A molecular genetic analysis of the response of basidiomycete communities associated with loblolly pine (Pinus taeda L.) to improved nitrogen availability

Ivan Philip Edwards, Purdue University

Abstract

The purpose of this work was to develop a below-ground surveying strategy for ectomycorrhizal (ECM) ecology based on the selective amplification of the basidiomycete nuclear ribosomal DNA Internal Transcribed Spacer (nrDNA ITS) from DNA extracted directly from soil. A below-ground molecular based strategy was desirable because (a) traditional surveying techniques such as sporocarp surveys inadequately reflect the diversity and distribution of the ectomycorrhizal communities within the forest floor and soil; and (b) molecular methods offer improved species-level resolution relative to morphological techniques. The strategy was used to examine the distribution of basidiomycete species along a gradient of N availability created by optimal fertilization in a loblolly pine plantation established on naturally nutrient poor sandy soils. The inter- and intraspecific resolution of nrDNA ITS Terminal Restriction Fragment Length Polymorphism (TRFLP) was assessed by computer-simulated restriction analysis of a collection of ECM nrDNA ITS sequences. With two to three restriction enzymes, sequences of <90% similarity produced distinct 5′ TRFLP-types, although species with sequences of 95% + similarity remained difficult to distinguish. For these species, our analysis revealed that two to three enzymes, and both 5′ and 3′ TRF fragment analysis improved resolution, and was comparable to conventional PCR-RFLP. TRFLP of soil, root and spore fraction samples revealed a high degree of congruence between the diversity of basidiomycete species actively colonizing roots and in the adjacent soil. Nevertheless, the results also revealed that DNA derived from basidiospores could also be detected. Basidiospore-derived DNA did not affect the diversity of species in the analysis, but contributed 1–10% to the TRFLP signal. TRFLP was used to examine the distribution of basidiomycete species in adjacent 18-year old unfertilized, and optimally fertilized loblolly pine stands. The diversity of the basidiomycete community was high, with in excess of 70 TRFLP-types recovered. The most abundant species were Tylopilus, Thelephora, Lactarius, and Russula spp. Tylopilus and Thelephora increased in abundance with increasing N availability, and Russula spp showed the opposite trend. Overall, the results show that TRFLP is a valuable addition to the array of molecular techniques employed in ECM ecology.

Degree

Ph.D.

Advisors

Turco, Purdue University.

Subject Area

Microbiology|Molecular biology|Ecology

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