- Previous studies have indicated that roots from five tree species (Picea sitchensis, Tsuga heterophylla, Pseudotsuga menziesii, Pinus ponderosa, and Pinus contorta) decompose at different rates across an environmental gradient in Oregon. Measurements of wood chemistry from each tree species as well as moisture and
temperature from each location do not explain the differences in decomposition
rates. Molecular techniques were employed to generate Internal Transcribed Spacer - Restriction Fragment Length Polymorphism (1TS-RFLP) patterns to examine saprotrophic fungi in roots of these tree species and to see if differences in the fungal communities might explain observed differences in decomposition rates. However, due to a large number of ITS-RFLP patterns recovered and low levels of similarity in ITS-RFLP patterns across samples, we were unable to explain root decomposition based upon the fungal community information. Consequently, the analysis focused on comparing levels of ITS-RFLP similarity at each sampling level, determining the extent to which the sampling methods captured the total fungal biodiversity, and examining samples with microscopy and gene sequencing techniques to identify fungi. Root samples were retrieved from trees cut seven to fifteen years prior to sampling. Two tree species were sampled at each of three locations across Oregon. DNA was extracted from hyphae samples collected directly from roots, and fungi cultured from root chips. DNA was also extracted from fungal herbarium specimens, field collected samples, and cultures of
saprotrophic fungi. To analyze genetic diversity of the samples, they were amplified using polymerase chain reaction (PCR) techniques, digested with endonucleases (Hinf I, Dpn II, and Hae III), and ITS-RFLP patterns were evaluated. Nei and Li similarity index analyses were used to compare differences in fungal composition based upon ITS-RFLP patterns between tree species, sites, and harvest stands. Over two hundred distinct ITS-RFLP patterns were recognized from fungal samples. Similarities in ITS-RFLP patterns of hyphal and cultured
samples ranged 0 to 1, where 0 indicated no overlap and 1 indicated 100% matching of ITS-RFLPs. When all ITS-RFLPs obtained from each stump were combined, similarities in patterns between sites ranged from 0 to 0.07, from 0 to 0.13 between tree species, from 0 to 0.11 between harvest stands, and from 0 to 0.67 between individual stumps. Linearly increasing JTS-RFLP sampling intensity curves indicate a large diversity of fungi. Using microscopy, cultured samples
were examined for hyphae and reproductive structures. In culture, zygomycetous
structures were prevalent. DNA gene sequences of the nuclear large and small
subunits were used to place unknown ITS-RFLP patterns into family and generic groups. Twenty-three common and five uncommon ITS-RFLP patterns were sequenced; most matched with the Mortierellaceae and Mucoraceae families of the zygomycetes. The large diversity of ITS-RFLP patterns indicates the coarse roots provided habitat to many fungi at the stage of decomposition when samples were collected.