Analysis of the dynamics of fungal communities in soil via fungal-specificPCR of soil DNA followed by denaturing gradient gel electrophoresis

A molecular method for profiling of fungal communities in soil was applied in experiments in soil microcosms, with two objectives, (1) to assess the p ersistence of two selected fungal species in soil, and (2) to analyze the r esponse of the natural fungal community to a spill of sulphurous petrol in the same soil. To achieve the aims, two soil DNA extraction methods, one or iginally designed for the direct extraction of bacterial community DNA and the other one aimed to obtain fungal DNA, were tested for their efficiency in recovering DNA of fungal origin from soil. Both methods allowed for the efficient extraction of DNA from introduced Trichoderma harzianum spores as well as Arthrobotrys oligospora mycelial fragments, at comparable rates. S everal PCR amplification systems based on primers specific for fungal 18S r ibosomal RNA genes were tested to design strategies for the assessment of f ungal communities in soil. The PCR systems produced amplicons of expected s ize with DNA of most fungi studied, which included members of the Ascomycet es, Basidomycetes, Zygomycetes and Chytridiomycetes. On the other hand, the 18S rRNA genes of Oomycetes (including key plant pathogens) were poorly am plified. Plant (Solanum tuberosum), nematode (Meloidogyne sp.) and bacteria l DNA was not amplified. For studies of soil fungal communities, a nested P CR approach was selected, in which the first PCR provided the required spec ificity for fungi, whereas the second (nested) PCR served to produce amplic ons separable on denaturing gradient gels. Denaturing gradient gel electrop horesis (DOGE) allowed the resolution of mixtures of PCR products of severa l different fungi, as well as products resulting from mixed-template amplif ications, into distinct banding patterns. The persistence of fungal species in soil was assessed using T. harzianum spores acid A. oligospora hyphal f ragments added to silt loam soil microcosms. Using PCR-DGGE, these fungi we re detectable for about 14 days and 2 months, respectively. Both singly-ino culated soils and soils that had received mixed inoculants revealed, next t o bands resulting from indigenous fungi, the expected bands in the DGGE pro files. The A. oligospora specific amplicon, by virtue of its unique migrati on in the denaturing gradient, was well detectable, whereas the T. harzianu m specific product comigrated with products from indigenous fungi. PCR-DGGE analysis of DNA obtained from the silt loam soil treated with dibenzothiop hene-containing petrol showed the progressive selection of specific fungal bands over lime, whereas this selection was not observed in untreated soil microcosms. Cloning of individual molecules from the selected bands and ana lysis of their sequences revealed a complex of targets which clustered with the 18S rDNA sequences of the closely-related species Nectria haematococca , N. ochroleuca and Fusarium, solani. Fungal isolates obtained from the tre ated soil on PDA plates were identified as Trichoderma sp., whereas those o n Comada agar fell into the Cylindrocarpon group (anamorph of Nectria spp). (C) 2000 Elsevier Science B.V. All rights reserved.