Molecular identification of ectomycorrhizal mycelium in soil horizons

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (> or = 99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had > or = 98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.     

Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (≥99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had ≥98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.     

Multiple species of ectomycorrhizal fungi are frequently detected on individual oak root tips in a tropical cloud forest

The ecological importance of ectomycorrhizal (EM) fungi in tropical ecosystems is increasingly recognized, but few studies have used molecular methods to examine EM fungal communities in tropical forests. The diversity and composition of the EM community on Quercus crassifolia in a tropical montane cloud forest in southern Mexico were characterized using DNA sequencing of single root tips. Individual root tips commonly harbored multiple fungal species that resulted in mixed polymerase chain reaction (PCR) products. By cloning and performing gel extractions on mixed PCR samples, we identified two or more EM fungi on 26% of the root tips. When non-EM fungi were considered, this figure increased to 31% of root tips. A total of 44 EM taxa and nine non-EM taxa were detected on roots from 21 soil cores (104 root tips). Taxa in the families Russulaceae, Cortinariaceae, Inocybaceae, and Thelephoraceae were frequent. This is the first study to characterize the belowground EM community in a tropical montane cloud forest. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ectomycorrhizal fungal associates of Pinus contorta in soils associated with a hot spring in Norris Geyser Basin, Yellowstone National Park, Wyoming.

Molecular methods and comparisons of fruiting patterns (i.e., presence or absence of fungal fruiting bodies in different soil types) were used to determine ectomycorrhizal (EM) associates of Pinus contorta in soils associated with a thermal soil classified as ultra-acidic to extremely acidic (pH 2 to 4). EM were sampled by obtaining 36 soil cores from six paired plots (three cores each) of both thermal soils and forest soils directly adjacent to the thermal area. Fruiting bodies (mushrooms) were collected for molecular identification and to compare fruiting body (above-ground) diversity to below-ground diversity. Our results indicate (i) that there were significant decreases in both the level of EM infection (130 +/- 22 EM root tips/core in forest soil; 68 +/- 22 EM root tips/core in thermal soil) and EM fungal species richness (4.0 +/- 0.5 species/core in forest soil; 1.2 +/- 0.2 species/core in thermal soil) in soils associated with the thermal feature; (ii) that the EM mycota of thermal soils was comprised of a small set of dominant species and included very few rare species, while the EM mycota of forest soils contained a few dominant species and several rare EM fungal species; (iii) that Dermocybe phoenecius and a species of Inocybe, which was rare in forest soils, were the dominant EM fungal species in thermal soils; (iv) that other than the single Inocybe species, there was no overlap in the EM fungal communities of the forest and thermal soils; and (v) that the fungal species forming the majority of the above-ground fruiting structures in thermal soils (Pisolithus tinctorius, which is commonly used in remediation of acid soils) was not detected on a single EM root tip in either type of soil. Thus, P. tinctorius may have a different role in these thermal soils. Our results suggest that this species may not perform well in remediation of all acid soils and that factors such as pH, soil temperature, and soil chemistry may interact to influence EM fungal community structure. In addition, we identified at least one new species with potential for use in remediation of hot acidic soil.     

Morphological and molecular analyses of ectomycorrhizal diversity in <Emphasis Type="Italic">Pinus densiflora</Emphasis> seedlings

Using ectomycorrhizal root tip morphotyping (anatomical and morphological identification) and molecular analysis (internal transcribed spacer region amplification and sequencing), we assessed diversity and composition of the ectomycorrhizal fungal community colonizing P. densiflora seedlings (from 1 to 5 years ) in a naturally regenerating area of a mature forest, in Dongning County of Hei Longjiang Province, China. A total of 22 ectomycorrhizal taxa were determined on the roots of 100 seedlings. The most frequent species was Cenococcum geophilum, which colonized 31.2% of the root tips and occurred in 93.8% of the all samples. It was followed by Clavulina cinerea, Thelephora sp.2, and Russula albidula. They colonized in 22.7%, 17.5%, 13.4% of the root tips and occurred in 63.4%, 42.6%, 29.3% of the samples respectively. These results suggested that ectomycorrhizal diversity increased in P. densiflora seedlings with increasing age.     

中国中部太白山不同海拔杜鹃兰根部内生真菌多样性

本研究以太白山自然保护区蒿坪站杜鹃兰Cremastra appendiculata为材料,采用菌丝团和根组织分离法进行真菌分离,并用ITS序列分子鉴定;用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)分析根部内生真菌多样性,研究海拔和根际土理化性质对真菌多样...     

The mycorrhizal community in a forest chronosequence of Sitka spruce [Picea sitchensis (Bong.) Carr.] in Northern England

Demography and fungal diversity of the belowground ectomycorrhizal community in a chronosequence of Sitka spruce [Picea sitchensis (Bong.) Carr.] in Northumberland, Northern England, were analysed; mycorrhizal root samples were taken from 6-, 12-, 30- and 40-year-old stands, and fungal fruiting bodies were collected in autumn to complement the survey. Naturally germinated seedlings less than 1 year of age (taken from the 30-year-old stand) were also examined. A total of 118,000 mycorrhizal root tips were extracted from 40 soil cores (ten per age class) and from the complete root systems of 25 seedlings and separated into active and senescent root tips according to their morphology and anatomy. Active tips were distinguished according to their mycobionts which were characterised and identified microscopically. Although almost 100% of all fine roots were mycorrhizal, EM fungal diversity throughout the chronosequence was low, consisting of a total of 16 species of which three were only found as fruiting bodies. Of the six mycobionts found most regularly below ground, Tylospora fibrillosa was the most common, colonising about 70% of all root tips and more than 90% of those of seedlings and young trees. Root density and mycorrhizal diversity increased, but percentage of vital root tips decreased with increasing tree age, levelling off in the 30- and 40-year-old stand. Among the five subdominant fungal species, Dermocybe crocea was found to have its peak of distribution in the 12-year-old stand and Russula emetica, Lactarius rufus, Hymenoscyphus ericae agg. and the unidentified Piceirhiza sulfo-incrustata in the 30- and 40-year-old stands. The possible correlations between the mycorrhizal community structure and biotic and abiotic factors are discussed.     

DGGE Fragments Oscillate with or Counter to Fluctuations in Cultivable Bacteria Along Wheat Roots

Previously, we showed that bacterial populations oscillate in response to a moving substrate source such as a root tip, resulting in moving wavelike distributions along roots. For this article, we investigated if bacterial communities fluctuate as a whole or if there is a succession in bacterial composition from peak to peak or within peaks. Rhizosphere microbial communities along roots of wheat Triticum aestivum L. were studied in detail (20–25 rhizosphere and bulk soil samples along the total root length) in two related soils by colony enumeration and culture-independent DNA analysis. Similar to our previous findings, the numbers of copiotrophic and oligotrophic bacteria oscillated with significant harmonics along each root, independent of soil moisture or lateral roots. Shifts in amplified eubacterial 16S rDNA fragments from denaturing gradient gel electrophoresis (DGGE) analysis were detected along the roots. The most abundant and intensively amplified fragments fluctuated in phase with colony-forming unit (CFU) oscillations; fewer amplified fragments with less intensive bands fluctuated out of phase or were restricted to certain root zones. The bacterial species richness along the root was negatively correlated with the numbers of oligotrophic bacterial CFUs. Discriminant analyses on DGGE patterns distinguished between soil types, rhizosphere and bulk soil, and waxing and waning phases in the oscillations along roots. Bacterial compositions shifted within oscillations but were repeated from oscillation to oscillation, supporting the idea that the most abundant bacterial taxa were growing and dying over time and consequently in space, whereas other taxa counterfluctuated or hardly responded to the substrate supplied by the passing root tip.     

Availability and function of arbuscular mycorrhizal and ectomycorrhizal fungi during revegetation of dewatered reservoirs left after dam removal

Revegetation following dam removal projects may depend on recovery of arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungal communities, which perform valuable ecosystem functions. This study assessed the availability and function of AM and EM fungi for plants colonizing dewatered reservoirs following a dam removal project on the Elwha River, Olympic Peninsula, Washington, United States. Availability was assessed via AM fungal spore density in soils and EM root tip colonization of Salix sitchensis (Sitka willow) in an observational field study. The effect of mycorrhizal fungi from 4 sources (reservoir soils, commercial inoculum, and 2 mature plant community soils) on growth and nutrient status of S. sitchensis was quantified in a greenhouse study. AM fungal spores and EM root tips were present in all field samples. In the greenhouse, plants receiving reservoir soil inoculum had only incipient mantle formation, while plants receiving inoculum from mature plant communities had fully formed EM root tips. EM formation corresponded with alleviation of phosphorus stress in plants (lower shoot nitrogen:phosphorus). Thus, revegetating plants have access to AM and EM fungi following dam removal, and EM formation may be especially important for plant P uptake in reservoir soils. However, availability of mycorrhizal fungi declines with distance from established plant communities. Furthermore, EM fungal communities in recently dewatered reservoirs may not be as effective at forming beneficial mycorrhizae as those from mature plant communities. Whole soil inoculum from mature plant communities may be important for the success of revegetating plants and recovery of mycorrhizal fungal communities.     

Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis.

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1. 4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.     

Ecological and molecular investigations of cyanotoxin production

This study investigates the effect of mercury contamination on the culturable heterotrophic, functional and genetic diversity of the bacterial community in soil. The changes in diversity were monitored in soil microcosms, enriched with 25 &mgr;g Hg(II) g(-1) soil, over a period of 3 months. The culturable heterotrophic diversity was investigated by colony morphology and colony appearance on solid LB medium. Functional diversity was analysed as sole carbon utilisation patterns in ECOplates. Genetic diversity was measured as bands on denaturing gradient gel electrophoresis (DGGE) gels obtained by purification of total soil DNA and amplification of bacterial 16S rDNA fragments by polymerase chain reaction. Concentrations of bioavailable and total mercury were measured throughout the experiment. The effect on the culturable heterotrophic and genetic diversity was very similar, showing an immediate decrease after mercury addition but then slowly increasing throughout the entire experimental period. Pre-exposure levels were not reached within the time span of this investigation. The DGGE band pattern indicated that a shift in the community structure was responsible for recovered diversity. When analysed by Shannon-Weaver indices, functional diversity was found to increase almost immediately after mercury addition and to remain at a level higher than the control soil for the rest of the experiment. The fraction of culturable heterotrophic bacteria increased from 1% to 10% of the total bacterial number as a result of mercury addition, and the mercury-resistant population increased to represent the entire heterotrophic population.     

Are Sebacinaceae common and widespread ectomycorrhizal associates of Eucalyptus species in Australian forests?

A molecular survey of basidiomycete ectomycorrhizal fungi colonising root tips at a site in Eucalyptus marginata (jarrah) forest revealed the presence of many fungal species which could not be identified from a database of ITS-PCR-RFLP profiles from morphologically identified species. Three of these unidentified taxa were among the six most frequently encountered profiles. Phylogenetic analyses of ITS and nuclear LSU sequences revealed a close relationship among the three fungi and that they belong to the family Sebacinaceae (sensu Weiss and Oberwinkler 2001). The possibility that DNA of non-ectomycorrhizal rhizosphere or endophytic fungi had been amplified selectively by the basidiomycete-specific primers was tested by amplification with fungal-specific primers. A single PCR fragment was amplified in all but two of the 24 samples tested and digestion with two restriction enzymes produced RFLP profiles which matched those from the Sebacinoid sequence. We conclude, therefore, that at least three species of Sebacinaceae are common ectomycorrhizal associates of E. marginata.     

Analysis of Fungal Diversity in the Wheat Rhizosphere by Sequencing of Cloned PCR-Amplified Genes Encoding 18S rRNA and Temperature Gradient Gel Electrophoresis

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1.4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.     

Evaluation of fungal aerosols using Temporal Temperature Gradient Electrophoresis (TTGE) and comparison with culture

Information obtained from fungal air samples can assist in the assessment of health hazards and can be useful in proactive indoor air quality monitoring. The objective of the present study was to evaluate the PCR-TTGE technique for the analysis of fungal diversity in the air. Eleven air samples were collected in five different sites using the bioimpactor CIP 10-M (Arelco). After a 2 hours sampling period, the collection liquid was recovered for subsequent cultivation and PCR-TTGE. A set of three fungi-specific primers (Fungcont 1, Fungcont 2+GC and Fungcont 3) was designed for the partial amplification of the 18S rRNA gene. The amplification was obtained in a single reaction tube by a semi-nested PCR. For identification, the TTGE bands were extracted and sequenced. PCR-TTGE allowed the clear separation of amplicons corresponding to distinct fungal species (both Ascomycota and Basidiomycota) that may be encountered in air. The number of fungal taxa detected after culture was systematically higher than the number of taxa found using PCR-TTGE. However, few fungal species were detected by PCR-TTGE and not by cultivation, suggesting that the combination of these approaches may provide a better analysis of fungal diversity in air samples than either method alone.     

A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus

In North America, asparagus (Asparagus officinalis) production suffers from a crown and root rot disease mainly caused by Fusarium oxysporum f. sp. asparagi and F. proliferatum. Many other Fusarium species are also found in asparagus fields, whereas accurate detection and identification of these organisms, especially when processing numerous samples, is usually difficult and time consuming. In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess Fusarium species diversity in asparagus plant samples. Fusarium-specific PCR primers targeting a partial region of the translation elongation factor-1 alpha (EF-1 alpha) gene were designed, and their specificity was tested against genomic DNA extracted from a large collection of closely and distantly related organisms isolated from multiple environments. Amplicons of 450 bp were obtained from all Fusarium isolates, while no PCR product was obtained from non-Fusarium organisms. The ability of DGGE to discriminate between Fusarium taxa was tested over 19 different Fusarium species represented by 39 isolates, including most species previously reported from asparagus fields worldwide. The technique was effective to visually discriminate between the majority of Fusarium species and/or isolates tested in pure culture, while a further sequencing step permitted to distinguish between the few species showing similar migration patterns. Total genomic DNA was extracted from field-grown asparagus plants naturally infested with different Fusarium species, submitted to PCR amplification, DGGE analysis and sequencing. The two to four bands observed for each plant sample were all affiliated with F. oxysporum, F. proliferatum or F. solani, clearly supporting the reliability, sensitivity and specificity of this approach for the study of Fusarium diversity from asparagus plants samples.     

Fine-scale distribution of ectomycorrhizal fungi colonizing Tsuga diversifolia seedlings growing on rocks in a subalpine Abies veitchii forest

Numerous species of ectomycorrhizal (ECM) fungi coexist under the forest floor. To explore the mechanisms of coexistence, we investigated the fine-scale distribution of ECM fungal species colonizing root tips in the root system of Tsuga diversifolia seedlings in a subalpine forest. ECM root tips of three seedlings growing on the flat top surface of rocks were sampled after recording their positions in the root system. After the root tips were grouped by terminal-restriction fragment length polymorphism (T-RFLP) analysis of ITS rDNA, the fungal species representing each T-RFLP group were identified using DNA sequencing. Based on the fungal species identification, the distribution of root tips colonized by each ECM fungus was mapped. Significant clustering of root tips was estimated for each fungal species by comparing actual and randomly simulated distributions. In total, the three seedlings were colonized by 40 ECM fungal species. The composition of colonizing fungal species was quite different among the seedlings. Twelve of the 15 major ECM fungal species clustered significantly within a few centimeters. Some clusters overlapped or intermingled, while others were unique. Areas with high fungal species diversity were also identified in the root system. In this report, the mechanisms underlying generation of these ECM root tip clusters in the root system are discussed.     

Dynamics of fungal communities in bulk and maize rhizosphere soil in the tropics

The fungal population dynamics in soil and in the rhizospheres of two maize cultivars grown in tropical soils were studied by a cultivation-independent analysis of directly extracted DNA to provide baseline data. Soil and rhizosphere samples were taken from six plots 20, 40, and 90 days after planting in two consecutive years. A 1.65-kb fragment of the 18S ribosomal DNA (rDNA) amplified from the total community DNA was analyzed by denaturing gradient gel electrophoresis (DGGE) and by cloning and sequencing. A rhizosphere effect was observed for fungal populations at all stages of plant development. In addition, pronounced changes in the composition of fungal communities during plant growth development were found by DGGE. Similar types of fingerprints were observed in two consecutive growth periods. No major differences were detected in the fungal patterns of the two cultivars. Direct cloning of 18S rDNA fragments amplified from soil or rhizosphere DNA resulted in 75 clones matching 12 dominant DGGE bands. The clones were characterized by their HinfI restriction patterns, and 39 different clones representing each group of restriction patterns were sequenced. The cloning and sequencing approach provided information on the phylogeny of dominant amplifiable fungal populations and allowed us to determine a number of fungal phylotypes that contribute to each of the dominant DGGE bands. Based on the sequence similarity of the 18S rDNA fragment with existing fungal isolates in the database, it was shown that the rhizospheres of young maize plants seemed to select the Ascomycetes order Pleosporales, while different members of the Ascomycetes and basidiomycetic yeast were detected in the rhizospheres of senescent maize plants.     

Dynamics of Fungal Communities in Bulk and Maize Rhizosphere Soil in the Tropics

The fungal population dynamics in soil and in the rhizospheres of two maize cultivars grown in tropical soils were studied by a cultivation-independent analysis of directly extracted DNA to provide baseline data. Soil and rhizosphere samples were taken from six plots 20, 40, and 90 days after planting in two consecutive years. A 1.65-kb fragment of the 18S ribosomal DNA (rDNA) amplified from the total community DNA was analyzed by denaturing gradient gel electrophoresis (DGGE) and by cloning and sequencing. A rhizosphere effect was observed for fungal populations at all stages of plant development. In addition, pronounced changes in the composition of fungal communities during plant growth development were found by DGGE. Similar types of fingerprints were observed in two consecutive growth periods. No major differences were detected in the fungal patterns of the two cultivars. Direct cloning of 18S rDNA fragments amplified from soil or rhizosphere DNA resulted in 75 clones matching 12 dominant DGGE bands. The clones were characterized by their HinfI restriction patterns, and 39 different clones representing each group of restriction patterns were sequenced. The cloning and sequencing approach provided information on the phylogeny of dominant amplifiable fungal populations and allowed us to determine a number of fungal phylotypes that contribute to each of the dominant DGGE bands. Based on the sequence similarity of the 18S rDNA fragment with existing fungal isolates in the database, it was shown that the rhizospheres of young maize plants seemed to select the Ascomycetes order Pleosporales, while different members of the Ascomycetes and basidiomycetic yeast were detected in the rhizospheres of senescent maize plants.