Genotypic Analysis of Variability in Zygomycetes a Mini-Review

Acta Biologica Hungarica 56 (3–4), pp. 345–357 (2005)

GENOTYPIC ANALYSIS OF VARIABILITY IN ZYGOMYCETES A MINI-REVIEW

M. TAKÓ* and Á. CSERNETICS

Department of Microbiology, Faculty of Sciences, University of Szeged P.O. Box 533, H-6701 Szeged, Hungary

(Received: February 21, 2005; accepted: March 11, 2005)

Different types of molecular markers are available for use in evolutionary and population studies of microscopic fungi. These approaches have proved their merits and have been successfully applied to a wide range of fungal species belonging in the Ascomycetes and Basidiomycetes. Species in the class Zygomycetes have been rather neglected from this aspect. This review discusses the information available from investigations of the genotypic variability in this group of fungi.

Keywords: Genetic variability – molecular markers – Zygomycetes

INTRODUCTION

The fungal class Zygomycetes comprises about 900 species; some of them are very widespread and abundant in nature. Currently there is an increasing interest in the exploitation of the biotechnological potential of some zygomycetes; other representatives of this group are known to be opportunistic pathogens of humans and animals. Certain other species are important as postharvest pathogens of agricultural products, or as spoilage microorganisms of certain foods. Molecular genotyping is of great potential for the provision of data for phylogenetic studies, taxonomic studies, diagnostic applications (i.e. the recognition and determination of defined taxonomic enti- ties), epidemiology and population genetics and monitoring for biotechnologically exploitable strains. Nonetheless, most of these fungi have hence received only limit- ed attention to date. Among the scientific problems that are awaiting solution, species identification is notoriously difficult in certain taxa of the Zygomycetes. Identifications below the genus level have traditionally been based on morphological observations (and sometimes on the results of mating experiments). However, such approaches are essentially hampered by the lack of good morphological markers and the rather unique mating behaviour of these fungi. The establishment of different types of molecular markers provides a new possibility with which to handle these

*Corresponding author; e-mail: [email protected]

0236-5383/$ 20.00 © 2005 Akadémiai Kiadó, Budapest 346 M. TAKÓ and Á. CSERNETICS questions [57, 59]. This review discusses the results and limitations of the molecular genotypic analysis in this fungal group.

Isoenzyme analysis

Isozyme analysis provides a well-established approach for the revelations of genetic variability in fungal populations. The protein polymorphisms detected reflect directly on the genetic background of the fungi; this approach therefore offers a relatively neutral means of determining genetic variation. As numerous different isoenzymes can be detected, it makes isoenzyme analysis an extraordinary flexible approach [25]; the technique can be useful in resolutions at population, intraspecific and species levels. The characteristics determined by means of this method are generally accepted as beeing of independent genetic origin [21]. As concerns the various isozyme studies carried out with the Zygomycetes, the results obtained by Stout and Shaw [56] were rather controversial. They investigated 28 isolates representing 20 Mucor species and found as much variation among M. racemosus isolates as among isolates representing different Mucor species. On the other hand, they did not observe any electrophoretic differences when 1 M. hiemalis and 1 M. mucedo strain were compared. Havens [16] carried out a similar survey in the section Hiemalis of the genus Mucor [50]: he coupled mating tests with isozyme analysis to reduce the possibility of misidentification. The strains investigated displayed relatively high levels of polymorphism; the isozyme similarity and sexual compatibility exhibited a correlation coefficient of 0.61. When 30 isolates of 10 Mucor species were studied with the involvement of 13 different isoenzyme systems, the lowest degree of variation was found in the isoenzyme markers of M. plumbeus, a Mucor species with very characteristic morphological traits [71]. Mucor piriformis is an important postharvest pathogen of some fruits and vegeta- bles. Isozyme polymorphisms (tested with 6 enzyme activities) among 59 isolates of M. piriformis from pears and nectarines were low: 7 electrophoretic phenotypes were identified for all the enzymes. However, there was a correlation between the esterase, glucose-6-phosphate dehydrogenase and malate dehydrogenase banding patterns and the mating potency of the isolates from pears; those with mating abilities (plus and minus isolates) were clearly different from those without mating abilities (neutral isolates). The 30 isolates from nectarines proved to be very homogeneous genetically; they did not reveal any polymorphisms [66]. Such a lack of isoenzyme polymorphisms has also been observed in Gilbertella persicaria (originally described as Choanephora persicaria), which is known to be a storage-rot microorganism pri- marily of peaches: however, in this case no correlation was found between the isoenzyme markers and the mating abilities [35, 39]. Homothallism is rather rare in the genus Mucor, where most of the known species are heterothallic. In a study, in which 10 strains of Mucor genevensis (a homothallic and dimorphic species), and representatives of 2 new homothallic species (Mucor meguroense and Mucor hachijyoensis) were investigated, substantial polymorphism

Acta Biologica Hungarica 56, 2005 Genotypic analysis of variability in Zygomycetes 347 of the isoenzyme markers was detected in M. genevensis [62]. Though both the M. meguroense strain and the M. hachijyoensis strain revealed characteristic differences, they grouped closer to the homothallic M. genevensis than to the heterothallic M. piriformis and M. hiemalis strains. These results are interesting in comparison with a study on Rhizomucor isolates [73]. These thermophilic fungi are of interest both as aetiological agents of zygomycosis and for their biotechnological abilities. As concerns the 2 ubiquitous species of the genus, R. miehei has been found to be homothallic, while R. pusillus is mainly heterothallic [51]. In an isoenzyme study (coupled with carbon source assimilation tests), 18 R. miehei and R. pusillus isolates were assayed. 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 5 enzyme systems investigated. One explanation could be that this phenomenon is connected with the homothallic and (mainly) heterothallic natures of R. miehei and R. pusillus, respectively. However, in this case the background of the high variability observed in M. genevensis remains unresolved. The relatively low polymorphism observed in these works suggest that isozyme analysis is not the most appropriate method of choice when individual isolates are to be characterized, but it demonstrates the value of this approach for the detection intraspecific diversity within a species.

Random amplified polymorphic DNA (RAPD) analysis

Among the molecular approaches available, RAPD analysis has proved to be a rapid and sensitive method. This is the reason why this technique has been used efficient- ly for the genotypic analysis of the Zygomycetes for various purposes. A biological control study demands the ability to distinguish released pathogens from locally occurring isolates of the same species. Zoophthora radicans (Entomophthorales) is a pathogen of the potato leafhopper. RAPD analysis was applied to Z. radicans isolates to determine the spread of fungi released into the envi- ronment. Besides verifying the successful establishment of experimental release, RAPD analysis revealed clear relationships among isolates derived from the same host taxon [18]. Entomophaga grylli is a pathogen that displays host-specific variance to grasshopper subfamilies. In the study by Bidochka et al. [4] 3 pathotypes of the E. grylli species complex were clearly differentiated by RAPD analysis (and two other molecular techniques). The pathotypes exhibited different amplification patterns: there was little or no interisolate variability in RAPD fragment patterns within each pathotype. RAPD analysis has been used to generate DNA fragments that are unique to isolates of 2 arbuscular mycorrhizal fungi: Glomus mosseae and Gigaspora margarita. Species-specific primer pairs have been established, allowing the subsequent specific identification of these fungi [1].

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The population structure of Basidiobolus (the number and distribution of the fungus in the soil and litter) was also studied via RAPD analysis. Isolates of Basidio- bolus and Conidiobolus (a related fungus) were obtained from different soil samples. RAPD analysis indicated that each Basidiobolus isolate is genetically unique; consequently, there is no evidence of clonal growth in soil. This study suggested that in some cases the intestines of reptiles and perhaps other animals may be a more significant reservoir than the soil for Basidiobolus [30]. Seven strains of the facultative mycoparasite Parasitella parasitica proved to be highly diverse when they were compared by RAPD analysis. This technique allowed their division into 2 groups, but they showed no morphological differences and the zygospores were found in all possible combinations either within or between the 2 groups [7]. RAPD analysis of 30 isolates of M. piriformis collected from infected fruit revealed 19 composite amplification types, indicating a much higher degree of variability than described in previous isoenzyme studies [38]. Numerical analysis revealed 3 clusters, which correlated with the mating competency of the isolates or their place of origin. These results demonstrate that RAPD analysis can identify isolates and subspecific populations of M. piriformis. Similar observations have been obtained with G. persicaria: in this case, RAPD markers were obtained which correlated with the mating types (plus or minus) of the isolates [31, 35, 41]. In a study on 23 Rhizomucor isolates, specific amplicons of value for the differentiation of R. miehei from R. pusillus could be identified by the RAPD technique [34, 74]. Similarly to izoenzyme analysis [73], RAPD analysis revealed that R. miehei isolates were more homogeneous genetically than the highly diverse R. pusillus strains. A taxonomic revision of the genus Rhizopus ruled out the independence of several described species (e.g. R. reflexus, R. circinans and R. niveus) from morphological and physiological considerations and differentiated them as only 2 varieties within the species: R. stolonifer var. stolonifer and R. stolonifer var. reflexus [52]. In contrast with this situation, RAPD analysis revealed a high degree of genetic heterogeneity within the Rhizopus stolonifer species suggesting practically interspecific distances between the isolates of R. stolonifer var. stolonifer and R. stolonifer var. reflexus [61]. The cluster analysis of the RAPD data demonstrated a species-level genetic distance between the R. stolonifer strain, CBS403.51, the type strain of the obsolete R. niveus, and all the other Rhizopus isolates. This work supported the need for a reconsideration of the original taxonomic distinction. In the same study, isolates of R. oryzae of very different origins formed a well-defined group, as an indication of the unity of this species. Lusta et al. [23] provided a practical application of RAPD-based diagnosis: a newly isolated fungal strain, which was found to secrete a high level of extracellular lipase, was successfully identified by RAPD, in combination with morphological- physiological methods, as R. microsporus var. rhizopodiformis.

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As an alternative to RAPD-PCR, a PCR fingerprint method wit a single consen- sus primer targeted to clustered tRNA genes was successfully employed by Carter et al. [8] to identify different strains of the species Mortierella alpina. In spite of the great advantages (e.g. non-radioactive detection, no prior sequence information required and experimental simplicity), problems inherent in the reproducibility and interpretation of the amplification patterns are generally mentioned as the main drawbacks of RAPD. Variations in the experimental design and conditions (e.g. a minor change of ion concentration) can affect the RAPD patterns observed, which reduces the reproducibility of a complex pattern as a whole. As such reproducibility problems mainly involve the faint bands, all amplifications should be repeated more than twice, and only amplicons that are reproducibly detected should be taken into account.

Restriction fragment length polymorphism (RFLP) analysis

Although RFLP is particularly valuable for taxonomy and strain typing, only a few data are available concerning the application of this method in Zygomycetes. Restriction fragments from genomic DNA successfully differentiated E. grylli pathotypes: genomic probes specific for each pathotype were isolated. These probes did not hybridize to DNA from Entomophaga aulicae or from grasshoppers [4]. Nested PCR with Entomophthora-specific primers targeted for the internal transcribed spacer (ITS) was used in combination with RFLP by Thomsen and Jensen [58]. Their analysis allowed the identification of resting spores and pointed to the need for more detailed studies to clarify host-pathogen specificity and interactions. When PCR-RFLP was applied to verify the taxonomic position of 5 Eryniopsis species and E. ptychopterae and E. transitans were found to belong in the genus Entomophaga [15]. The single known isolate of R. tauricus has been investigated by PCR-coupled RFLP of the ITS region. This study (and the accompanying isoenzyme analysis) suggested extensive similarity between R. tauricus and R. pusillus. Accordingly, the isolate described originally as R. tauricus does not represent a true species, but is a devi- ating heterothallic R. pusillus strain [68]. ITS-RFLP and microsatellite PCR fingerprinting was performed on several clinical isolates of Apophysomyces elegans, a rare agent of human zygomycosis with an increasing number of cases in India [9]. ITS-RFLP was successfully applied to dif- ferentiate A. elegans from other species of the Zygomycetes, while microsatellite PCR fingerprinting was utilized for the analysis of the intraspecific genetic polymorphism. Similarly, a differentiation of clinical isolates of Cunninghamella bertholletiae, C. echinulata and C. elegans at a subspecies level could be achieved by ITS- RFLP and PCR fingerprinting [22].

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Comparison of DNA sequence data

Comparison of ribosomal RNA genes (rDNA) is the method most frequently applied for phylogenetic and molecular taxonomic studies [13]. While the 18S rDNA and domains D1 and D2 of the 28S rDNA are commonly used for the phylogeny of major groups of filamentous fungi (i.e. at a genus level or above), the ITS are of great potential as targets for the characterization and identification of closely related species and intraspecific groups. In the recent past, phylogeny and evolutionary relationships of the Zygomycetes have been analysed in detail on the basis of 28S and 18S rDNAs and some protein coding gene sequences, such as actin and translation elongation factor EF-1α genes [32, 75, 76], and several taxonomic problems could be resolved. These works focused mainly on the order Mucorales and revealed that the traditional, morphologically based classification schemes are highly unnatural at the family level. These studies demonstrated that the genera Micromucor- Umbellopsis (previously placed in the order Mortierellales) are in fact members of the Mucorales, and highlighted the need for the establishment of the new order Mortierellales (including the genera Mortierella, Dissophora and Echinosporan- gium); some families and genera (i.e. Mucor and Absidia) of the Mucorales proved to be polyphyletic. Other protein coding sequences, such as orotidine 5’-monophosphate decarboxylase of M. circinelloides, Phycomyces blakesleeanus and Blakeslea trispora [44, 45], glyceraldehyde-3-phosphate dehydrogenases (gpd) of M. circinelloides and R. miehei [2, 72], HMG-CoA reductase (hmg) of R. miehei [64] and many others have been compared with members of other major groups of fungi (e.g. the Ascomycetes and Basidiomycetes). The findings suggest that the Zygomycetes are situated some- what closer to the Basidiomycetes than to the Ascomycetes. In contrast with the abundance of phylogenetic studies on higher taxonomic levels, sequence comparisons have rarely been applied to analyse the genetic variability of this fungal group at a subgenus level or especially at a subspecies level. Sequencing of the ITS region has been employed to identify 7 of 9 Cunninghamella isolates as C. bertholletiae and 2 as C. echinulata, and for the first time C. echinulata could be identified as an agent of zygomycosis in humans [22]. The recent taxonomic position of the genus Gilbertella was analysed by sequencing and comparison of the ITS regions and the 5.8S rDNA of 12 mucoralean isolates belonging in 6 genera [33]. Gilbertella was found to be closely related to Choanephora and Blakeslea, while a relatively low level of homology was detected between the ITS regions of R. miehei and R. pusillus, supporting the results of earlier isoenzyme and carbon source utilization studies [73]. At the same time, the ITS sequences of the thermophilic Rhizomucor strains did not exhibit similarity with the sequences of the other mucoralean strains. Analysis of partial gpd sequences from several strains belonging in the genera Mucor, Blakeslea, Choanephora, Poitrasia, Gilbertella and Rhizomucor led to similar results [40]. In spite of the paucity of data concerning the members of the order Mucorales, rDNA sequence-based methods are frequently used for species and strain identifica-

Acta Biologica Hungarica 56, 2005 Genotypic analysis of variability in Zygomycetes 351 tions for the arbuscular mycorrhizal fungi belonging in the order Glomales. In this group, much effort has been focused on molecular identification based on rDNA sequences, for morphological analysis is hampered by the ecological, physiological and methodological conditions [47–49]. PCR primers have also been employed to amplify exclusively glomalean rDNA directly from colonized roots while eliminat- ing amplification of plant or other fungal DNA [17, 46, 55, 69]. Phylogenetic data and methods of molecular identification of the Entomophtho- rales, which contain the insect-pathogenic zygomycete species, are important from the point of view of planning possible biocontrol strategies based on these fungi [14]. Jensen et al. [19] published a phylogeny inferred from 18S rDNA sequences, where the order proved to be monophyletic with the exception of Basidiobolus ranarum, which is related to the Chytridiomycota rather than to Zygomycota as proposed previously. The Mucorales and Entomophthorales formed a sister group separated from the Mortierellales in accordance with the above-mentioned works [32, 75, 76]. The ITS region was sequenced and compared to assess the genetic variation among several isolates of the aphid pathogen Pandora neoaphidis and related species [60]. The species-specific primers designed by using the resulting sequences proved to be useful tools for the identification of P. neoaphidis and P. kondoiensis from infected aphids without isolation of the fungus.

Extrachromosomal genetic elements: mtDNAs, introns, dsRNAs and VLPs

One of the first RFLP techniques widely used in fungal taxonomy involves comparison of the restriction patterns of the mitochondrial DNA (mtDNA), which indicates differences below the species level. However, as regards the zygomycetous fungi, there have been only reports concerning the mtDNA organization of individual isolates from a few species, i.e. M. racemosus [53], M. piriformis [37], R. stolonifer [43], R. oryzae, Mortierella verticillata and Smittium culisetae [54]. Schramke and Orlowski [53] described the special structure of mtDNA in M. circinelloides: the mitochondrial chromosome was found to exist in the form of two flip-flop isomers with inverted repeat sequences encoding both rRNA genes. However, such structures have not been observed in M. piriformis and other mucoralean fungi. Seif et al. [54] reported on the presence of a variety of insertion elements in the zygomycetous mtDNA, such as double hairpin elements, numerous mobile group I introns and a new mobile endonuclease element. Their phylogenetic analysis of the closely related group I introns of the cox1 genes revealed that these intronic ORFs are closest to an intron that also invaded angiosperm mtDNAs. Mycoviruses have been reported from all major groups of microscopic fungi: it is estimated that virus-like particles (VLPs) occur in about 30% of all fungal species. The term VLP is used for particles which are morphologically similar to viruses, but for which no information on particle composition is available [5, 12]. Although the presence of mycoviruses can induce phenotypic consequences ranging from hypovir-

Acta Biologica Hungarica 56, 2005 352 M. TAKÓ and Á. CSERNETICS ulence to hypervirulence, and from an unaltered host organism to one with an abnor- mal phenotype, in most cases the fungal hosts that harbour them do not exhibit dis- cernible alterations in their phenotypes [70]. The presence of fungal viruses can therefore generally be revealed only through detection of their genomes and/or the VLPs in the cell. The vast majority of mycoviruses are isometric particles 25–50 nm in diameter and contain undivided, or segmented double-stranded RNA (dsRNA) genome. The presence of a specific dsRNA pattern in a fungal nucleic acid extract is therefore a good indicator of mycovirus infection. In spite of the growing number of mycoviruses detected in fungal hosts, there have been few reports on their presence in fungi belonging in the Zygomycetes. VLPs have been detected in 3 zygomycete species: electronmicroscopic ultrastructure studies revealed enveloped bacilliform VLPs in the entomoparasitic fungus Strongwell- sea magna [11], pleomorphic particles in the similarly entomopathogenic Entomo- phaga aulicae [26] and isometric VLPs in the trichomycete Paramoebidium arcua- tum [24]. However, these particles have not been characterized biochemically, and accordingly their viral nature remains uncertain. The occurrence of mycoviruses was recently reported in some genera of Muco- rales: dsRNA molecules and isometric VLPs were detected in some isolates of 4 Mucor species (M. aligarensis, M. hiemalis, M. corticolus and M. mucedo) and also in Micromucor rammaniana [63, 67]. Isolates belonging in 4 Rhizopus species were also screened for the presence of dsRNA molecules: of the 27 isolates examined, 2 R. stolonifer, 2 R. microsporus and 1 R. oryzae harboured such genetic elements [36]. The molecular sizes corresponding to these bands were in the range 2.2–14.8 kb, while electronmicroscopy revealed polyhedral VLPs 40 nm in diameter. In one of the R. microsporus isolates, the occurrence of an uncapsidated large dsRNA segment (14.8 kb) was probable. Such 12–14 kb dsRNA elements have been demonstrated in some G. persicaria isolates screened for the presence of dsRNA elements. No phenotypic effect or VLPs in the fungal cytoplasm have been detected [35, 42].

Pulsed field gel electrophoresis (PFGE)

Fungal genetics achieved a significant progress when electrophoretic karyotype analysis using different PFGE techniques became available. This methodology allowed the separation of DNA molecules even larger than 10 megabase pairs (Mb) [77]. As a result, the physical karyotypes of numerous previously genetically unchar- acterized fungal species have been established, but among them there have been only a few reports on the genome analyses of zygomycetes fungi. In early experiments, orthogonal field alteration gel electrophoresis (OFAGE) karyotyping resolved 5 chromosomal mobility groups: as a known underestimate, a genome size of 30 Mb was determined for M. circinelloides f. lusitanicus [65]. The improved separation of chromosomal size DNA molecules through contour-clamped homogeneous electric field (CHEF) gel electrophoresis revealed the presence of 8 bands for a strain of M. circinelloides f. lusitanicus, and 4, presumably multiple

Acta Biologica Hungarica 56, 2005 Genotypic analysis of variability in Zygomycetes 353 bands for a M. circinelloides f. gryseo-cyanus strain. The approximate sizes of the resolved chromosomal DNA bands ranged from 2.3 to 8.1 Mb, giving estimated genome sizes of 38.7 and 32.6 Mb, respectively [29]. The presence of chromosomal length polymorphism (CLP) in Mucor became evident from the study by Diaz- Minguez et al. [10] when 10 strains of Mucor circinelloides f. lusitanicus were analysed by CHEF gel electrophoresis. Most of the strains analysed displayed polymorphisms, but a different main karyotype pattern could be correlated with each mating type. Hybridization experiments confirmed the similarity between the mating- type (minus) strains and indicated some heterogeneity among the mating-type (plus) strains. Further studies established electrophoretic karyotypes for other Mucor species (M. bainieri, M. mucedo, M. plumbeus and M. racemosus) [27]. The sizes of the chromosomal DNA were estimated to be between 2.5 and 8.7 Mb., while the total genome sizes were calculated to be between 30.0 and 44.7 Mb. The fact that their karyotypes differ was not surprising, but the results also showed that M. plumbeus strains have very similar karyotypes, with only 2 different chromosomal mobility groups. These observations harmonize well with the results of an earlier isoenzyme study [71]. Chromosomal size DNA from the zygomycete Absidia glauca have been resolved by rotating field gel electrophoresis (RFGE) [20]. As observed in M. circinelloides, strains with different mating types exhibited considerable differences in their electrophoretic karyotypes. The total genome sizes of Absidia glauca were calculated to be between 33.3 and 42 Mb. A different situation was observed when 7 strains of P. parasitica were analysed by RFGE (and also with RFLP and RAPD-PCR). These strains were highly diverse at the molecular level and could be divided consistently into 2 different groups: the chromosome sizes were highly divergent, ranging from 3 to 6.5 Mb in one group and between 2 and 4.5 Mb in the other. However, these differences did not correlate with the mating types [7]. Electrophoretic karyotypes have also been revealed from 2 Micromucor species (M. isabellina and M. ramanniana). The most surprising result of these experiments was that the Micromucor chromosomal DNAs are rather small (between 2.60 and 0.4 Mb); most of them are comparable in size to those detected, for example in Saccharomyces. All other zygomycetes genomes investigated to date revealed larger chromosomal DNAs and consequently larger genome sizes. The chromosomal banding patterns likewise revealed substantial variability among the isolates: 11 to 14 chromosomal mobility groups were resolved and the minimum total genome sizes were estimated to be between 24.19 and 24.9 Mb [28]. The experimental results reviewed here demonstrate the merits of molecular approaches in resolving taxonomic problems, diagnostic applications and strain identification of fungal taxa when investigation of the morphological traits does not sup- ply sufficient concrete information. The high genetic variability observed within some species can be explained in terms of either existing intraspecific variations or the inadequacy of species identifications. From this respect, genotypic analysis with different types of molecular methods, critical analysis and comparison of their results are of special importance in this fungal group.

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