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ŠUMARSKI LIST 11-12/2021 str. 43     <-- 43 -->        PDF

precipitation amount per season is as follows: winter 180 mm, spring 198 mm, summer 252 mm, and autumn 161 mm, the average amount of precipitation in the vegetative period is 443.6 mm and average annual amount of precipitation is 791.4 mm (Babić 2015).
Soil sampling was performed at the end of October 2017, in the absence of snow cover, and ten soil samples were taken per site. The sampling was performed with a standardized soil corer (Kraigher 1999) (4 cm in diameter and 18 cm in length, making a total sample volume of 274 mL), 0.5-1 m from the target tree, as far as possible from non-target trees. Soil samples were stored at 4°C for up to two months. Prior to analyses, each sample was submerged in cold tap water to loosen the soil structure. All roots were carefully washed from the soil. Using a dissecting microscope Olympus SZX 10 (Olympus Corp., Tokyo Japan) with magnifications 10-63× (light source: Olympus Highlight 3100, daylight filter), all fine roots were separated as vital ECM root tips or as old, nonturgescent and unidentifiable. Vital ECM root tips were categorized into different morphotypes of ectomycorrhizae based on their morphological and anatomical characteristics. Later ones were assessed using a microscope (Olympus BX 53®, Olympus Corp., Tokyo Japan) with magnifications 100-1000×. Morphotypes of ectomycorrhizae were described following the methodology given by Agerer (1991) and Kraigher (1996). If it was possible, a fungal partner was identified by comparison with published descriptions in Agerer et al. (2006), Agerer (2008), or Agerer and Rambold (2020).
Morphotypes of ectomycorrhizae were also classified into the ETs as proposed by Agerer (2001). All vital ECM root tips were counted. The granulometric and chemical composition of soil were analyzed as well.
Molecular identification of ectomycorrhizal fungi
Confirmation of fungal partners in ectomycorrhiza using molecular methods was based on PCR amplification of fungal nuclear rDNA internal transcribed spacer (ITS) region. Total genomic DNA was extracted from ECM root tips using a DNeasy® Plant Mini Kit (Qiagen, Hilden, Germany). If DNA extraction of representative root tips of some morphotype of ectomycorrhiza was not successful and morpho-anatomical identification was insufficient to determine the ECM fungus, this ECM morphotype was labeled as an “unidentified” type. Amplifications were performed with ITS-1F (Gardes and Bruns 1993) and ITS 4 primer pair (White et al. 1990). The amplification reaction was performed in Eppendorf Master cycler (Eppendorf AG, Hamburg, Germany). Negative controls with no fungal DNA were run for each experiment to check for any contamination. The PCR mixture for one sample was composed of 2.5 µL of 10× Gold Buffer, 2 µL of deoxynucleotide triphosphates (0.2 mM each), 0.6 µL of each primer (10 µM each), 2µL of MgCl2 (2.0 mM), 15 µL of sterile distilled water, 0.3 µL of Taq polymerase (5 U µL-1), and 2 µL of a DNA extract. Thermal cycling conditions were as follows: initial denaturation and polymerase activation at 95°C for 5 min; 13 cycles at 94°C for 45 s, 55°C for 55 s and 72°C for 45 s.; 13 cycles at 94°C for 45 s, 55°C for 55 s and 72°C for 120 s; 12 cycles at 94°C for 45 s, 55°C for 55 s and 72°C for 180 s and a final extension at 72°C for 10 min. Amplified DNA fragments were separated and purified from the agarose gel using the QIAquick gel extraction kit and QIAquick PCR purification kit (Qiagen, Valencia, CA, USA) and sent for sequencing in Macrogen Europe B.V. Species, genus, or family of ECM fungi were determined by comparing the sequences to those deposited in GenBank (NCBI 2020) and UNITE (Nilsson et al. 2018) database.
Data analysis
Diversity indexes were calculated per sample and per site (i. e. by pooling the ECM community data) following the formulas given by Atlas and Bartha (1981): (i) Species richness (d) = (S-1)/log10N, where S is the number of ECM fungal taxa and N is the number of all mycorrhizal tips; (ii) Shannon-Weaver’s diversity index (H) = C/N(NlogN-&931;nilogni), where C=2.3, N is the number of all mycorrhizal tips and ni is the number of mycorrhizal tips of an individual ECM fungal taxon.
According to the granulometric and chemical composition of soil samples taken in studied sessile oak stands, soil on