Ectomycorrhizal status of Norway spruce seedlings from bare-root forest nurseries

Forest Ecology and Management 236 (2006) 375–384 www.elsevier.com/locate/foreco

Ectomycorrhizal status of Norway spruce seedlings from bare-root forest nurseries Maria Rudawska a,*, Tomasz Leski a, Lidia K. Trocha a, Roman Gornowicz b a b

Institute of Dendrology, Polish Academy of Sciences, 5 Parkowa St., 62-035 Ko´rnik, Poland Agriculture University, Faculty of Forestry, 28 Wojska Polskiego St., 60-637 Poznan´, Poland

Received 8 October 2005; received in revised form 4 July 2006; accepted 21 September 2006

Abstract We examined the ectomycorrhizal communities associated with Norway spruce seedlings grown in 16 bare-root nurseries in northwest Poland. One through four-year-old seedlings were examined and compared. We found 11 morphotypes in total with an average of 2.7 morphotypes per nursery. Among these 11 morphotypes, RFLP-analysis detected 17 distinct RFLP-patterns. By comparison with the available reference material, 12 of these could be identified to genus or species. These were Amphinema byssoides, Hebeloma crustuliniforme, Hebeloma longicaudum, Paxillus involutus, Thelephora terrestris, Cenococcum geophilum, Phialophora finlandia, Tuber sp., Wilcoxina mikolae, Wilcoxina sp. 1, Wilcoxina sp. 2 and Tricharina ochroleuca. The five unidentified morphotypes were: basidiomycetes ITE.5 and four ascomycetes designated as IDPAN.1, IDPAN.2, IDPAN.3 and IDPAN.4 that in morphotyping were classified as an E-strain. The ascomycete W. mikolae was predominant, whereas the basidiomycetes were less common. ECM community of Norway spruce seedlings from bare-root nurseries were not structured in association with soil pH and nutrient status of the host plant. The consistent presence of inocula of ascomycete symbionts in the substrate from all nurseries is quite notable and may reflect their adaptation and/or resistance to highly transformed nursery soil substrates compared to symbionts from the Basidiomycota. # 2006 Elsevier B.V. All rights reserved. Keywords: Picea abies; Ectomycorrhiza; Morphotype; PCR–RFLP; Nursery

1. Introduction Norway spruce (Picea abies (L.) Karst.) has an extensive geographic range in Europe, growing from Scandinavia to the Balkans, the Alps and the Carpathians. In Poland, Norway spruce is a native species and after Scots pine, the most commonly planted conifer, accounting for 5.8% of Polish forests. As with most tree species in boreal and temperate forests, ectomycorrhizal (ECM) fungi extensively colonize the fine roots of Norway spruce. These ectomycorrhizal associations are ubiquitous and are believed to be critical to successful seedling establishment and tree growth, facilitating both nutrient and water uptake, increasing resistance to certain root diseases, and enhancing the tolerance of the tree to stress (Harley and Smith, 1983; Allen, 1991). Mycorrhizal formation is usually adequate under natural conditions. ECM fungi live in the forest soil and litter layer and readily colonize new tree seedlings. Determination of the ECM community structure of

* Corresponding author. Tel.: +48 61 8170033; fax: +48 61 8170166. E-mail address: [email protected] (M. Rudawska). 0378-1127/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.foreco.2006.09.066

young and mature Norway spruce stands has received much attention recently (Egli et al., 1993; Mehmann et al., 1995; Ka˚re´n and Nylund, 1996; Kraigher et al., 1996; Dahlberg et al., 1997; Erland et al., 1999; Kraigher, 1999; Mahmood et al., 1999; Fransson et al., 2000; Jonsson et al., 2000; Peter et al., 2001a,b; Haug, 2002; Kieliszewska-Rokicka et al., 2003). As an alternative to natural regeneration, Norway spruce seedlings are grown for 3 or 4 years in nurseries before outplanting. Nursery managers have long recognized the importance of well-developed mycorrhizas for healthy seedling growth in the nursery and desired performance after outplanting. There are currently around 1200 bare-root forest nurseries in Poland, producing close to 200 million Norway spruce seedlings each year for reforestation and afforestation (Lenart, 2000). Very little is known about the ECM symbionts that associate with spruce seedlings in nurseries and how this relates to seedling and ecosystem function during the first years following outplanting. Only a few reports describe the ECM community structure of several commercially important Picea species originating as bare-root or containerized seedling nursery stock, such as Picea sitchensis or Picea glauca (Thomas and Jackson, 1979; Danielson and Visser, 1989;

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Grogan et al., 1994; Krasowski et al., 1999; Kernaghan et al., 2003). The first record of nursery mycorrhizas of P. abies appears to be that of Weiss and Agerer (1988) who described three unidentified mycorrhizas of Norway spruce cuttings grown in a Bavarian nursery. Recently, fungal community structure in fine roots of P. abies seedlings under different nursery cultivation system in Lithuania was demonstrated by Menkis et al. (2005). Identification of the indigenous mycorrhizal populations on seedlings produced in bare-root nurseries is complicated because nursery management practices such as fertilization, fumigation, and use of fungicides are known to affect mycorrhizal colonization (Molina and Trappe, 1982). The intensive cultivation of soil (i.e. ploughing, weeding, root pruning, etc.) breaks up the delicate fungus–soil network, leading to a significant reduction in ECM colonization and makes sporophores production near unfeasible (Xavier and Germida, 1999). Thus, the species composition and relative abundance of fungi that colonize seedlings in bare-root nurseries have to be estimated directly by observing roots. Molecular techniques have greatly advanced the opportunities to identify fungal species and strains from even single ectomycorrhizal root tips (Gardes and Bruns, 1993). Using this approach, we have examined the species richness and abundance of ECM community associated to spruce seedlings grown in bare-root nurseries in Poland. The aims of this study were (1) to obtain comprehensive insight into the ECM fungi that form mycorrhizal associations in the condition of nursery and (2) to determine whether the detected ECM fungi diversity would differ according to the nursery stock sample, age of the seedlings, pH of the nursery bed soil and nutritional status of seedlings, estimated as the foliar content of macroelements (N, P, K, Ca, and Mg). 2. Materials and methods

five to 10 ha, and separated into several compartments with four to six standard nursery seedbeds each. Norway spruce seedlings were all precision seeded by machine and fertilized following a schedule designed to satisfy their nutrient requirements and based on soil analysis of each nursery. Depending on the current nursery production, P. abies stock (1-, 2-, 3- and 4-year-old) or transplants (1-, 1.5- and 2-year-old trees transferred to transplant beds) were harvested, between the beginning of September and middle of October during 3 consecutive years (2001, 2002, 2003). Each stock was classified according to seedling age and growing location. For example, a 1 + 0 classification represents stock that has been grown for 1 year in a seedbed and 0 years in a transplant bed; a 1 + 1 is grown for 1 year in a seedbed and 1 year in a transplant bed, etc. 2.2. Analysis The pH of the nursery-bed soil was determined by mixing 20 ml of soil substrate with 40 ml of de-ionized water. After 1 h, the pH was determined with a calibrated pH meter equipped with a glass electrode. The foliar content of macronutrients (N, P, K, Ca, and Mg) was determined using three composite samples. Each sample consisted of five seedlings lifted from the Norway spruce seedbeds of a given age. Milled samples of spruce needles, of approximately 2.5 g dry mass each, were digested in a mixture of spectrally pure concentrated acids: HNO3 and HClO4 in a proportion of 4:1 (v:v) and diluted with bi-distilled water to make 25 ml. Nitrogen was analyzed by the micro-Kjeldahl method. The remaining macroelements were measured by atomic absorption spectroscopy (Varian 220 FS) with atomization in an air–acetylene flame. The accuracy of the analyses was checked against standard reference material, namely pine needles 1575 and tomato leaves SRM 1573a (National Institute of Standards and Technology, USA).

2.1. Nurseries and seedlings sampled 2.3. Ectomycorrhizal assessment We surveyed 16 nurseries that were each active in supplying planting stock for the restocking of forests and the reforestation of post-agricultural land. The sampled nurseries belonged to Forest Districts located in northwestern Poland (Fig. 1). They are all rather large provincial nurseries, ranging in size from

Four subsamples, each comprised of five seedlings lifted from the Norway spruce seedbeds of a given age were randomly sampled. The base unit for data analysis was the average of these four replicates (=nursery stock sample, NSS). A total of

Fig. 1. Location of sampled nurseries. The names of nurseries are labeled as: Ch, Chojna; Dr, Dobrocin; Dw, Dabrowa; Iw, Ilawa; Kt, Kartuzy; Mk, Mieszkowice; Mm, Milomlyn; Mr, Miradz; Ok, Okonek; Ol, Olsztynek; Pr, Przymuszewo; Rn, Runowo; SK, Solec Kujawski; Sw, Swidnica; Wr, Wronki; Zt, Zlotow.

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32 nursery stock samples and 640 seedlings were analyzed. Seedlings were removed from the nursery together with an adjacent soil sample and transported immediately to the laboratory in plastic bags. The root system was gently washed in tap water to remove most of the soil and organic debris, minimizing any damage to the ectomycorrhizas. Tightly adhering materials were removed with forceps. Because of the large number of root tips present on each seedling (usually 300–600, but up to 1000 per seedling), assessments of morphotype frequencies by counting all root tips was too time consuming. Therefore, the clean roots were cut into approximately 2.5-cm long sections and placed in a Petri dish filled with water. Sections were randomly selected and the numbers of all active root tips colonized by each morphotype were counted. Successive root sections were examined until 300 root tips had been counted in each of the replicates. Different morphotypes were distinguished by their macroscopic and microscopic characteristics and named if matched to published descriptions (Agerer, 1987–2002; Danielson and Visser, 1989; Ingleby et al., 1990; Massicotte, 1994; Shihido et al., 1996). Each root tip was examined under a microscope (6–90 magnification) for features such as color, shape, size, texture, presence or absence of mycelial strands or rhizomorphs; presence of cystidia, etc. Mycorrhizal colonization was confirmed by microscopic (500) examination of whole mounts of root tips to determine the presence of a mantle and a Hartig net. Each distinct mycorrhizal morphotype was described and photographed for further reference. For DNA analysis, fragments of the root system with the same mycorrhizal types were placed in Eppendorf-tubes in cetyltrimethyl ammonium bromide (CTAB) buffer and stored at room temperature until processing. 2.4. Molecular identification of ectomycorrhizas For molecular identification, three to six root tips per morphotypes from each NSS were analyzed. In case of morphotypes that in preliminary analysis represented mixtures of taxa (different RFLP-types) additional analysis were performed consisting five root tips of this morphotypes from each subsample (n = 20) within NSS. Obtained results were a basis to calculate relative abundance of mycorrhizal species.

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Ectomycorrhizal fungi on mycorrhizal root tips were identified using restriction fragment length polymorphism (RFLP) of the PCR-amplified internal transcribed spacer of DNA. Each sample consisted of a single mycorrhiza. DNA was extracted using the miniprep method developed by Gardes and Bruns (1996); the amplification followed the protocol of Henrion et al. (1994) as modified by Ka˚re´n et al. (1997). RFLPpatterns were obtained using the restriction enzymes MboI, HinfI, and TaqI. Restriction fragments were separated using 2% agarose gel electrophoresis (4 h; 100 mV), stained with 0.5% ethidium bromide, and recorded on black and white PolaroidTM film. Morphotypes were identified by matching the sample and reference specimens (obtained from a regional collection of sporocarps or pure culture) using the Taxotron1 software package (Pasteur Institute, Paris, France). Different restriction fragment length polymorphism (RFLP) patterns were denoted as separate species if the fragments varied above 4%. 2.5. Data analysis The diversity of the ectomycorrhizas on the seedlings was expressed as the number of identified ECM species (species richness). The relative abundance of previously identified ECM fungal species was calculated as number of tips of given ECM species per total number of mycorrhizal tips extracted in NSS. Because the mycorrhizal species richness and relative abundance values were not normally distributed and typical transformations did not correct the distribution, nonparametric Kruskal–Wallis tests were used to test the effects of nursery stock sample, age and seedling transplant type. The test could be applied only for the nine fungal species that occurred in four or more of the examined NSSs. The remaining taxa occurred too infrequently to apply statistical tests. ECM species richness in relation to macroelement contents in foliage of Norway spruce seedlings and pH of the nursery-bed soil were correlated using the Spearman rank correlation test. Macronutrient status between Norway spruce seedlings of varying age and pH of the nursery soil substrate were compared by ANOVA (Table 1). When a significant difference was observed, Tukey’s post hoc test was applied. Canonical correspondence analysis (CCA) was used to check if relative abundance is related with tested variables (nutritional status of Norway spruce seedlings and nursery-bed

Table 1 Macroelement concentration (%) in foliage of Norway spruce seedlings of varying age and pH of the nursery soil substrate (means from 33, 27 or 36 of composite samples for 1-, 2-, 3–4-year-old seedlings, respectively; SE in brackets) 1-year-old

N P K Ca Mg pHH2 O pHKCl

2-year-old

Min–max

Mean

1.62–2.85 0.12–0.38 0.43–1.26 0.51–1.57 0.10–0.24 4.18–8.14 3.57–7.84

2.24 0.27 0.81 0.79 0.14 6.06 5.87

(0.34) (0.06) (0.22) (0.35) (0.04) (1.18) (1.29)

a a a a a a a

3–4 year-old

Min–max

Mean

1.76–2.30 0.10–0.40 0.19–0.91 0.32–1.16 0.08–0.13 4.22–7.07 3.77–6.75

2.04 0.25 0.59 0.82 0.10 5.86 5.49

Mean values within a row followed by the same letter do not differ at P < 0.05 (Tukey test).

(0.21) (0.10) (0.24) (0.30) (0.02) (0.80) (0.88)

a,b a b a b a a

Min–max

Mean

1.12–2.66 0.18–0.28 0.31–0.72 0.74–1.72 0.07–0.14 4.72–7.93 4.26–7.59

1.96 0.24 0.53 0.94 0.10 6.07 5.84

(0.39) (0.03) (0.14) (0.26) (0.02) (1.05) (1.26)

b a b a b a a

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soil pH). An unrestricted Monte Carlo permutation test (999 permutations) was performed to determine if tested relationships were statistically significant for the first canonical axis and for all four of the extracted axes combined (ter Braak and Sˇmilauer, 1998). Relative abundance values were transformed (log10 + 1) before analysis, and the CCA was performed with CANOCO 4 software.

These were basidiomycete ITE.5 (Ingleby et al., 1990) and four ascomycetes designated as IDPAN.1, IDPAN.2, IDPAN.3 and IDPAN.4 that were classified as an E-strain in morphotyping (Table 2, Fig. 2). The RFLP-patterns of these morphotypes did not match the ECM fungal species in our database.

3. Results

ECM species richness among the tested nurseries was quite variable and ranged from 1 to 8 per NSS with an average of 2.7 species per NSS (Table 3). Statistics based on the Kruskal– Wallis tests indicated no effect of seedling age and transplant type on ECM species richness of Norway spruce seedlings; however, significant differences (P = 0.0127) were found among NSS (Table 3). The relative abundances and distributions of ECM taxa in the screened nurseries are presented in Table 3. The Kruskal– Wallis test for nine most frequent fungal species indicated that relative abundance of this species differed significantly among NSS (P < 0.001). W. mikolae was a dominant ECM species on 1- and 2-year-old seedlings (mean relative abundance of 56.5 and 37%, respectively), and less abundant on 3- and 4-year-old seedlings (mean relative abundance of 21%); however, age effect was statistically not significant (Table 3). W. mikolae was very often the only species found on seedlings from the tested nurseries (100% relative abundance). Several infrequently observed taxa (Wilcoxina sp.2, IDPAN.1, IDPAN.2, IDPAN.3)

3.1. Mycorrhizal community composition The mycorrhizal colonization of all tested samples was nearly 100%. Very small proportions of the root tips appeared dark and less turgid and were omitted in the analysis. Overall, 11 distinct ECM morphotypes were recorded. Among these 11 morphotypes, RFLP-analysis distinguished 17 unique RFLPpatterns. Upon comparison with the available reference material, twelve of these could be identified at least to genus. These were Amphinema byssoides, Hebeloma crustuliniforme, H. longicaudum, Paxillus involutus, Thelephora terrestris, Cenococcum geophilum, Phialophora finlandia, Tuber sp., Wilcoxina mikolae, and Tricharina ochroleuca. Two RFLPpatterns associated with an E-strain morphotypes that matched closely, but not identically with W. mikolae (similarity level with data base less than 96%), were designated as Wiloxina sp. 1 and Wiloxina sp.2. Five morphotypes remained unidentified.

3.2. Species richness, frequency and relative abundance

Table 2 Description and identification of Norway spruce ECM morphotypes originating from bare-root forest tree nurseries in Poland ECM morphotype

Description

Identification

ECM species code

1. Amphinema byssoides

Comparable with published descriptions for A. byssoides (Ingleby et al., 1990; Agerer, 1987–2002) Comparable with descriptions for Hebeloma (Ingleby et al., 1990; Agerer, 1987–2002) Comparable with published descriptions for Paxillus involutus (Ingleby et al., 1990; Agerer, 1987–2002) Comparable with published descriptions for Thelephora terrestris (Ingleby et al., 1990; Agerer, 1987–2002) Conforms to the published descriptions for ITE.5 (Ingleby et al., 1990) Comparable with published descriptions for Cenococum geophilum (Ingleby et al., 1990; Agerer, 1987–2002) Conforms to the published descriptions for ITE.3 (Ingleby et al., 1990) Monopodial–pyramidal, lemon-yellow or pale brown, mantle surface smooth and spiny Conforms to the published descriptions for Humaria hemispherica (Ingleby et al., 1990) Conforms to the published descriptions for Tricharina gilva (Ingleby et al., 1990) Mycorrhizas single to pinnate, orange brown, pale yellowish-brown to dark reddish-brown or blackish-brown with whitish-brown emanating hyphae, moderately thick-walled, verrucose, septate, no clamps, rhizomorphs not observed

A. byssoides

Ab

Hebeloma crustuliniforme Hebeloma longicaudum P. involutus

Hc, Hl

T. terrestris

Tt

ITE.5

ITE.5

C. geophilum

Cg

Phialophora finlandia

Pf

Tuber sp.

Tsp.

Wlicoxina mikolae Wilcoxina sp.2, ID PAN.1, ID PAN.2 Wilcoxina sp. 1 Tricharina ochroleuca

Wm, Wsp.2, ID PAN.1 ID PAN.2 Wsp.1 To

ID PAN.3, ID PAN.4

ID PAN.3 ID PAN.4

2. Hebeloma-like 3. Paxillus-like

4. Thelephora-like

5. Dark brown 6. Cenococum geophillum

7. Mycelium radicis atrovirens 8. Tuber-like 9. E-strain-like 1 10. E-strain-like 2 11. E-strain-like 3

Pi

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379

Fig. 2. Ectomycorrhizas observed on Norway spruce seedlings from bare-root forest tree nurseries. The coding follows that shown in Table 2. Bars, 1 mm.

reached very high (>60%) relative abundances in certain nurseries. Other rare species such as H. longicaudum, P. involutus, and ITE.5 were less abundant (<20%). The frequencies of ECM species observed on roots of screened nursery stock are presented in Fig. 3. The most frequent species on 1- and 2-year-old seedlings was W. mikolae, which was noted in more than 70% of the NSS’s but was less frequent on 3and 4-year-old seedlings. Some ECM taxa such as T. terrestris, P. finlandia, Wilcoxina sp. 1, A. byssoides, C. geophilum, and Tuber sp. were consistently present in samples of all seedling age classes (Fig. 3). During the study, these species collectively occurred at a frequency of 10%. Basidiomycete taxa P. involutus and ITE.5 were exclusively found on 1 + 0 seedlings. Both Hebeloma species (H. crustuliniforme and H. longicaudum) were absent on 1 + 0 stock, but present on older

seedlings. The remaining five ascomycete taxa (IDPAN.1– IDPAN.4 and T. ochroleuca) were unevenly distributed among the tested nursery stock. 3.3. Foliage nutrient concentration and pH of the nurserybed soil Table 1 summarizes the minimum, maximum and mean contents of macronutrients in the foliage of Norway spruce seedlings as well as the pH of the nursery-bed soil. The mean concentration of nitrogen (N) decreased with age of seedlings; however, significant difference was found only for the 3–4year-old plants. Regardless of seedling age, phosphorus (P) and calcium (Ca) concentration in the needles of the tested seedlings did not differ significantly. The mean concentration of

380

Table 3 Species richness and relative abundance (%) of ECM fungi species found on 1-, 2-, and 3–4-year-old Norway spruce seedlings from bare-root forest nurseries (n = 20 per NSS, SE in brackets) Richness A. byssoides H. crustuliniforme Hebeloma P. involutus T. terrestris ITE.5 longicadum

1-year-old Dr 1–0 Iw 1–0 Kt 1–0 Mm 1–0 Ok 1–0 Rn 1–0 SK 1–0 Sw 1–0 Sw 1–0 Wr 1–0 Ch 1–0

1 1 2 1 2 3 3 4 4 3 1

0 0 0 0 76 (11.2) 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 18 (12.1) 0 0 0

0 0 0 0 0 10 (7.1) 8 (3.7) 34 (11.2) 0 0 0

0 0 0 0 15 (3.3) 0 0 0 0 0 0 0 0 0 0 0 0 10 (5.5) 0 0 0 0

0 0 0 0 0 0 3 (3.0) 0 4 0 0

0 0 0 0 0 7 (2.2) 0 0 0 0 0

100 (0.0) 100 (0.0) 85 (12.5) 100 (0.0) 0 0 89 (10.2) 45 (10.0) 38 (7.8) 65 (1.1) 0

2-year-old Dw 2–0 Iw 1–1 Mk 2–0 Mm 1.5–0.5 Mr 2–0 Pr 2–0 Rn 2–0 Ok 2–0 Wr 2–0

2 2 4 2 1 5 3 8 3

0 0 0 0 0 0 0 10 (3.5) 25 (9.2)

0 0 0 0 0 15 (2.4) 0 0 0

0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

0 0 0 0 0 14 (1.1) 20 (2.3) 15 (5.6) 0

0 0 0 0 0 0 0 0 0

0 6 (2.1) 0 0 0 2 (5.3) 0 5 (2.2) 0

0 0 0 0 0 4 (2.1) 0 3 (1.1) 0

0 0 5 (4.5) 5 (3.3) 0 0 15 (8.3) 45 (7.3) 0

20 94 13 0 0 63 65 17 60

3 and 4-year-old Dr 2–1 Iw 1–2 Mm 2–1 Mr 2–1 Mr 3–0 Ot 1.5–1.5 SK 1–2 Ch 3–0 Wr 3–0 Zt 3–0 Mm 4–0 Pr 2–2

1 1 2 1 4 2 4 2 3 4 3 4

0 0 0 0 0 37 (3.3) 0 0 0 0 0 0

0 0 0 0 0 0 3 (13.3) 84 (2.2) 0 0 0 62 (5.1)

0 0 0 0 0 0 0 0 0 0 14 (10.3) 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 65 (11.3) 0 77 (7.8) 0 0 0 0 14 (4.5)

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 3 0 19 (6.2) 0 0 7 (2.3) 0 0

0 0 0 0 4 (2.2) 0 4 (2.4) 0 0 30 (6.7) 0 0

0 100 (0.0) 0 100 (0.0) 0 0 0 0 0 0 63 (11.1) 0 0 0 16 (2.2) 0 0 56 (8.8) 60 (14.3) 3 (4.2) 0 0 5 0

***

nt nt nt

nt nt nt

nt nt nt

nt nt nt

***

***

***

***

***

ns ns

ns ns

ns ns

ns ns

ns ns

Source * NSS Age ns Transplant type ns Kruskal–Wallis test:

ns ns ***

P < 0.001; *P < 0.05; ns, not significant, P > 0.05; nt, not tested.

Cenococcum P. finlandia Tuber sp. Wilcoxina Wilcoxina sp. 1 Wicoxina sp. 2 T. ochroleuca IDPAN.1 IDPAN.2 IDPAN.3 IDPAN.4 geophillum mikolae 0 0 0 0 24 (12.2) 0 0 0 0 20 (3.3) 0

0 0 0 0 0 83 (11.5) 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 48 (11.1) 0 100 (0.0)

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

80 (21.2) 0 0 0 0 0 0 2 (3.3) 0

0 0 0 0 0 0 0 0 0

0 0 0 0 0 35 (4.5) 95 (4.6) 0 0 100 (0.0) 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 25 (4.9) 0 63 (17.4) 0

0 0 0 0 0 0 0 0 0 0 23 (6.5) 19 (7.7)

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 28 (6.8) 72 (15.3) 0 100 (0.0) 0 28 (9.1) 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 19 (3.5) 0 0 0

nt nt nt

nt nt nt

nt nt nt

***

ns ns

nt nt nt

ns ns

(6.6) (5.9) (7.8)

0 0 42 (5.5) 0 0 (5.9) 0 (9.9) 0 (2.3) 3 (2.2) (16.2) 15 (5.7)

nt nt nt

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 3 (2.4) 0 15 (5.5) 0 0 0 0 0 0 0 0 0 0

***

M. Rudawska et al. / Forest Ecology and Management 236 (2006) 375–384

Nursery symbol/stage

M. Rudawska et al. / Forest Ecology and Management 236 (2006) 375–384

potassium and magnesium was significantly higher in 1-yearold seedlings compared to the older ones. The mean pH of the nursery-bed soil ranged from slightly acidic (4.18 in H2O and 3.57 in KCl) to slightly alkaline (8.14 in H2O and 7.84 in KCl) and did not differ in dependence on the age of the seedlings. 3.4. Relationships between the pH of the nursery-bed soil, foliar macroelement concentration, and ECM richness and abundance The Spearman rank correlation coefficient was used to examine the association of the plant and soil variables (foliar macroelement concentration and pH of the nursery-bed soil) with the species richness of the ECM fungi. The only positive rank correlation was found for magnesium concentration in the needles of the tested spruce seedlings (P = 0.002, Table 4).

381

Table 4 Spearman’s rank correlations between ECM species richness and the macroelement concentration of foliage and pH of the nursery soil Rs N P K Ca Mg pHH2 O pHKCl

0.13 0.13 0.13 0.12 0.37 0.13 0.15

t (N 1.07 1.03 1.05 0.93 3.16 1.06 1.21

2)

P 0.29 0.31 0.29 0.35 0.002 0.29 0.23

The abundances of nine fungal species that occurred in four or more of the examined NSSs and their corresponding CCA species scores were plotted together with the examined plant and soil variables of foliar nutrient concentration and pH of the nursery-bed soil. The first two axes of the CCA, with eigenvalues of 0.234 and 0.198, explained 14.4% of the species variance and 64% of the species–environmental relationship. Along with the low percentage of species variance explained, a not significant relationship between species distributions and the measured environmental variables was revealed for the first axis (F = 1.86, P = 0.87) and all four axes combined (F = 0.914, P = 0.58) (figure not shown). 4. Discussion 4.1. Ectomycorrhizal diversity

Fig. 3. Frequency of occurrence of mycorrhizal species in nursery stock samples (NSS) of 1-year-old (a); 2-year-old (b); and 3–4-year-old; (c) Norway spruce seedlings (n = 11, 9 or 12 for 1-, 2-, and 3–4-year-old seedlings, respectively). The coding follows that shown in Table 2.

While natural mycorrhizal colonization is common in bareroot nurseries, the fungal diversity of colonization can be quite variable (Danielson and Visser, 1989; Letho, 1989; Ursic and Peterson, 1997; Ursic et al., 1997; Rudawska et al., 2001; Menkis et al., 2005). We found that 17 fungi species might contribute to the ECM community structure of 1–4-year-old Norway spruce seedlings. Due to the more precise method of identification in the present study, based on molecular analysis, the total number of mycorrhizal species in the screened nurseries was higher than reported in previous studies based on morphotyping or microscopic examination (e.g. Thomas and Jackson, 1979; Danielson and Visser, 1990; Ursic and Peterson, 1997) but comparable with molecular studies of Menkis et al. (2005) for Lithuanian nurseries. Given the fact that we found no statistically significant effect of seedling age on ectomycorrhizal species richness of Norway spruce seedlings (Table 3), it might be interpreted that one to 4-year-old seedlings have a comparable physiological potential to host mycorrhizal symbionts. The fact that 17 ECM species were found in the tested nurseries likely reflects the high potential of Norway spruce seedlings to form ectomycorrhiza. However, individual nursery stock samples significantly differed in terms of species richness and abundance and varies from 1 to 8 per nursery stock sample with an average of 2.7. It raises the question about the causes of such a limited species associations of ECM fungi with spruce seedlings of particular NSS. Kranabetter (2004) studied pioneer ECM community of hybrid spruce seedlings and indicate a better growth response by the less diverse, more

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unevenly distributed pioneer ECM communities in the earliest stand conditions. Spearman rank correlation test (Table 4) show that the ECM richness were not structured in association with soil pH and nutrient status (N, P, K, Ca concentration) of the host plant in the conditions of the bare-root nursery. The only weak correlations with magnesium may be connected with magnesium shortage in the foliage of nursery seedlings. Positive correlations between species richness and increasing magnesium concentration were recorded by Humphrey et al. (2003) for Sitka spruce stands. Ordering of the data by CCA also did not indicate linkage of abundance of some fungal species to tested variables (figure not presented). Similarly, Flynn et al. (1998) found no significant correlations between ECM colonization and soil pH, loss-on-ignition, or water content on Sitka spruce seedlings in a Scottish plantation forest. Krasowski et al. (1999) concluded that the differences in the composition and abundance of ectomycorrhizas on white spruce seedlings appeared to be related more to root vigor than to differences in root growth media and fertilizer treatments. Although several authors have reported that high fertilizer levels decrease mycorrhiza formation (for review see Smith and Read, 1997), Molina and Chamard (1983) and Danielson et al. (1984) reported that ectomycorrhiza formation was not affected by fertilization treatments. As shown by Brunner and Brodbeck (2001) Norway spruce can tolerate N at rather high application rates. The highest application rates of N influenced mycorrhization, but at levels (800 kg N ha 1 year 1) never used in Polish bare-root nurseries (Rudawska, unpublished data). Regardless of seedling age, the mean concentration of nitrogen (N) in the needles of the tested seedlings fell within the range considered optimal for spruce seedlings (1.80–2.40%) (Ingestad, 1962). The inconsistency in the ECM community structure of planting stock from different nurseries may be the variation among the different trials in fertilizer formulas, stock types, site-specific soil characteristics, host physiology, and the availability of inoculum among others (Perry et al., 1987). The processes that control ECM diversity in bare-root forest tree nurseries are still poorly understood. Other factors, including competitive interactions among different ECM species and the availability of different sources of inoculum (Bruns, 1995), may well have contributed to the observed differences in the mycorrhizal fungi communities among the different nurseries. 4.2. Ecology and epidemiology of nursery fungi W. mikolae was the most common ascomycete mycobiont detected in our study, present in high abundances on more than 60% of the nursery stock samples. Wilcoxina spp. are known as common colonizers of nursery seedlings (Danielson, 1991; Egger, 1995). Presumably, W. mikolae, classified into the ‘Estrain’ fungi, was also the most common mycorrhizal type found by Grogan et al. (1994) in Irish nurseries on Sitka spruce. Mycorrhizas present on Sitka spruce in forest tree nurseries, morphologically resembling W. mikolae from our studies, were classified by Ingleby et al. (1990) as Humaria hemisphaerica and Tricharina gilva. According to Fay and Mitchell (1999), mycorrhizas of H. hemisphaerica are commonly associated

with conifer seedlings in nurseries, but do not persist on the roots after outplanting onto forest sites. In our studies a small proportion of the mycorrhizas from the E-strain group matched T. ochroleuca, but none of E-strain morphotypes matched the fruitbodies of H. hemisphaerica. This fungus was also absent in containerized P. glauca seedlings from four nurseries in northern Alberta, Canada (Kernaghan et al., 2003). W. mikolae was quite frequent there instead. It seems that W. mikolae and several other closely related species comprise a group of symbionts best suited to nursery conditions. Wilcoxina sp. is a member of the ascomycete fungi, which contribute to the resistant propagule community, and in terms of life strategy correspond with the ruderal model in plants (Taylor and Bruns, 1999). These fungi do not compete well in the stable environment of a mature forest, but they have persistent propagules and respond rapidly to disturbance. Mycorrhizas of Wilcoxina have been shown to persist for longer periods in habitats where competitors are reduced (Danielson and Pruden, 1990). The observation that certain types of E-strain fungi produce thick-walled chlamydospores (Danielson, 1982) that remain viable in the soil for extended periods of time support this designation. The high abundance of W. mikolae and several closely related fungi (Wilcoxina sp. 1 and 2) indicate that most nursery soils may be considered a disturbed habitat. W. mikolae is also the most common mycorrhizal symbiont found on other conifers such as Scots pine and European larch in Polish and Lithuanian bare-root nurseries (Iwan´ski et al., 2006; Menkis et al., 2005; Rudawska, unpublished data). Other ECM taxa consistently present in the tested nurseries were P. finlandia, C. geophilum, Tuber sp., A. byssoides, and T. terrestris. P. finlandia conforms to the published descriptions for ITE.3 and the fungus associated with this mycorrhiza is a member of the Mycelium radicis atrovirens group. According to Ingleby et al. (1990) some fungi of the M. radicis atrovirens group may be pathogenic. Their persistence on nursery-grown seedlings may be associated with higher abundance of moribund roots due to mechanical injury of the root system arising from mechanized nursery-bed practices (Ingleby et al., 1990). C. geophilum is distinguished morphologically by black, club-shaped mycorrhizas and thick emanating hyphae. Cenococcum is one of the most abundant EM fungal taxa in many studies and apparently has a high fitness or competitive ability relative to other EM fungal species (LoBuglio, 1999). Its lack of known sexual reproductive structures suggests that this species can spread effectively through mycelial growth or asexual soilborne sclerotia (Jonsson et al., 2000). The Tuber sp. was also widespread, but considerably less abundant than other ascomycetes from the genus Wilcoxina. This may reflect limited distribution of the field inoculum of Tuber sp. or its poor competitive ability relative to other species in nursery soil. Different authors have consistently identified Tuber-type mycorrhizas on young seedlings of various host species (e.g. Ingleby et al., 1990; Ursic and Peterson, 1997). The Tuber-type mycorrhizas we found in forest nurseries bear a strong morphological resemblance to that of T. puberulum, as described by Agerer (1987–2002) on mature Norway spruce trees. However, our molecular identification did not match this

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species but other from our data base not identified to genera. Trappe (1969) described T. maculatum in Pinus strobus nursery beds and T. maculatum colonized P. strobus in northern Italian nurseries (Fassi and De Vecchi, 1963); however, T. maculatum has not been found in Poland (Ławrynowicz, 1988). Recently Menkis et al. (2005) reported close matching of Tuber-type mycorrhiza from nursery Norway spruce seedlings with Tuber rapaeodorum. The resolution of taxonomic position of our Tuber-type mycorrhiza needs further studies. In total, 11 ascomycete RLFP types were detected among the tested seedlings. The consistent presence of inocula of ascomycete symbionts in the substrate from all nurseries is quite notable and may reflect their adaptation and/or resistance to highly transformed nursery soil substrates compared to symbionts from the Basidiomycota. This result is in contrast with the findings of Kernaghan et al. (2003) on containerized P. glauca seedlings where the ectomycorrhizal basidiomycetes Thelephora americana and A. byssoides were dominant and the ascomycetes were less common. ECM symbionts from the Basidiomycota (H. crustuliniforme, H. longicaudum, A. byssoides P. involutus, T. terrestris and ITE.5) occurred infrequently in the surveyed nurseries. They are all classified as pioneer or multistage species, occurring in nurseries and young forest plantations with a low humus content and in disturbed habitats (Last et al., 1983; Ingleby et al., 1990; Deacon and Fleming, 1992; Kranabetter, 2004; Marmeisse et al., 2004). The PCR–RFLP procedure is a promising method for identifying ectomycorrhizal fungal partners. However, the number of mycorrhizas identified to the species level in the Ascomycota is still limited owing to a lack of fungal reference material. It is highly probable that additional species, especially those of the Ascomycota group, remain to be identified at bareroot forest tree nurseries, which in the present study were surveyed over a limited 3-year period. Continued observation of ectomycorrhizas and ectomycorrhizal fungi in forest tree nurseries is necessary before the effects of forest nursery practices on particular ectomycorrhizas can be predicted and managed. Acknowledgements The authors wish to thank the Board of State Forest District in Gdansk, Olsztyn, Pila, Szczecin, Torun and Wroclaw for encouragement to these studies and providing the plant material. We thank Dr. Mark Tjoelker for critical reading of the manuscript and English correction and Dr. Marek Kasprowicz for statistical advice. We also acknowledge the helpful comments of two anonymous reviewers. This research was supported by KBN Grant no. 6 PO6L 027 22. References Agerer, R., 1987–2002. Colour Atlas of Ectomycorrhizae, 1–12th ed. Einhorn– Verlag, Schwa¨bisch Gmu¨nd, Germany. Allen, M.F., 1991. The Ecology of Mycorrhizae. Cambridge University Press, Cambridge.

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