RESUMO
The old Zn-Pb-contaminated (calamine) tailings in southern Poland are spontaneously colonized by metal-tolerant Anthyllis vulneraria L. (Fabaceae), which can form simultaneously symbiotic association with nitrogen-fixing rhizobia and phosphorus-acquiring arbuscular mycorrhizal fungi (AMF). So far, fungal colonization and the AMF diversity of calamine-inhabiting legumes have been poorly studied. Thus, we determined AMF spore density in the substratum and the mycorrhizal status of nodulated A. vulneraria plants occurring on calamine tailings (M) and on a reference non-metallicolous (NM) site. The results indicate the presence of the Arum-type of arbuscular mycorrhiza in the roots of both Anthyllis ecotypes. Despite the presence of AM fungi in M plant roots, the dark septate endophyte (DSE) fungi (hyphae and microsclerotia) were occasionally also detected. Metal ions were accumulated mainly in the nodules and intraradical fungal structures rather than thick plant cell walls. Mycorrhization parameters (frequency of mycorrhization and intensity of root cortex colonization) for M plants were markedly higher and differed in a statistically significant manner from the parameters for NM plants. Heavy metal excess had no negative effect on the number of AMF spores, the amounts of glomalin-related soil proteins and AMF species composition. Molecular identification of AMF using PCR-DGGE analysis based on the 18S rDNA ribosomal gene by nested-PCR with primers AM1/NS31 and NS31-GC/Glo1 revealed similar genera/species of AMF in the roots of both Anthyllis ecotypes: Rhizophagus sp., R. fasciculatus, and R. iranicus. The results of this work indicate the presence of unique fungal symbionts, which may enhance A. vulneraria tolerance to heavy metal stress and plant adaptation to extreme conditions on calamine tailings.
