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The zygomycoses comprise a diverse group of rare mycotic diseases; the term mucormycoses is preferred for zygomycoses caused by some members of the order Mucorales (e.g., Absidia, Rhizomucor, Rhizopus, and Mortierella [7, 10]). These infections are most frequently associated with diabetic ketoacidosis, immunosuppressive conditions, extreme malnutrition, or neutropenia (7). Though these mycoses are relatively rare, they attract special attention, as they are rapidly progressive and frequently fatal (12, 14).

The genus Rhizomucor includes three species: Rhizomucor pusillus, Rhizomucor miehei, and Rhizomucor tauricus; these are clearly distinct from Mucor by virtue of their thermophilic nature and some morphological features (8). While the legitimacy of regarding R. tauricus as an autonomous species (represented by a single isolate only) is sometimes questioned, the other two Rhizomucor species are well represented in nature (8).

Fungi belonging in this genus are found among the etiological agents of human and animal mucormycosis. The role of R. tauricus in such infections is unknown, but there are both clinical and experimental data concerning infections caused by R. pusillus or R. miehei (7, 11). However, there are two reasons why the exact number of infections caused by one or another of the Rhizomucor species can only be guessed at. One is that in several cases the fungal pathogen has not been identified properly at low taxonomic levels (genus and species), and the other is that there is some uncertainty in the differentiation of R. miehei and R. pusillus isolates. Though R. miehei has been found to be homothallic, while R. pusillus is mainly heterothallic, the discovery of the rarely occurring homothallic R. pusillus isolates did not help to simplify the scheme (8). Certain approaches, such as determination of the number of nuclei in the sporangiospores (15) or mating studies coupled with determination of the morphological traits of the zygospores (8, 18), could supply characteristics for the delimitation of these species. However, the differences are not clear-cut in every case (15), or the procedure requires a prolonged time (8).

Both isoenzyme analysis and the determination of carbon source utilization patterns have proved to be valuable tools in the handling of taxonomic questions in the genus Mucor (16, 17). Analysis of proteases by immunoelectrophoretic techniques underlined the connection of the homo- and heterothallic strains of R. pusillus and their difference from R. miehei (2). Assimilation abilities on a limited number of compounds have also been checked: the growth-stimulative effect of thiamine and the inability to assimilate sucrose were found to be characteristic of R. miehei (10, 11), although these differences seem to be insufficient to allow species delineation in every case. These characteristics, together with the colony color (brownish for R. pusillus and grayish for R. miehei) and the sizes of the zygospores (the diameter is below 50 µm for R. miehei and over 50 µm for R. pusillus), are now used to identify Rhizomucor isolates (8, 11).

The purpose of the present study was to broaden the basis of knowledge, affording a methodically simple, quick, and more unambiguous identification of the two Rhizomucor species. As part of this, carbon source assimilation patterns were determined and isoenzyme analysis was carried out with 19 Rhizomucor strains obtained from various sources and three other zygomycetous strains (Table 1).

The abilities of the Rhizomucor and selected Absidia, Mucor, and Rhizopus strains to utilize 87 individual compounds as their sole carbon source were tested basically as described earlier for Mucor isolates (16), except that the incubation was at 37°C (20°C for Mucor).

Of the 87 carbon substrates tested, 13 yielded uniformly positive results; these included D-lyxose, L-xylose, maltose, lactose, melibiose, starch, sorbitol, L-alanine, L-proline, L-tyrosine, L-asparagine, raffinose, and glycerol--monoacetate. Thirteen other compounds gave only negative results; these were xylan, -butyrolactone, vanillin, -methyl-D-xyloside, ascorbic acid, L-glutamine, L-malic acid, methanol, orotic acid, L-lysine, protocatechuic acid, inulin, and gallic acid. Certain carbon substrates led to ambiguous results with low reproducibility. These were L-rhamnose, L-isoleucine, L-serine, cytosine, thymine, dihydroxyacetone, -methyl-D-galactoside, D-glucosamine, fumaric acid, succinic acid, gluconic acid, L-ornithine, and uracil.

The remaining 48 carbon substrates were found to be utilized to various extents by the investigated strains (Table 2). Most of these variations were intergeneric and related to the differences observed between Rhizomucor, Absidia, Mucor, and Rhizopus strains. Among them, however, four compounds whose utilization showed a clear difference between the R. miehei and R. pusillus isolates were identified. These were sucrose, glycine, phenylalanine, and -alanine; this is the first example of the distinctive value as biochemical markers of the last three of these. The difference in sucrose utilization was described earlier (10); this and the stimulative effect of thiamine on the growth of R. miehei were the only two biochemical markers of some practical value for the differentiation of these species (11). The use of these four carbon sources allows a clear delineation of these two species.

To provide further characteristics of distinctive value, isoenzyme analysis of the isolates was also carried out. Sporangiospores collected from malt extract agar slants incubated at 37°C for 5 days were inoculated (10⁷ spores/150 ml) in yeast extract-glucose liquid medium. Cultivation was carried out in 500-ml Erlenmeyer flasks at 37°C on a rotary shaker at 200 rpm. Protein extraction and electrophoresis were performed as described earlier (17). The resolvable enzyme systems used were acid phosphatase (EC 3.1.3.1) (4), -esterase (EST) (EC 3.1.1.2) (4), glutamate dehydrogenase (EC 1.4.1.4) (1), glucose-6-phosphate dehydrogenase (G6D) (EC 1.1.1.49) (5), and malate dehydrogenase (EC 1.1.1.38) (3). All incubations took place at 21°C, in the dark. The gels were then rinsed, first in distilled water and next in 7% acetic acid, and read.

The highest variability was found for G6D; eight different electrophoretic patterns (electromorphs) were detected, while for EST three electromorphs were found. The least variable were the acid phosphatase, glutamate dehydrogenase, and malate dehydrogenase staining patterns; these each showed two different electromorphs. These two electrophoretic patterns correlated perfectly with the two investigated Rhizomucor species. A similar correlation was found in the case of the EST pattern; however, this is less easily scored because of the more complex pattern of this activity stain (Fig. 1).

View larger version (35K): [in this window] [in a new window]   FIG. 1.   Isoenzyme patterns observed for various Rhizomucor, Absidia, Rhizopus, and Mucor isolates. The direction of migration is towards the anode. RP, heterothallic R. pusillus strains; RM, R. miehei strains. For other strain codes, see Table 1.

This study was started with 18 different Rhizomucor strains. However, after preliminary experiments, it turned out that the strain NRRL 2543 (isolated from an animal mycosis) is not homogeneous; it contains two morphologically slightly different fungi. Otherwise, this strain clearly illustrates the problems with the species identification in the genus; it is maintained under different names in two international collections (as R. pusillus in the Northern Regional Research Laboratory Agricultural Research Service Culture Collection, Peoria, Ill. [NRRL], and as R. miehei in the American Type Culture Collection, Rockville, Md. [ATCC]). On the basis of our results, it was identified as R. pusillus. While the carbon source assimilation patterns of these separated strains were the same, their G6D patterns were different.

Besides determining easily countable characteristics for the identification of Rhizomucor isolates, the experimental data provide preliminary information concerning the genetic variability of these species. After enzyme staining, each independent band with a defined relative mobility was coded with a binary state character; this was 1 if the band was present and 0 if the band was absent for an isolate (variations in staining intensity were not taken into account). The results of the carbon source utilization experiments were coded as follows: 0, negative; 0.5, weak, ambiguous reaction; 1, positive reaction. Matrices created from these data were used for the calculation of Jaccard coefficients. Dendrograms were produced with an unweighted pair-group method by using arithmetic averages (UPGMA) (13) linkage. All computations were performed with the SYNTAX 5.0 software package (6).

There was no basic difference between the dendrograms created from the unified data matrix or from the matrix of carbon source utilization data (results not shown) and those created from the isoenzyme data alone (Fig. 2). All these dendrograms revealed the presence of three clusters. One of these (cluster C) corresponds to the isolates used as outgroups in this study (Ab1, N6, and Rp11), while clusters A and B contain the investigated R. pusillus and R. miehei isolates, respectively (Fig. 2). While the reading of the extent of colony growth inevitably involves some inconsistencies (resulting in a higher variation of characteristics), the evaluation of isoenzyme patterns is less problematic. Therefore, we suggest that this discloses the intraspecific variability of R. miehei and R. pusillus more precisely than the other two dendrograms. Three important features could be observed in this dendrogram. First, all R. pusillus isolates segregate from the R. miehei isolates at a very high level of dissimilarity (D = 0.90); this is practically the same as the level of dissimilarity found for the representatives of the other three genera involved in the outgroup (Absidia, Mucor, and Rhizopus; D = 0.87, 0.89, and 0.92, respectively). The second feature is that the extent of genetic polymorphism is different in the two Rhizomucor species: while substantial polymorphism was found among the R. pusillus strains, the investigated R. miehei strains proved to be homogeneous; no difference was revealed with the five enzyme systems investigated. One explanation could be that this phenomenon is connected with the different, homothallic and (mainly) heterothallic natures of R. miehei and R. pusillus, respectively. In this respect, it would be interesting to examine if there is any difference in genetic variability in the homo- and heterothallic R. pusillus strains. Unfortunately, homothallic R. pusillus strains are rare; e.g., of the strains investigated, only R2A and R2B (of common origin) were found to be homothallic. This number of strains is too limited to provide a basis for any conclusion about their different genetic variability. However, as a third observation, it could be seen that the homo- and heterothallic R. pusillus strains segregate into two subclusters at a rather high level (D = 0.56). This clustering is caused by their different EST and G6D patterns. Further investigations, with the involvement of more R. pusillus isolates, should be performed to clarify the background of this high degree of intraspecific polymorphism and its connection (if any) with the sexual characteristic.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   UPGMA dendrogram of the Rhizomucor and outgroup strains produced by UPGMA linkage of the Jaccard coefficients. It was based on the 57 characteristics obtained from their isoenzyme analysis. The scale represents dissimilarity (squared distance). The cophenetic correlation was 0.9975. The labels A, B, and C mark clusters containing the R. pusillus, the R. miehei, and the outgroup strains, respectively.