Zastosowanie regionu ITS1/2 rDNA i 18S rDNA do badania mykobioty gleby leśnej

The aim of the studies was to check the usefulness of ITS1/2 rDNA and 18S rDNA regions in the molecular investigation of forest soil microbiota structure. Soil studied, originated from a 1−year−old plantation and a 40−year old stand of Scots pine located in Bierzwnik and Międzychów forest districts located 200 km apart. The hypothesis assumed that both approaches lead to the discovery of abundant microbiota communities with different structures and with rare common species. The environmental DNA was extracted with a Power Soil ® DNA Isolation Kit from two soil samples in each site. The ITS1/2 rDNA was amplified with specific primers ITS1 and ewfitsrev 1, and 18S rDNA with universal primers NS1 and NS2. PCR products were cloned into pGEM−T Easy. Inserts were primarily selected in blue/white screening on a X−gal medium. Representative clones were further selected in two separate RFLP analyses with HhaI and BsuRI restriction enzymes. Representative clones purified and sequenced using the Sanger Method in the DNA Research Centre (Poznań). Each sequence was identified to the lowest taxonomic rank. Ninety to 233 clones with DNA of 5−44 taxa including 3−37 taxa of fungi were obtained from 4 samples of soil. After application of ITS1/2 rDNA and 18S rDNA, the fungal DNA was detected respectively in 89,60−100,00% and 11,77−64,8% clones and the number of fungal species detected was respectively 12−37 and 3−19. Fungi were represented by four orders: Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota. Both primers also amplified also DNA of other organisms (mostly from Animalia and Protista Kingdom) represented by 0−9 taxa. If compared, the application of forest soil microbiota structure with ITS1/2 rDNA and 18S rDNA led to detect a lower abundance of fungi and a bigger abundance of other organisms. Considering the higher number of clones and taxa recognized, the region of ITS1/2 rDNA was more effective in the studies of the soil microbiota structure. The region of 18S rDNA was efficient in local detection of Chytridiomycota and Zygomycota and of rare species of fungi from Ascomycota and Basidiomycota. Despite the deficiency of NCBI database the use of the 18S rDNA region in studies on fungal community the region should be included in molecular studies of fungal diversity. It is concluded that studies on the biodiversity of soil microorganisms need the application of a few independent methods of detection and identification.