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

Abstract

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.

FIG. 1.

DGGE profiles showing the comparison of the fungal rhizosphere communities of the maize cultivars Nitroflint (Nf) and Nitrodent (Nd) at different stages of plant development (20, 40, and 90 days). The fingerprints of fungal communities were generated by separation of 18S rDNA fragments amplified with primers NS1 and FR1-GC. The following fungal species, from top to bottom, were used as standards (lanes M): Colletotrichum sp., Sclerotium tuliparum, Trichoderma harzianum, Myrothecium cinctum, Ustilago nuda, Myrothecium leucotrichum, and Penicillium simplicissimum.

FIG. 2.

Dendrogram constructed with the fungal community fingerprints of the maize cultivars Nitroflint (Nf) and Nitrodent (Nd) at different stages of plant development (20, 40, and 90 days). The differences between the profiles are indicated by percentage of similarity. The dendrogram was based on the Pearson correlation index and cluster analysis by the unweighted pair group method using arithmetic averages.

FIG. 3.

DGGE profiles showing the fungal community fingerprints of rhizosphere samples from young (R20) and senescent (R90) plants (Nitrodent) and their corresponding bulk soil fingerprints. Two lanes each represent a typical DGGE profile from the first and second growth periods. The relative band position of the more dominant bands and the cloned DNA fragments are shown. The following fungal species, from top to bottom, were used as standards (lanes M): Colletotrichum sp., Sclerotium tuliparum, Trichoderma harzianum, Myrothecium cinctum, Ustilago nuda, Myrothecium leucotrichum, and Penicillium simplicissimum.

FIG. 4.

Dendrogram constructed with the fungal community fingerprints of rhizosphere (R) samples from young (R20) and senescent (R90) plants (Nitrodent) and their corresponding bulk soil (S20 and S90) fingerprints. Two full growth periods (I and II) were analyzed. The differences between the profiles are indicated by percentage of similarity. The dendrogram was based on the Pearson correlation index and cluster analysis by the unweighted pair group method using arithmetic averages.

FIG. 5.

Phylogenetic tree for partial sequences of cloned 18S rDNA fragments and the most closely related fungi. The clones are indicated by their code and accession number (National Center for Biotechnology Information), respectively.