Mycopathologia 134: 6549, 1996.

65
9 1996Kluwer Academic Publishers. Printed in the Netherlands.

The utility of mitochondrial DNA restriction analysis in the classification of

strains of Chrysosporium (hyphomycetes)

J. C a n o 1, J.M. G u i l l a m d n 1, R Vidal 2 & J. G u a r r o 2

1Unidad de Microbiologia, Escuela de Enologia, Universidad Rovira i Virgili, Rambn i Cajal s/n,
43005-Tarragona, Spain; 2Unidad de Microbiologia, Facultad de Medicina, Universidad Rovira i Virgili, San
Lorenzo 21, 43201-Reus, Spain

Received 18 September1995;acceptedin finalform 17 July 1996

Abstract

The taxonomy of the fungal genus Chrysosporium is mainly based on morphological features. In our current studies
we have found several Chrysosporium species which showed intermediate morphological characteristics between
several species. For this reason, we have carried out an analysis of the mitochondrial DNA restriction fragments of
these strains that have permit us to classify each isolate strain in a species.

Introduction and its mode of inheritance independent of the nuclear

The fungal genus Chrysosporium is composed of a
large group of species frequently isolated from soil [ 1-
3]. The majority of them are anamorphs of dermato-
phytes and have keratinophilic properties. The identifi-
cation of these species is simple when the teleomorph
xs present. However, a number of them have not been
associated with any teleomorph and the characteris-
tics used for their identification are very similar. Their
conidial structures are very simple and the distinc-
tion between the species are based mainly on obscure
observable morphological features, such as conidi-
al shape and size, wall ornamentation, abundance of
intercalary conidia, etc. The differences of Chrysospo-
riurn with other similar genera such as Myceliophtho-
ra, Malbranchea or Geomyces, seem to be sometimes
subjective.
Different molecular approaches provide a useful
tool in elucidating systematic ambiguities in difficult
fungal groups, such as the valuation of fatty acids of
the cell [4], wall polysaccharides [5, 6], isoelectric
focusing studies of somatic extracts [7] and mainly
nucleic acids [8]. Recently several molecular markers,
based on DNA, have been used successfully in fun-
gal taxonomy, such as nuclear base composition [9],
DNA homology [9] and restriction fragment length
polymorphisms (RFLP) of nuclear and mitochondrial
DNA (mtDNA) [ 10]. The small size of the mtDNA
genome make this molecule attractive for studies of
fungal biology, taxonomy and evolution [ 11-14]. This
technique can be used to assess relationships between
genera [15], species [16-20] or inclusive isolates of
the same species [21-26]. The digestion of total fun-
gal DNA with '4-base cutter' enzymes such as HaeIII,
CfoI and MspI produces a small number of distinct,
high molecular weight fragments, which have been
presumed to correspond to mtDNA [26-28].
In our current studies on clinical and environmental
keratinophilic fungi we have found several Chrysospo-
rium strains which are difficult to identify as one of the
known species but lacking enough distinctive features
to be described as new species. They show interme-
diate characteristics between several species, C. ker-
atinophilum Frey ex Carmichael, C queenslandicum
Apinis & Rees, C. tropicurn Carmichael and C. zona-
turn A1-Musallan & Tan. In the present study we have
carried out an analysis of the mitochondrial DNA
restriction profiles of these strains for assessing the
possible use of this technique as an aid in their identi-
fications.
Materials and methods
Strains. The strains used in this study are listed in
Tables 1 and 2.
66
Table 1. Type and reference strains of Chrysosporiurn species
DNA restriction analysis. DNA samples of between
Species Strain number Isolation source 3 and 7 #g were digested overnight at 37 ~ with
restriction endonucleases: HaelII (GG/CC) and CfoI
C. keratinophilum FMR 5147R River sediment sample (GCG/C) (Boehringer Marmheim) in 20 #1 volumes.
C queenslandicum IMI* 121675 T Feathers sample Samples were separated by electrophoresis in 0.7%
C tropicum CBS 171.62r Wollen overcoat
agarose in Tris-sodium borate-EDTA buffer (TBE,
C zonatum CBS* 340.89 T Soil sample
[30]). Gels were stained for 30 rain, in 0.5 mg/ml
RReference strain. ethidium bromide.
TType strain.
IMI* International Mycologycal Institute culture collection, Sur- Estimates of similarities between electrophoretic pat-
rey, UK. CBS* Centraalhureau voor Schimmelculturen, Delft, the
Netherlands.
terns. The similarities between patterns were deter-
mined by the fraction of shared bands (Dice coeffi-
cient). Clusters were was calculated by the unweight-
Table 2. Strains analyzed in the present study ed pair group method using arithmetic average linkage
(UPGMA method) included in the NTSYS package
Strain Isolation source Identification based (Numerical Taxonomy and Multivariate Analysis Sys-
on this study tem, Exeter Publishing Ltd.)
FMR* 4435 River sediment sample Chrysosportum zonatum
FMR 4436 Dung sample Chrysosportum zonatum
FMR 4437 Dung sample Chrysosportum zonatum Results
FMR 4438 Dung sample Chrysosportum zonatum
FMR 4439 Dung sample Chrysospormrn zonatum Restriction profiles of mtDNA digested with HaeIII
FMR 4440 Dung sample Chrysosporiurn zonatum and CfoI are shown in Figures 1 and 2. Digestion of
FMR 5140 River sediment sample Chrysosportum zonatum mtDNAs with HaelII (Figure 1) yielded an exclusive
FMR 5141 River sediment sample Chrysosporium tropicum restriction pattern for the four Chrysosporium refer-
FMR 5142 River sediment sample Chrysosporium tropicum ence strains. C. queenslandicum showed eight bands of
FMR 5143 Beach sediment sample Chrysosponum tropicum approximately 16.8, 7.4, 6.7, 5.5, 4.7, 3.9, 3.5 and 2.6
FMR 5144 Beach sediment sample Chrysosponum zonatum kb in length. C. zonatum type strain showed six bands
FMR 5145 River sediment sample Chrysosponum zonatum of approximately 8.0, 5.3, 4.7, 3.5, 2.6 and 2.3 kb. C.
FMR 5146 River sediment sample Chrysosponum zonatum keratinophilum showed five bands of approximately
*FMR, Faculty of Medicine of Reus, fungi culture collection, Uni- 16.8, 6.6, 3.5, 3.3 and 2.1 kb. C. tropicum showed only
versity Rovira y Vkgili, Tarragona, Spain. four bands of approximately 16.8, 4.0, 3.8 and 3.5 kb.
Similarly with CfoI, these strains yielded an exclusive
restriction pattern (Figure 2). This enzyme produced a
smaller number of bands than the other one, except in
DNA isolation. Fungal DNA was isolated as C. queenslandicum with nine bands. The C. zonatum,
described by Estmch et al. [29] with some modifi- C. keratinophilum, C. tropicum patterns gave only two
cations. Each strain was grown in Sabouraud's broth fragments of approximately 14.2 and 11.9; 14.2 and
at 28 ~ with constant shaking (200 rpm). Myceli- 9.4; 14.2 and 7.6 kb respectively.
um was recovered by filtration through nytal mesh The patterns of the problem strains were practi-
(pore diam. approximately 50 #m), washed with dis- cally identical to that of C. tropicum or C. zonatum.
tilled water, blotted with paper towels and ground to Both enzymes gave similar results. With HaelII, three
a fine powder with mortar and pestle in liquid nitro- strains (FMR 5141, FMR 5142 and FMR 5143) gave
gen. The powder was incubated for 1 h at 65 ~ in identical restriction profile as C. tropicum. The rest of
1-2 ml of extraction buffer (TrisHC1 200 raM, NaC1 strains showed an identical pattern with C. zonatum,
250 raM, EDTA 25 raM, SDS 0.5%, pH 8). The lysate with only slight differences, e.g. the FMR 5140 pro-
was extracted with phenol/chloroform/isoamyl alco- duced an extra band of 7.7 kb. With CfoI the results
hol (25 : 24 : 1) and DNA was recovered by isopropanol were practically identical. All the strains tested had the
precipitation. The pellet was washed with 70% ethanol, same band patterns as C. tropicum or C. zonatum.
dried under vacuum and resuspended in TE (TrisHC1
10 mM, EDTA 1 mM, pH 8).

Figure 1. TotaI DNA cleaved by HaeIII and separated on a 0.7%
agarose gel. Lanes 1 and I2 are a mixtureof Aphage DNA digested
with HindIII-EcoRI and HindlII used as size markers. Lane 2: C.
zonatum CBS 340.89T. Lane 3: C. tropicum CBS 171.62T. Lane 4:
C. queenslandicum IMI 121675T. Lane 5: C zonatum FMR 5140.
Lane 6: C. zonatum FMR 4440. Lane 7: C. zonatum FMR 4438.
Lane 8: C zonatum FMR 4435. Lane 9: C. tropicum FMR 5141,
Lane 10: C tropic'urn FMR 5142. Lane 11: C keratinophilurn FMR
5147R.
Discussion
The polymorphisms resident in the mtDNA molecule
have been widely reported in filamentous Amgi [8].
These polymorphism patterns have been used as inter-
specific as well as intraspecific level [21-26]. In the
present study, we have obtained RFLPs of the mtDNA
with the use of '4 base-cutter' enzymes. These restric-
tion enzymes have permitted us to carry out mtDNA
analysis without the necessity of purifying the mtD-
NA. This analysis is less time-consuming and permits
the routine analysis of a large number of samples [8, 20,
26]. These enzymes recognize a large number of sites
in the fungal nuclear DNA and few sites in the mtDNA.
Hence, mitochondrial restriction fragments are easily
observed by agarose gel electrophoresis. This gives
an advantage for our study because the production of
few bands permits us to establish an exclusive mtDNA
pattern for each species.
67
Figure2. TotalDNAcleavedby CfoI and separatedon a 0.7%agarose
gel. Details as in Figure 1.
In the present study, we have utilized this technique
for the classification of 13 isolates of Chrysosporium
which according to their morphological and cultur-
al characteristics cannot be assigned to any species,
although they showed features shared by one or sever-
al of the following four species: C. keratinophilum,
C. queenslandicum, C. tropicum and C. zonatum.
Morphologically, C. zonatum is closely related to C.
queenstandicum; the species can be differentiated by
the colony colour, white in C. queenslandicum and
buff in C. zonatum. However, very old cultures C.
queenslandicum also become buff in colour. In C.
queenslandicum, arthroconidia occur sporadically but
are abundant in C. zonatum. C. keratinophilum can
be distinguished from C. tropicum by the larger coni-
dia, occasional presence of intercalary conidia and the
often echinate conidia. The problem was investigat-
ed and solved using the enzymes HaeIII and CfoI and
comparing the mtDNA band patterns obtained from
the four reference strains. Then, the 13 isolates were
assigned to a species: three to C. tropicum and ten to
C, zonatum (Table 2). From these results, we conclude
that the use of restriction enzymes with G-C four base
recognition may be a rapid and simple method to solve
taxonomic problems of closely related species.
 68

o.oo
t '
o.~ ~ .... o .I ~ 1.oo

Ch. zonatum

Ch. queensla.ndlcum

Ch. tropIcum

Ch. keratlnophllum

Figure 3. UPGMA dendrogram of the four reference strains. Dendrogram was obtained by using Dice coefficient (fraction of shared restriction
fragments).

Table 3. Similarities between mtDNA restriction patterns of

To quantify the degree of relation between the four the type and reference Chrysosporium strains studied a
species involved in the study, we performed a clus-
ter analysis on the basis of the fraction of shared Strain Similarity to strain:
restriction fragments (Table 3). The resulting dendo- CBS CBS IMI FMR
gram, depicted in Figure 3 shows the relation among 340.89 T 171.62 121675 5147
mitochondrial restriction patterns of the four reference
CBS 340,89 1.0000
strains: two different branches with C. zonatum clus- CBS 171,62 0.1428 1.0000
tered with C. queenslandicum and C. tropicum with IMI 121675 0.3076 0.2500 1.0000
C. keratinophilum (this result agrees with the mor- FMR5147 0.1333 0.4615 0.1600 1.0000
phological similarities existing between these species).
Although the method used here is valuable for tax- aSimilarities were determined as the fraction of shared restric-
tion fragments (Dice coefficient).
onomic purposes, its phylogenetic value is limited
because the number of characters (fragments of mtD-
NA) studied are few.
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Adclressfor correspondence: J. Cano, Unidad de Microbiologia,
Escuela de Enologia, Universidad Rovira i VirgiIi, Ram6n i Cajal
s/n, 43005-Tarragona; Spain.
Phone: 34-77-229596.