Norway spruce (
Norway spruce (
In Serbia, Norway spruce is the most represented coniferous tree species with a forest gene pool making up 5.2% of the total volume (National Forest Inventory of Republic of Serbia -
Besides its importance as a quality wood source, Norway spruce is known to host several ectomycorrhizal species (
Norway spruce forests of Europe have been thoroughly studied for ectomycorrhizal diversity (
The isolated populations of Norway spruce on mountains in Serbia remain unexplored for their ectomycorrhizal community. To bridge this knowledge gap, we selected sites at three protected areas at mountains where spruce reaches its southernmost distribution range: Stara planina, Kopaonik and Tara, located in Southeast, South and Southwest Serbia, respectively. The study focused on the diversity of ectomycorrhizal fungi at Norway spruce natural sites and on potential influences of two contrasting seasons (spring and autumn) and three different sites on ectomycorrhizal communities of spruce.
The sampling sites were selected in spruce stands located in protected areas in Serbia. Sites were selected aiming at covering the distribution patches of spruce at its southernmost natural distribution range on mountains in Serbia (
Soil sampling (four per site) was performed in the absence of snow cover, in September 2013 and June 2014, resulting in eight samples per site. A standardised soil corer with 4-cm diameter and 18-cm length (total volume 274 ml) was used for soil core sampling at 0.5 m from the tree trunks (
Soil samples were stored at 4 °C for up to 6 weeks. Prior to analyses, each sample was submerged in cold tap water to loosen the soil structure. All roots were carefully washed from soil. Using a binocular (Kruss GmbH, Hamburg, Germany) with magnifications 10-45× (light source: Olympus Highlight 3100, daylight filter), fine roots were separated into vital ectomycorrhizal root tips, old and non-turgescent fine roots, or non-mycorrhizal vital fine roots.
The ectomycorrhizal species were identified in a two-step procedure combining morphological and anatomical characterisation of ectomycorrhizal root tips to a level of an individual anatomorphotype. Each anatomorphotype was further analysed by molecular analysis of nuclear rDNA ITS region.
Microscopic characteristics of ectomycorrhizal root tips were assessed using an Olympus BX 51® (Olympus Corp., Tokyo Japan) with magnifications 100-2000×. Anatomorphotypes of ectomycorrhiza were identified by comparison with published descriptions in
Ectomycorrhizal types were also classified into the exploration types based on the presence and abundance of emanating elements as proposed by
Coarse roots in each soil sample were checked for tree roots species confirmation following the anatomical characteristics of wooden parts (
Molecular confirmation of fungal partners in ectomycorrhiza using molecular methods was based on a PCR amplification of fungal nuclear rDNA ITS region from each separated anatomorphotype. This molecular marker is currently considered as the best for fungi barcoding and differentiation at the species level (
Amplifications were performed with ITS 1f (
Amplified DNA was separated and analysed as described by
To compare diversity at sample, site, and season level, Species richness (d), Shannon Weaver diversity index (H), Evenness (e) and Equitability (J) were calculated following
Data of an individual soil sample was used as statistical unit. The two-way ANOVA and Fisher’s LSD test were used to analyse seasonal and site differences for the measured parameters. In order to obtain normal distribution of data sets for these tests, square root transformation was performed (
Overall, we found 29 different types of ectomycorrhizae at the three selected sites,
The lowest total number of ectomycorrhizal types was recorded for Tara in the autumn (5), while the highest number (9) was recorded in the spring for the same site and for Kopaonik. The lowest number of vital ectomycorrhizal root tips per site was counted for Tara in the autumn (735), while the highest number was counted for Stara planina in the spring (2060). Also, the highest total number of fine roots and the lowest percentage of vital ectomycorrhizal root tips (8%) were counted at Stara planina in the spring. On the other hand, the highest percentage of vital ectomycorrhizal root tips was recorded for Kopaonik in the autumn (46% -
Mean number of ectomycorrhizal types per soil sample was significantly higher for Stara planina in the autumn (4.75) compared with Tara in the same season (2.5 -
Presence and abundance of exploration types (ET) differed among the investigated spruce stands. Stara planina was dominated by a medium distance exploration type (subtype smooth) with abundance that decreased in the autumn compared to the spring. On the other hand, a number of short distance exploration types increased from the spring to the autumn (
Abundance of contact ET and medium distance ET (both smooth and fringe subtypes) were significantly influenced by site, while the season had no significant impact (
This insight into the ectomycorrhizal diversity and fine root parameters in Serbia is the first example of a Norway spruce ectomycorrhizal community study from the southernmost edge of the species’ range. Although all sites were located in protected, unpolluted areas on mountains, the examined ectomycorrhizal communities differed substantially in their structure and abundance of ectomycorrhizal types.
In general, the most widespread ectomycorrhizal symbiont in the southernmost natural distribution of Norway spruce was
Tomenteloid fungi are also known to be frequent and widespread ectomycorrhizal partners of deciduous and evergreen tree species in the forests of Europe and North America (
From 29 ectomycorrhizal anatomorphotypes recorded at the investigated spruce stands in Serbia, 9 types were found on Stara planina, 13 on Kopaonik and 12 types were recorded on Tara mountain.
The lowest number of ectomycorrhizal types was recorded at Stara planina where
It is worth noting that there were differences in the age and character of spruce trees in forest stands among the different sites. Namely, the Norway spruce stand on Kopaonik was homogenous and 50 to 70 years old, while the Tara forest stand was comprised of 40- to 50-year-old spruce trees mixed with beech and silver fir, and on Stara planina spruce trees were 10 to 20 years old and planted in natural beech forest. There is a well known division between early- and late-stage fungi that form mycorrhizae with tree roots grown in soils with different physical and chemical properties, especially different accumulations of recalcitrant leaf litter (
Investigating the diversity of ectomycorrhizae in four mature spruce stands in Poland,
In a 100-year-old Norway spruce forest in Sweden,
Dominance of some species in ectomycorrhizal communities is indicated by the Shannon Weaver index (
The most abundant ectomycorrhizal types, representing more than 10% of ectomycorrhizal communities of spruce stands from Stara planina, Kopaonik and Tara were:
The edge of the southernmost distribution of Norway spruce is under constant external pressures, such as pests and unfavourable conditions due to the warming climate (
In this study, the three selected sites did not significantly differ in the number of ectomycorrhizal types and vital ectomycorrhizal root tips nor diversity indices. These results were in accordance with
According to previous studies (
In the current study, exploration types characteristic for mineral horizons dominated all tested sites in both seasons. The exception was the short-distance exploration type known to prefer organic layers, which was the most abundant at Tara in the autumn. It is important to highlight that no ectomycorrhizal fungi belonging to long-distance ET were found at all examined sites in both seasons. Abundance of contact- and medium-distance ETs was affected by site. Short-distance ET was present at all sites and the influence of site on its abundance was not statistically significant. Since ectomycorrhizal types belonging to short-distance ET prefer humus-rich environments and its abundance was not influenced by site, it could be assumed that all studied sites have similar characteristics concerning humus content.
In the first study of ectomycorrhiza on spruce in its southernmost natural distribution area, 29 different types of ectomycorrhizae were identified. The ectomycorrhizal communities investigated at Stara planina, Kopaonik and Tara differed in the composition of ectomycorrhizal types and abundance of contact- and medium-distance exploration types, but not in the mean number of ectomycorrhizal types, number of vital ectomycorrhizal root tips, nor diversity indices. Season had no influence on the parameters assessed nor on the abundance of exploration types. In order to obtain a broader picture of ectomycorrhizal communities in Norway spruce forests in Serbia, further research supplemented with metagenomic approaches should be conducted on more sites.
This work is a part of the project III 43002 “Biosensing technologies and global systems for continuous research and integrated management of ecosystems”, financed by the Ministry of Education and Science of the Republic of Serbia, the Research programme P4-0107 of the Slovenian Research Agency and STSM under COST Action FP1305 for M. Katanić. Dr. Ronald S. Zalesnyis is gratefully acknowledged for revising the manuscript for English style.
Relative abundance of ectomycorrhizal types at examined sites Stara planina, Kopaonik, and Tara in the spring and autumn.
Mean abundance of exploration types (ET) at examined sites Stara planina (top), Kopaonik (middle) and Tara (bottom) in the spring and autumn.
Coordinates of sites, altitude, climate, mean annual temperature and precipitation (
Site | Stara planina | Kopaonik | Tara |
---|---|---|---|
Coordinates | 43°10′ 34.6″ N22°43′ 25.0″ E | 43°18′ 16.8″ N20°50′ 54.3″ E | 43°55′ 03.8″ N19°25′ 51.6″ E |
Altitude (m a.s.l.) | 950 | 1490 | 1060 |
Climate | Temperately continental submontanum, montanum | Temperately continental montanum, subalpine | Temperately continental montanum |
Mean annual temperature (°C) * | 10.0 | 3.6 | 7.7 |
Mean temperature in June 2014 (°C) | 14.5 | 11.0 | 12.6 |
Mean temperature in September 2013 (°C) | 12.8 | 9.1 | 9.3 |
Mean annual precipitation (mm) | 624.7 | 984.4 | 1017.3 |
Soil type | Brown soil | Ranker | Brown soil |
Species | Natural forest of |
Natural |
Natural mixed forest of |
Age of spruce trees (years) | 10-20 | 50-70 | 40-50 |
Summarised values for total number of ectomycorrhizal types, vital ectomycorrhizal root tips, total number of fine roots, and percentage of vital ectomycorrhizal root tips on spruce from different sites in Serbia in two examined seasons (based on 4 soil cores per site and per season).
Variable | Stara planina | Kopaonik | Tara | |||
---|---|---|---|---|---|---|
spring | autumn | spring | autumn | spring | autumn | |
Total number of ectomycorrhizal types | 6 | 8 | 9 | 8 | 9 | 5 |
Total number of vital ectomycorrhizal root tips | 2060 | 1273 | 1463 | 1469 | 893 | 735 |
Total number of fine roots | 28175 | 4239 | 5274 | 3174 | 4347 | 2683 |
% of vital ectomycorrhizal root tips | 8 | 32 | 28 | 46 | 41 | 27 |
Mean number of ectomycorrhizal (ECM) types, vital ECM root tips, total number of fine roots, percentage of vital ECM root tips, and diversity indices on spruce from different sites in Serbia in the spring and autumn, based on 4 soil samples. Differences among values of a particular variable marked with the same letter are not significant (p > 0.05), according to Fisher’s LSD test.
Variable | Sites by Season | Sites | Seasons | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Stara planina | Kopaonik | Tara | Staraplanina | Kopaonik | Tara | Spring | Autumn | ||||
spring | autumn | spring | autumn | spring | autumn | ||||||
Mean number of ECM types | 3.5 ab | 4.75 b | 3.75 ab | 3.00 ab | 3.25 ab | 2.5 a | 4.12 a | 3.37 a | 2.87 a | 3.50 a | 3.42 a |
Number of vital ECM root tips | 515 a | 318.7 a | 365.7 a | 369.7 a | 223.2 a | 226.5 a | 416.8 a | 367.7 a | 224.8 a | 367.9 a | 304.9 a |
Total number of fine roots | 7043.7 b | 1547 a | 1327 a | 806.5 a | 1186.7 a | 671.7 a | 4295.3 b | 1066.7 a | 929.2 a | 3185.8 b | 1008.4 a |
% of vital ECM root tips | 8.7 a | 27.2 ab | 29.4 ab | 45.8 b | 23.0 b | 36.1 b | 17.9 a | 37.5 b | 29.5 ab | 20.4 a | 36.3 b |
Species richness index (d) | 0.925 ab | 1.507 b | 1.087 ab | 0.826 ab | 0.950 ab | 0.629 a | 1.216 a | 0.955 a | 0.785 a | 0.988 a | 0.982 a |
Shanon Weaver index (H) | 0.736 a | 1.216 a | 1.024 a | 0.649 a | 0.872 a | 0.581 a | 0.976 a | 0.835 a | 0.725 a | 0.875 a | 0.815 a |
Evenness (e) | 1.158 a | 1.748 a | 1.843 a | 1.239 a | 1.695 a | 1.007 a | 1.453 a | 1.540 a | 1.350 a | 1.563 a | 1.333 a |
Equitability (J) | 0.503 a | 0.756 a | 0.800 a | 0.538 a | 0.736 a | 0.437 a | 0.630 a | 0.670 a | 0.590 a | 0.681 a | 0.579 a |
Berger-Parker index | 0.319 a | 0.544 a | 0.413 a | 0.270 a | 0.417 a | 0.309 a | 0.432 a | 0.340 a | 0.364 a | 0.382 a | 0.375 a |
F-test of two-way ANOVA for number of ectomycorrhizal types, vital ectomycorrhizal root tips, total number of fine roots, and percentage of vital ectomycorrhizal root tips. (*): p < 0.05.
Variable | Factors | ||
---|---|---|---|
Sites (A) | Seasons(B) | Interaction (A×B) | |
Mean number of ectomycorrhizal types | 1.417 | 0.050 | 1.262 |
Number of vital ectomycorrhizal root tips | 1.428 | 0.385 | 0.320 |
Total number of fine roots | 16.891* | 16.600* | 6.348* |
% of vital ECM root tips | 3.616* | 6.485* | 0.156 |
F-test of two-way ANOVA for exploration types (ET). (*): p < 0.05.
Variable | Factors | ||
---|---|---|---|
Sites(A) | Seasons(B) | Interaction(A×B) | |
Contact ET | 8.175* | 0.213 | 0.35 |
Short distance ET | 2.205 | 0.184 | 0.455 |
Medium distance ET-subtype fringe | 9.203* | 2.374 | 0.966 |
Medium distance ET-subtype smooth | 67.757* | 0.539 | 0.539 |