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65
9 1996Kluwer Academic Publishers. Printed in the Netherlands.
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.
Figure 1. TotaI DNA cleaved by HaeIII and separated on a 0.7% Figure2. TotalDNAcleavedby CfoI and separatedon a 0.7%agarose
agarose gel. Lanes 1 and I2 are a mixtureof Aphage DNA digested gel. Details as in Figure 1.
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. In the present study, we have utilized this technique
Lane 8: C zonatum FMR 4435. Lane 9: C. tropicum FMR 5141, for the classification of 13 isolates of Chrysosporium
Lane 10: C tropic'urn FMR 5142. Lane 11: C keratinophilurn FMR which according to their morphological and cultur-
5147R.
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,
Discussion C. queenslandicum, C. tropicum and C. zonatum.
Morphologically, C. zonatum is closely related to C.
The polymorphisms resident in the mtDNA molecule queenstandicum; the species can be differentiated by
have been widely reported in filamentous Amgi [8]. the colony colour, white in C. queenslandicum and
These polymorphism patterns have been used as inter- buff in C. zonatum. However, very old cultures C.
specific as well as intraspecific level [21-26]. In the queenslandicum also become buff in colour. In C.
present study, we have obtained RFLPs of the mtDNA queenslandicum, arthroconidia occur sporadically but
with the use of '4 base-cutter' enzymes. These restric- are abundant in C. zonatum. C. keratinophilum can
tion enzymes have permitted us to carry out mtDNA be distinguished from C. tropicum by the larger coni-
analysis without the necessity of purifying the mtD- dia, occasional presence of intercalary conidia and the
NA. This analysis is less time-consuming and permits often echinate conidia. The problem was investigat-
the routine analysis of a large number of samples [8, 20, ed and solved using the enzymes HaeIII and CfoI and
26]. These enzymes recognize a large number of sites comparing the mtDNA band patterns obtained from
in the fungal nuclear DNA and few sites in the mtDNA. the four reference strains. Then, the 13 isolates were
Hence, mitochondrial restriction fragments are easily assigned to a species: three to C. tropicum and ten to
observed by agarose gel electrophoresis. This gives C, zonatum (Table 2). From these results, we conclude
an advantage for our study because the production of that the use of restriction enzymes with G-C four base
few bands permits us to establish an exclusive mtDNA recognition may be a rapid and simple method to solve
pattern for each species. 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).
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