-Gluconate D-Gluconate D-Glucuronate D-Galacturona te DL-Lactate Succinate
Melezitose Inulin Sol. starch Glycerol meso-Erythritol Ribitol Xylitol L-Arabinitol D-Glucitol D-Mannitol
Sucrose Maltose a,a-Trehalose methyl-a-D-Glucoside Cellobiose Salicin Arbutin Melibiose Lactose Raffinose
D-Glucose D-Galactose L-Sorbose D-Glucosamine D-Ribose D-Xylose L-Arabinose D-Arabinose L-Rhamnose
+ +
+ + + + + + + +
+ + + +
+ +
+ +
+ + + + + + + +
+ + + +
+ +
+ + + + + + +
w
w
+
w
?
+ +
+ + +
w w
w w
+
w
+ + + +
w
+ +
+
+
+
?
w
+ + + + + +
w
w
+ + + +
w
+
w
+ +
+
+
+
+ +
w
+ +
+
+
+ +
+ +
+
+
+
+ + + + +
+
+
w
+ + + +
w
w
+ + + +
+
+
+
+ + w
+
+
+ + + + + +
+ + + + +
+ + + + +
+
+
+ + + + + +
+ + + + +
+
+
w
+ + + + + +
+ + + + +
+
+
+
+
+ + + + +
+
w
+ + +
160.89
+ + + + + +
+ + + + + + + + +
150.93
+ + + + + +
w
+ + +
217.79
+ + + + + +
w
+ + +
510.81
+ + +
w
+ + +
660.76
+ + + + + +
w
+ +
+ + +
w
256.92
191.87
Table 4. Growth reactions and other tests carried out with the isolates.
+ + + + + + + + + +
+ +
w
+ + + +
w
+
+ + +
+ + + + + +
+
+ + + +
w
+ + +
159.89
+ + + + + + + + + + +
+ + + + + + + + + + + +
+
+ + + +
w
+ +
+
+
+ + + + + +
+ + + + +
w
+ + +
661.76
+ + + + w
+
+
+
+ + + + + +
+ + + + + + + + +
515.76
+ + + + + + + + + +
+ + + + +
w
+ + + +
+ + +
+/w
+ + + + +
+ + + +
w
+ + + +
+ + + + + + +
w
+
w
+ + + + + + +
w
+ +
+ + +
+ + + + + +
+ + + + + + + + +
dH9733 157.67
w
+
+
+ + + +
+ +
w
+ + + +
+ +
+
+ w
w
+ + + + +
+ + + + +
w
+ + +
100.68
+
w
w
w
+ +
w
+ +
w
+ +
w
+ +
+ + + +
w
+ +
+
+ w
+ + + + + +
+ + + + + + + + +
537.73
+ + + + + + +
+ +
+ + + +
+ +
w
+ +
+ + + + + +
+ + + + + + + + +
665.76
+
+ +
w
+
+ + +
+ + +
+ +
+ +
w
+
+ + +
+ + + + + +
+ + + + + + + + +
587.66
v.
~
~
o
",
~
~
~
~
00 00
40C Fermen ta tion Gelatine Source Status
5 % MgCh 10% MgCh 5% NaCI 10% NaCI 0.01 % Cycloheximide 0.1 % Cycloheximide Mycosel Urease 30C 37C
Citrate Methanol Ethanol Nitrate Nitrite Ethylamine L-Lysine Cadaverine Creatine Creatinine
Table 4. Continued
+
+ +
+
T
? Fish
+ +
+
+
Fish
w
w w
+
w w
+
+ + +
+
+
w
+ + + + + +
+ + +
w
256.92
w
191.87
w
+
Soil
+ +
+
+
w
?
+
+ + + + +
+
+
+
+ + +
Compost Soil
w
+ +
+ +
+
+ +
+ +
+ + + + + + + +
+ + + + + +
w
w
+
217.79
510.81
660.76
+
Soil
+ + + + +
w
Soil
+ + + +
w
w
+
w
+ + + + + + + +
w
+ + + + + + + + +
160.89
150.93
+
Soil
? Soil
+
+
+
w
w
+ +
+
+
+ +
+ + + + +
+
515.76
+ +
+
w w
+ + + + + + + +
+
159.89
+
Soil
+
+ +
+
+
w w
+
+ + + + + + +
+
661.76
+
+ + + +
+
+
w
+ + + + + + + +
+
? Fish T
+ + + +
w
+ + + + + + + +
+
dH9733 157.67
+
Pisum
+
+ + +
w
w
w
+ + +
+ +
100.68
+
? Fish
+
+ +
+
w
w
+
+ + + + + +
+
665.76
+
+
T
+
Fish
+
+ + +
+ w w
+ + + + +
+ +
w
537.73
T
+
Litter
+ + + + +
+ w w
+ + + + + + + +
w
587.66
~
00
v.
'"
!:l
~
'"o·
ciQ.
~
(l)
S -<
~
0-
'<:
;¥ '" o·
..,p;'" !:l 0-
~
()
~ o
(b
590
J.M. J.UIJTHOFetal.
I I
1665.76 1
!
100.68 404.81
1587.661 157.67 T
I
1 510.8~ I 217.79
150.93 661 .76
537.73 T
515.76
191.87 T
159.89
256.92
160.89
260.80
dH9733
11660.761 1
Fig. 1. Graphical representation of ITSI-ITS4 restriction profiles (see Tab. 3). For each restriction enzyme used, the isolates showing identical patterns are surrounded with a line.
preliminary identification of the studied isolates was based on these key features. However, these criteria are insufficient for a reliable species identification. In the course of this study some of the CBS strains listed under these names proved to be degenerate cultures of other Exaphiala species, and were therefore excluded. Length variations in the NS1-NS24 amplicon containing the 18S rRNA gene sequence are a common feature in Exaphiala species. In E. dermatitidis these variations were supposed to be due to Intron Group I elements (HAASE et al., 1995) which have been found in many other fungi (DEPRIEST, 1993; GARGAS et al., 1995). They occur in isolates of E. dermatitidis sensu stricta, which had nearly identical ITS1 sequences (UIJTHOF et al., 1996). Their taxonomic significance is as yet unclear. The RFLP patterns of the SSU rDNA of the isolates, with an amplicon length of 1800 bp, were similar. In fact these were patterns observed in most other Exaphiala species as well (Patterns A, Table 2; UUTHOF and DE HOOG, 1995). Strains CBS 660.76 and 587.66 showed unique patterns with HaeIII. The ITS1-ITS4 amplicons, containing the relatively variable ITS regions, had a constant length, but gave more heterogeneous RFLP patterns. The type strains of the three species analyzed yielded clearly different patterns with all enzymes (Fig. 1). Identical patterns were observed with all enzymes in isolates CBS 776.76, 515.76, 159.89, 160.89 and dH9733; the type strain of E. salman is was relatively close to this group (Fig. 1, Table3). Phylogenetic analysis of ITS 1 sequences confirmed the close affinity of E. salmanis to the group of five identical isolates, which also includes CBS 150.93 at 5 bp distance (Fig. 1). All strains of
this group, except CBS 157.67, ongmate from soil or rhizospheres. Given the relatively large distance of CBS 157.67 to these soil isolates, it is doubtful whether these strains all belong to a single species. CBS 256.92 and 260.80 proved to be identical to the type strain of E. psychraphila, CBS 191.87. As this small group clearly differs from the remaining strains, E. psychraphila is considered to be a separate species. CBS 537.73, the type strain of E. pisciphila, took a rather isolated position. In this representation the isolate CBS 665.76 was found in the same branch as E. salmanis, CBS 157.67, but the terminal branch lengths were quite long. DE HOOG (1977) synonymized E. brunnea, CBS 587.66 (PAPENDORF, 1969) with E. salmanis, CBS 157.67, but this is not supported by the results obtained in the present study. ITS1 sequencing data suggest that this species should be maintained as a separate taxon. Physiological data in main traits corresponded well with phylogenetic analyses on the basis of ITS1 sequencing and ITS1-ITS4 RFLP. The five isolates that were closely related (Fig. 3) were phenetically similar to each other, with strain CBS 150.93 at some distance (Fig. 4) . Isolates CBS 256.92 and 191.87, which were found to be identical on the basis of ITS1 sequencing data were also the nearest neighbours in the physiological comparison (Fig.4). The strains CBS 256.92 and 191.87 (E. psychraphila) do not grow with lactose, D-glucuronate and D-galacturonate while E. salmanis, CBS 157.67 and E. pisciphila, CBS 537.73 are positive for all these compounds. E. pisciphila contrasts from the others by its ability to grow on L-arabinitol. DE HOOG and GUARRO (1995) listed E. pisciphila as negative for ethanol, while in the present study it was found to be
Molecular and Physiological Investigations 10 •••
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TCATTAACG AGTTAGGGTC CCCCTCTGGG CCCGACCTCC CAACCCTTTG TCTACTTGAC CATC-GTTGC .... . . . . . . . . . . . . . . .
--T . . . . . . -
--T ...... - -T . . . . . . -- T .... .. - -T . . . . . . --T . . . . . . - -T.A.C .. - -T.A .C .. --T ...... - -T.A .C .. - -T.A.C .. T -T . . - A. . .. T. -GC .. TTTTATA . .
.... .. C ..
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TCGGCGAGC CCGTC----.........
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.. . . .....
- ..... T .. .. - ..... T ... . - ..... T .. .. - .T ... . ... .
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- - CTC - ----
.... - ..... .. .. - ..... .T ... CAA ..
110 ••••
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. T .. . CC. .. 120 ••
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. C - AC .... .
130 ••••
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-C ... G- - - -C ... G - - --- .. . G --- -
•... G ... -. .... G ... -.
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I
••••
. A . . . . . .. . . A ....... .
.... G ... - T T .. - - --- - G A . . . . . . . . .
.... G ... -T T .. --- --- G A .. .... .. .
--T .. -- --.... G . . - - . .. . G-T . . A- .. A .. C ... . - - - -TGTA- - - - .T . . . . . . . . . . G ... T . . . . . -G .. C..... TCTCC CT .. . CGGGG GGG .A . . . . . . ... G ... -- .... CA .AC - C .T .. .. .. .
150 •
••
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160 ••••
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170 ••••
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.. .... ..
.. . . . . . . . . . . T . . . . . . . . . . T-. - .. ... T .. .... .... T-.- ..
.. .... ..
- - .... A .. -
220 •••
I
I
•••
•
I
I
.. .... .. ..
.... T-.- . . . . . . . . ..
- - ... . . .. T - - ..... . . T - - .... . . . T - - .. ..... T - - ....... T GA . . G .... . - - ....... T - - .. G .... .
230 ••••
I
I
••••
I
I
••••
,
I
TATTTGTTA AATAAAAGCA AAACT A .. . A- . . . A ... A- .. .
A ... A- .. . A . .. A - .. . A ... A- .. . A ... A - .. .
. ... - ... .
.. .... ...
- .... C ... - ... T. . . . .. . T .. TG. - .. - ... . C ... - ... T. . . . .. . T .. TG . - .. A ......... . .. T. . . . .. . .... - . A . ' . . . . . . . .. C .. T . . . . .. . T . GTG . - .. - . T . .. .. N - C .. T. . . . .. . T . GTG . - " - ... T .... - . .. T . . . . .. . T . - - - . - -. . . . . . . . . . . . . . . . . . . . T ... -.- .. C ... T .. .. - .. TT ...... . T .. - - .C - .
salmonis, T species pisciphila pisciphila pisciphila pisciphila pisciphila psychrophila, T pisciphila pisciphila species pisciphila brunnea, T pisciphila, T lecanii-corni, T
I
. . . . . . . . . . . . . . A.... .. .. . A . . ..
CC .. - T . .. CC .. - T ... CC .. A . C - .. CT .. AA ... CTT. AA .. C - A . . . ... . -A ... T ... -
157 .67 dH97 33 661.76 515.76 1 59.89 160. 89 150.93 191.87 256.92 660.76 510.81 217.79 587.66 537.73 123.33
I
. . . . . . . . . . . . . . A....
· .. A . A. . . . · .. A . A. . .. · .. A. A. . . . · .. ACA . T . . · .. ACA ... T · . . A. . . . .. ... A.A ... -
salmonis, T species pisciphila pisciphila pisciphila pisc iphil a pisciphila psychrophila, T pisciphila pisciphila specie s pisciphila brunnea, T pisci phil a , T lecanii-corni, T
210 ••••
CCGCGCTCG TCGGTGGCCC - -AACCTTTA TAAAATCTTT AACCAAACGT GCCTC-A-AT - -CTAAGTAC . . ..... . . . . . . . A.... . . ..... . . " .. T ...... . ... T-. -. . . . . . . .. . .. ...... . . . .. . A.... .. . . . . . . . . . . T ...... .... T-. -.. .. .... ..
. . . . . A ....
157.67 dH9733 661.76 515.76 159.89 16 0 .89 150.9 3 191.87 256.92 660.76 510.81 217.79 587.66 537.73 123 . 33
I
... . G .. . - - T ... T- - - -G TA ... CT .. .
.. . A .--- --
salmo nis,T species pisciphila pisciphila pisciphila pisciphila pisciphila psychrophila, T pisciphila pisciphila species pisciphila brunnea,T pisciphila,T lecanii-co rni, T
I
.... - .. CCT ... - .. CC T ... - .. CC -
157.6 7 dh9 733 661.76 515.76 159.89 160 .89 150.93 191.87 256.92 660.76 510.81 217.79 587.66 537.73 123.33
140 ••••
- - ACGGACCG CCGGAGGG -A CCTT-CATT- GGCCCTCTGG .. . . . . . . . . . . . . . . . T ... -T.C . - ........ .. T . . . - T.C. T . .. - T.C......... - . T ... -T .C. -. T ... -T . C.-
..... ... - .
591
....... A .. . . . . . . . A .. ..... . . A .. . . . . . . . A ..
.. .... . A ..
C . A-.. T . . . . . . . . . . . . . . . . . G . . . . . T . . . . .. ..
..... T . . . . T ...... ..
1 57 .67 dH9733 661 .7 6 515 . 76 159.89 160.89 150.93 191.87 256 .92 660.76 510.81 2 17 . 79 587.66 537 .7 3 123.33
salmonis, T species pisciphila pisciphila pisciphi la pisciphila pisciphila psychrophila, T pisciphila pisciphila species pisciphila b runnea , T pisciphila, T lecanii-c orni, T
Fig.2. Alignment of ITS1 sequences from the isolates studied. Strain CBS123.33 Exophiala jeanselmei var. lecanii-corni (type) is introduced as outgroup. The first six nucleotides represent the 3' end of the 185 small subunit ribosomal DNA and the last four nucleotides represent the 5' end of the 5.85 ribosomal RNA gene. A dot indicates identity to the leading strand, a "-" a gap introduced for optimal alignment.
592
J. M. J. UIJTHOF et
al.
Distance 0.1
r--
dH9733 spec
515.76 pisciphila 160.89 pisciphila
~ 661.76 ¢"iphil. 84
159.89 pisciphila 150.93 pisciphila
87
157.67 salm onis T
-
100 87
100 100
217.79 pisciphila 510.81 Spec 660.76 pisciphila
Y
1001 256.92 pisciphila
I 191.87 psychrophila T 537.73 pis ciphila T
587.66 brunnea T 123.331ecanii-comi Fig. 3. Neighbor joining tree constructed with Treecon, using the ITSI sequence alignment in Fig. 1. The tree was bootstrapped 100 times. Bootstrap values over 75 are indicated.
r - - - - - CBS 191.87 L..-_ _ _ _ CBS 256.92 r----CBS 510.81
' - - - - - CBS 537.73 CBS 160.89 CBS 159.89 ~-CBS
661.76
"--CBS 515.76 '------CBS 150.93 .......- - - - - - C B S 587.66 ~----CBS
157.67
L..-_ _ _ _ CBS 665.76 ~----CBS
217.79
' - - - - - - C B S 660.76 L..-------CBS 100.68 Fig. 4. Phenogram based on similarity of physiological properties (UPGMA) of isolates studied.
positive. This discrepancy is explained by a late growth reaction with some compounds. The remaining strains can be discriminated on the basis of assimilation of lactose, inulin, L-arabinitol, galactitol, D-glucuronate and Dgalacturonate, their tolerance of 5% NaCl and elevated temperatures. These characters are among the most important physiological features in diagnostics of Exophiala (DE HOOG et aI., 1995). This confirms the conclusion, based on molecular differences, that the initially distinguished groups represent individual taxa. Thus, in contrast to the situation in E. dermatitidis (UIJTHOF et aI., 1996), the species from fish seem to form a heterogeneous assemblage. Black yeasts in fish mainly affect salmon and trout held in hatcheries (RICHARDS et aI., 1978; CARMICHAEL, 1966; OTIS et aI., 1985; LANGVAD et aI., 1985; LANGDON and McDONALD, 1987), fish in pond water (FIJAN, 1969) or aquaria (BLAZER and WOLKE, 1979). All infections reported thus concerned fish raised under artificial conditions which may predispose them for infection by opportunistic pathogens (OTIS et aI., 1985). This is further underlined by histopathological data, the prevalent form of growth of the fungus in internal organs being loose hyphae. A possible source of infection are biological filter systems with garden soil which are used for recirculation of water for the containers in which the young fish are maintained in anticipation of their transfer to seawater (LANGVAD et aI., 1985). In the present study the strains from soil indeed were found in the same ITS 1 branch as strains from fish. Some of the soil strains were isolated from nematode cysts. MORGAN-JONES et al. (1984) sup-
Molecular and Physiological Investigations posed that E. pisciphila may be a regular invader of nematode eggs. IWATSU and UDAGAWA (1984), however reported several strains matching E. pisciphila from rotten plant material and food. A moderate degree of psychrophily seems to be a common ecological factor, since none of the strains were able to grow at temperatures above 30°C, except for a strain from compost (T able3). The fish inhabiting strains comprise two distinct types of pathogenicity. All are systemic, but may be either cranial and neurotropic (e. g. CARMICHAEL, 1966; represented by strain CBS 157.67) or visceral, particularly in the kidney (e. g. FIJAN, 1969, represented by strain CBS 537.73; LANGVAD et ai., 1985, represented by strain CBS 191.87). Symptoms are consistent within each epidemic. These three strains turned out to be phylogenetically distinct (Fig. 3). Thus, separate species with different pathology might be concerned. In conclusion, the possibility is not excluded that the fish branch comprises a number of species different from those found in other ecological niches, in addition to primary fish pathogens. This might explain the apparent heterogeneity of the group under study. In contrast, the mainly human pathogenic E. dermatitidis was remarkably homogeneous (UIJTHOF et ai., 1996). In order to establish whether there are well-adapted fish pathogens among the strains studied in this paper, or whether just a wide array of moderately psychrophilic opportunists from soil are concerned, a larger set of strains will have to be studied.
References BLAZER, V. S., WOLKE, R. E.: An Exophiala like fungus as the cause of a systemic mycosis of marine fish. J. Fish Dis. 2,
145-152 (1979)
CARMICHAEL, J. W.: Cerebral mycetoma of trout due to a Phialophora like fungus. Sabouraudia 5, 120-123 (1966) DE HaaG, G. S.: Rhinocladiella and allied genera. Stud. Mycol.
15, 1-140 (1977)
593
GARGAS, A. , DEPRIEST, P. T., TAYLOR, 1. W.: Positions of multiple insertions in SSU rDNA of lichen-forming fungi. Mol. BioI. Evol. 12,208-218 (1995) GOTO, S., AONO, R., SUGIYAMA, J., HORIKOSHI, K.: Exophiala alcalophila, a new black yeast-like hyphomycete with an accompanying Phaeococcomyces alcalophilus morph, and its physiological characteristics. Trans. Mycol. Soc. Japan. 22,
429-439 (1981)
HAASE, G ., SONNTAG, L., VAN DE PEER, Y., UIJTHOF, J. M. J., PODBIELSKI, A., MELZER-KRICK, B.: Phylogenetic analysis of ten black yeast species using small subunit rRNA gene sequences. Antonie van Leeuwenhoek 68, 19-33 (1995) IWATSU, T., UDAGAWA, S.: Materials for the fungus flora of Japan. Trans. Mycol. Soc. Japan 25, 389-394 (1984) KAWASAKI, M. , ISHIZAKl , H. , NISHIMURA, K., MIYAJI, M.: Mitochondrial DNA analysis of Exophiala jeanselmei and Exophiala dermatitidis. Mycopathologia 110, 107-112 (1990) KAWASAKI , M., ISHIZAKI, H., NISHIMURA, K., MIYAJI, M.: Mitochondrial DNA analysis of Exophiala moniliae. Mycopathologia 121, 7-10 (1993) LANGDON, J . S., McDONALD, W. L.: Cranial Exophiala pisciphila infection in Salmo solar in Australia. Bull. Eur. Assoc. Fish Pathol. 7, 35-37 (1987) LANGVAD, F., PEDERSEN, 0., ENGJOM, K.: A fungal disease caused by Exophiala sp. nova in farmed Atlantic salmon in Western Norway, pp. 323-328. In: Fish and Shellfish Pathology; First Int. Conf. Eur. Assoc. Fish Pathol. (A. E. ELLIS, ed.) London, Academic Press 1985 MASCLAUX, F., GUEHO, E., DE HOOG, G. S.: Phylogenetic relationships of human pathogenic Cladosporium (Xylohypha) species inferred from partial LSU rRNA sequences. J. Med. Vet. Mycol. 33, 327-338 (1995) MASUDA, M., NAKA, W., TAJIMA, S., HARADA, T ., NISHIKAWA, T., KAUFMAN, L., STANDARD, P.: Deoxyribonucleic acid hybridization studies of Exophiala dermatitidis and Exophiala jeanselmei. Microbiol. Immunol. 33, 631-639 (1989) MCGINNIS, M. R., AJELLO, L.: A new species of Exophiala isolated from channel catfish. Mycologia 66, 518-520 (1974) MOLLER, E. M., BAHNWEG, G., SANDERMANN , H ., GEIGER, H. H.: A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissue. Nucleic Acids Res. 20, 6115-6116
(1992)
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