The Method Research on Tuber spp. DNA in Soil
Truffles are ascomata of ectomycorrhizal hypogeous fungi associated with endemic Pinus tabuliformis in Mount Helan National Nature Reserve in Inner Mongolia, China, was investigated f in soil cubes taken from pure P. tabuliformis stands. The objectives of
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The Method Research on Tuber spp. DNA in Soil Yong-jun Fan, Fa-Hu Li, Yan-Lin Zhao and Wei Yan
Abstract Truffles are ascomata of ectomycorrhizal hypogeous fungi associated with endemic Pinus tabuliformis in Mount Helan National Nature Reserve in Inner Mongolia, China, was investigated f in soil cubes taken from pure P. tabuliformis stands. The objectives of this study were (i) to develop a molecular method to detect mycelia of Tuber spp. in soil and (ii) to test for mycelial distribution around two truffle-bearing P. tabuliformis trees in a truffle orchard. Isolation of total DNA from soil was performed, followed by PCR amplification with Tuber spp.-specific primers and restriction analysis. To address the detection sensitivity level, soil samples were inoculated with known amounts of gleba of Tuber spp. Mycelium was detected primarily within the area defined by the truffle burn and within the top 35 cm of the soil in all directions from the trees. Keywords Tuber spp
Soil mycelium PCR–RFLP Truffle orchard ITS
390.1 Introduction Tuber spp. hyphae are not visible to the naked eye, but its DNA can be selectively amplified by PCR from total soil DNA extracts using specific primers. Several authors have designed Tuberspp.-specific primers [1–4], but these primers were not adequate for our purposes because of weak or nonspecific amplifications. The objectives of this study were (i) to establish an accurate DNA isolation protocol applicable to soil samples (ii) to develop a PCR primer pair specific for Tuberspp., Y. Fan (&) F.-H. Li Y.-L. Zhao W. Yan The Biology Science and Technology Department of Baotou Teacher’s College, Baotou, China e-mail: [email protected]
S. Li et al. (eds.), Frontier and Future Development of Information Technology 3059 in Medicine and Education, Lecture Notes in Electrical Engineering 269, DOI: 10.1007/978-94-007-7618-0_390, Springer Science+Business Media Dordrecht 2014
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and (iii) to test the ability of these primers to detect the presence and distribu-tion of Tuberspp. Mycelium in soil surrounding two Pinus tabuliformis trees in a commercial truffle orchard.
390.2 Materials and Methods 390.2.1 Source of Fungal Material Fruit bodies from 1 Tuber species and from 1 subspecies belonging to same genera were collected from under the Pinus tabuliformis in Mount Helan National Nature Reserve in Inner Mongolia, China. Fresh fruit bodies were freeze-dried and stored at -20 C before use.
390.2.2 Soil Samples Four soils were used to test the extraction procedure: (1) from nursery pots of truffle-inoculated Pinus tabuliformis; (2) from a wild truffle bed; (3) from a productive truffle orchard; and (4) from a cereal field adjacent to the truffle orchard. All samples were stored at -20 C until use. Roots from nursery seedlings were examined under a light micro-scope to confirm the presence of Tuber Spp. ectomy-corrhizae, which were characterized by spinulae surface of the mantle and sheath ornamentations (Rauscher et al. [5]). Soil samples from the wild truffle
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