Effects of prescribed burning upon mycorrhizal fungal diversity inhabiting the roots of two and a half-year old black spruce (Picea mariana) : molecular characterization of ectomycorrhizal fungi via PCR/RFLP analysis

Out-planted black spruce seedlings were harvested from unburned as well as low and high intensity prescribed burned sites to assess the effects of burning and fire intensity upon ectomycorrhizal (ECM) fungal diversity. The polymerase chain reaction (PCR), in conjunction with universal and fungal specific primers, was used to amplify a fragment spanning the two internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA belonging to ECM fungi. Sampling allowed for the harvesting of both mycorrhizal and non-mycorrhizal (NM) root tips. Despite the fact that some tips were classified as being NM, a PCR fragment was amplified from them. Staining of NM root tips showed there to be numerous types of endophytic hyphae surrounding and penetrating the root cortical cells. Restriction fragment length polymorphisms (RFLP) were used to classify specific fungal genotypes. Molecular data indicated that root tips harboring ECM fungi with distinct mantles (mantled tips), produced distinct RFLP genotypes compared to those root tips presumed to be non-mycorrhizal, or supporting mycorrhizas with thin/patchy mantles (exposed tips). Both NM tips, along with tips supporting thin/patchy-mantled fungi, displayed a wide variety of RFLP genotypes. -- Both the Shannon-Wiener and the Simpson indices were used to assess diversity based upon the RFLP patterns. It was found that neither fire, nor its intensity, caused a significant change in the ECM fungal diversity and/or community structure. Mantled tips showed distinct RFLP clades, which corresponded to crude morphotype groups. Some of these clades showed several intraspecific polymorphisms representing a particular genotype. It appears that the Glide Lake study site has a very high degree of ECM, as well as endophytic, fungal diversity. The level of diversity within the endophytic genotypes was comparable to that of ECM genotypes. The study also indicates that traditional diversity indices (Shannon-Wiener and Simpson) are not well-suited for molecular data. Thus an index based upon phylogenetic distances, the 'Phi index', was constructed. The greater the degree of variability among the distances then the greater the diversity. -- It would appear that foresters need not be overly concerned with prescribed burning, nor the intensity of these burns, affecting the ectomycorrhizal diversity of outplanted black spruce. However, this does not mean that other ECM fungi, which do not associate with black spruce, are not affected. Further studies are needed in order to investigate the effects of fire upon other hosts and their associated mycorrhizal fungi. As well, more rigorous testing is needed before the Phi index can be declared a better measure of diversity when using RFLP data