(2019). Ectomycorrhizae of Norway Spruce from Its Southernmost Natural Distribution Range in Serbia
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Research Article ii FF o o r r e e s s t t doi: 10.3832/ifor2729-011 Biogeosciences and Forestry vol. 12, pp. 43-50 Ectomycorrhizae of Norway spruce from its southernmost natural distribution range in Serbia Marina Katanić (1), Norway spruce (Picea abies Karst.) reaches its southernmost limit in the Saša Orlović (1), mountainous regions of south Serbia and Bulgaria. The species is a regionally (2) important timber species for the wood industry and a significant host for vari- Tine Grebenc , ous ectomycorrhizal fungi, including edible species. We analysed ectomycor- Marko Bajc (2), rhizal community and fine root parameters of high continental / subalpine Nor- Saša Pekeč (1), way spruce stands at three sites (Stara planina, Kopaonik, Tara) located in pro- (1) tected areas in Serbia. In addition, we assessed the potential effects of alti- Milan Drekić , tude and growing season on the ectomycorrhizal diversity and fine root param- (2) Hojka Kraigher eters. Using standardised sampling in combination with morpho-anatomical and molecular identification of ectomycorrhizae, we recorded 29 different anatomorphotypes. None of the identified fungi belonged to commercial edi- ble fungal species. Compared to other Norway spruce ectomycorrhiza studies in central Europe, sites in Serbia exhibited lower species diversity and differ- ent dominant species composition, with Cenococcum spp. and Russula spp. as the dominant ectomycorrhizal fungi. A number of ectomycorrhizal types and the value of the species richness index differed between Stara planina and Tara in the autumn, but the influence of site and season on the studied diver- sity indices was not significant. The total number of fine roots increased in the spring, while percentage of vital ectomycorrhizal root tips increased in the au- tumn. This study was the first examination of Norway spruce ectomycorrhizal communities at the edge of the natural geographical range of the species. Keywords: Ectomycorrhiza, Picea abies Karst., Community Structure, Diver- sity, Fine Roots Introduction on high mountains and mountain ranges been thoroughly studied for ectomycor- Norway spruce (Picea abies Karst.) is eco- with cold and humid climate or in frost rhizal diversity (Taylor et al. 2000). Ectomy- nomically important, being the main spe- sinkholes (Jovanović 1991, Ballian et al. corrhizal communities in Norway spruce cies in the Boreal and subalpine conifer for- 2007). The local populations are likely to be populations were well studied from the ests, distributed from Central Europe to relicts from the last glaciation period (Le- Central and Southern Alps, namely Slove- Northern and Eastern Europe. The species wandowski et al. 1997, Ravazzi 2002). nia (Kraigher 1999), Austria (Wang et al. reaches its southernmost natural distribu- Besides its importance as a quality wood 2015) and Germany (Baier et al. 2006), as tion in the Dinaric Alps, the Balkan Moun- source, Norway spruce is known to host well as in the Carpathian mountains for tains, and the Carpathian Mountains (Cau- several ectomycorrhizal species (Agerer & Central European populations (Peter et al. dullo et al. 2016). Rambold 2017) that are renowned for their 2008) and the Northern populations in the In Serbia, Norway spruce is the most rep- culinary value, for example Boletus edulis boreal region (Dahlberg et al. 1997, Osto- resented coniferous tree species with a for- Bull., Cantharellus cibarius Fr. and Hydnum nen et al. 2011, Ostonen et al. 2013). est gene pool making up 5.2% of the total rufescens Pers. These and other ectomycor- The isolated populations of Norway volume (National Forest Inventory of Re- rhizal fungi are commonly collected by lo- spruce on mountains in Serbia remain un- public of Serbia – Banković et al. 2009). As cals and either consumed or sold at re- explored for their ectomycorrhizal commu- in other southern alpine areas, Norway gional markets (Boa 2004). nity. To bridge this knowledge gap, we se- spruces is distributed above the beech area Norway spruce forests of Europe have lected sites at three protected areas at mountains where spruce reaches its south- ernmost distribution range: Stara planina, (1) University of Novi Sad, Institute of Lowland Forestry and Environment, Antona Cehova Kopaonik and Tara, located in Southeast, 13, 21000 Novi Sad (Serbia); (2) Slovenian Forestry Institute, Večna pot 2, 1000 Ljubljana South and Southwest Serbia, respectively. (Slovenia) The study focused on the diversity of ecto- mycorrhizal fungi at Norway spruce natural @ Marina Katanić ([email protected]) sites and on potential influences of two contrasting seasons (spring and autumn) Received: Jan 18, 2018 - Accepted: Oct 26, 2018 and three different sites on ectomycor- rhizal communities of spruce. Citation: Katanić M, Orlović S, Grebenc T, Bajc M, Pekeč S, Drekić M, Kraigher H (2019). Ectomycorrhizae of Norway spruce from its southernmost natural distribution range in Serbia. Methodology iForest 12: 43-50. – doi: 10.3832/ifor2729-011 [online 2019-01-10] Sites and sampling procedures Communicated by: Alberto Santini The sampling sites were selected in spruce stands located in protected areas in © SISEF https://iforest.sisef.org/ 43 iForest 12: 43-50 Katanić M et al. - iForest 12: 43-50 y r Serbia. Sites were selected aiming at cover- lowing the methodology given by Agerer best product in the reaction. Amplification t ® s ing the distribution patches of spruce at its (1991) and Kraigher (1996). reaction was performed in GeneAmp PCR e r southernmost natural distribution range Ectomycorrhizal types were also classi- System 9700 (Applied Biosystems, Foster o on mountains in Serbia (Skrøppa 2003). De- fied into the exploration types based on City, CA, USA) according to the procedure F tailed characteristics of the selected areas the presence and abundance of emanating explained by Sulzbacher et al. (2016). Nega- d n are provided in Tab. 1. elements as proposed by Agerer (2001). All tive controls with no fungal DNA were run a Soil sampling (four per site) was per- vital ectomycorrhizal root tips, old and for each experiment to check for any con- s e formed in the absence of snow cover, in non-turgescent fine roots and non-mycor- tamination. The PCR mixture for one sam- c n September 2013 and June 2014, resulting in rhizal vital fine roots were counted under a ple was composed of 5 µl of 10× Gold Buf- e i eight samples per site. A standardised soil binocular. Total number of fine roots was fer, 4 µl of deoxynucleotide triphosphates c s corer with 4-cm diameter and 18-cm length obtained by summing all of these cate- (0.2 mM each), 1.2 µl of each primer (0.32 o (total volume 274 ml) was used for soil gories of roots. µM each), 4µl of MgCl2 (2.0 mM), 30.3 µl of e g core sampling at 0.5 m from the tree Coarse roots in each soil sample were sterile distilled water, 0.3 µl of Taq poly- o -1 i trunks (Kraigher 1999). At mixed sites, the checked for tree roots species confirma- merase (0.03 U µl ), and 4 µl of DNA ex- B areas with pure Norway spruce were tar- tion following the anatomical characteris- tract. Thermal cycling conditions were as – geted for sampling. When possible, soil tics of wooden parts (Mrak et al. 2016). All follows: initial denaturation and polymer- t s cores were taken from locations with trees extraneous coarse roots with attached fine ase activation at 95 °C for 5 min; 13 cycles at e r of different age to obtain potentially wider roots were eliminated from further analy- 94 °C for 45 sec, 55 °C for 55 sec and 72 °C o F diversity of ectomycorrhizal community. sis. for 45 sec.; 13 cycles at 94 °C for 45 sec, 55 i Soil samples were stored at 4 °C for up to °C for 55 sec and 72 °C for 120 sec; 12 cycles 6 weeks. Prior to analyses, each sample Molecular identification of at 94 °C for 45 sec, 55 °C for 55 sec and 72 °C was submerged in cold tap water to loosen ectomycorrhizal fungi for 180 sec and a final extension at 72 °C for the soil structure. All roots were carefully Molecular confirmation of fungal part- 10 min. washed from soil. Using a binocular (Kruss ners in ectomycorrhiza using molecular Amplified DNA was separated and ana- GmbH, Hamburg, Germany) with magnifi- methods was based on a PCR amplification lysed as described by Grebenc et al. (2009). cations 10-45× (light source: Olympus High- of fungal nuclear rDNA ITS region from Amplified DNA fragments were separated light 3100, daylight filter), fine roots were each separated anatomorphotype. This and purified from the agarose gel using the separated into vital ectomycorrhizal root molecular marker is currently considered Wizard SV® Gel and PCR Clean-Up System® tips, old and non-turgescent fine roots, or as the best for fungi barcoding and differ- (Promega Corporation, Madison, WI, USA) non-mycorrhizal vital fine roots. entiation at the species level (KõlJalg et al. and sent for Sanger sequencing to Macro- The ectomycorrhizal species were identi- 2013). Total genomic DNA was extracted gen Korea (Seoul, Korea). Sequencher® ver. fied in a two-step procedure combining from ethanol-stored ectomycorrhizal root 5.1 (Gene Codes Corporations, Ann Arbor, morphological and anatomical characteri- tips using a Plant DNeasy® Mini Kit (Qiagen, MI, USA) was used to identify the consen- sation of ectomycorrhizal root tips to a Hilden, Germany). If DNA extraction of rep- sus sequence from the two strands of each level of an individual anatomorphotype. resentative ectomycorrhizal root tips of isolate. Species, genus, or family of ecto- Each anatomorphotype was further ana- some anatomorphotype was not success- mycorrhizal fungi were determined by lysed by molecular analysis of nuclear rDNA ful and morpho-anatomical identification comparing the sequences to those de- ITS region.