Mycorrhiza DOI 10.1007/s00572-014-0555-x

ORIGINAL PAPER

Growth of mycorrhizal jack pine (Pinus banksiana) and white spruce (Picea glauca) seedlings planted in oil sands reclaimed areas Nnenna E. Onwuchekwa & Janusz J. Zwiazek & Ali Quoreshi & Damase P. Khasa

Received: 4 July 2013 / Accepted: 2 January 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract The effectiveness of ectomycorrhizal inoculation at the tree nursery seedling production stage on growth and survival was examined in jack pine (Pinus banksiana) and white spruce (Picea glauca) planted in oil sands reclamation sites. The seedlings were inoculated with Hebeloma crustuliniforme strain # UAMH 5247, Suillus tomentosus strain # UAMH 6252, and Laccaria bicolor strain # UAMH 8232, as individual pure cultures and in combinations. These treatments were demonstrated to improve salinity resistance and water uptake in conifer seedlings. The field responses of seedlings to ectomycorrhizal inoculation varied between plant species, inoculation treatments, and measured parameters. Seedling inoculation resulted in higher ectomycorrhizal colonization rates compared with non-inoculated control, which had also a relatively small proportion of roots colonized by the nursery contaminant fungi identified as Amphinema byssoides and Thelephora americana. Seedling inoculation had overall a greater effect on relative height growth rates, dry biomass, and stem volumes in jack pine compared with white spruce. However, when examined after two growing seasons, inoculated white spruce seedlings showed up to 75 % higher survival rates than non-inoculated controls. The persistence of inoculated fungi in roots of planted seedlings was examined at the end of the second growing season. Although the inoculation with N. E. Onwuchekwa : J. J. Zwiazek (*) Department of Renewable Resources, University of Alberta, Edmonton, Canada e-mail: [email protected] A. Quoreshi Aridland Agriculture and Greenery Department/Food Resources and Marine Sciences Division, Kuwait Institute for Scientific Research (KISR), Kuwait City, Kuwait D. P. Khasa Centre for Forest Research and Institute of Integrative and Systems Biology, Université Laval, Québec City, QC, Canada G1V 0A6

H. crustuliniforme triggered growth responses, the fungus was not found in the roots of seedlings at the end of the second growing season suggesting a possibility that the observed growth-promoting effect of H. crustuliniforme may be transient. The results suggest that the inoculation of conifer seedlings with ectomycorrhizal fungi could potentially be carried out on a large scale in tree nurseries to benefit postplanting performance in oil sands reclamation sites. However, these practices should take into consideration the differences in responses between the different plant species and fungal strains. Keywords Conifer seedlings . Mycorrhizas . Oil sands . Reclamation

Introduction Oil sands mining in northeastern Alberta, Canada causes severe disturbance to boreal ecosystems since it involves complete removal and reconstruction of landforms. In the next several decades, oil sands mining activities will annually disturb approximately 2,000 ha of land that will have to be reclaimed to form self-sustainable ecosystems, largely boreal forests (Government of Alberta 2009). Following bitumen extraction, mine tailings are partly solidified, capped with the tailings sand and overburden (the layer of soil, mineral material and muskeg which was present on the top of the oil sands deposit before removal and stockpiling). Landform reconstruction involves placement of a thin soil layer which had been stripped from the site prior to mining and stock-piled (OSVRC 1998). Reclamation of mining sites involves planting of seedlings of the dominant boreal forest tree species. However, land reclamation efforts are hampered by challenging site conditions including low nutrient availability, high soil pH, salinity, and elevated levels of fluorides, boron, and naphthenic acids (Danielson and Visser 1989; Marx 1990;

Mycorrhiza

Renault et al. 2000; Franklin et al. 2002; Kamaluddin and Zwiazek 2002) It has been well established that plant growth can be improved in nutritionally imbalanced and contaminated soils by the presence of mycorrhizal associations (Brundrett 1991). Mycorrhizal associations can increase plant tolerance to salinity (Muhsin and Zwiazek 2002; Calvo-Polanco and Zwiazek 2011), fluorides (Calvo-Polanco et al. 2009; Calvo-Polanco and Zwiazek 2011). Ectomycorrhizal (ECM) associations have also been found to modify plant water uptake responses to soil pH, likely due to their effect on the aquaporin mediated cell-to-cell transport in roots (Siemens and Zwiazek 2011). Coniferous tree species commonly used for oil sands reclamation are known to form ECM associations with numerous fungal species (Molina et al. 1992). However, since oil sands reclamation sites have been reported to have low soil mycorrhizal inoculum potential, seedlings may not be readily colonized by mycorrhizal fungi in planting sites and may require fungal inoculation prior to planting (Danielson and Visser 1989; Bois et al. 2005). Despite abundant evidence for the beneficial role of mycorrhizal fungi in plants, the results of mycorrhizal studies in disturbed sites have been mixed. While some studies reported positive effects of tree nursery mycorrhizal inoculation on seedlings planted in disturbed sites (Ortega et al. 2004; Parlade et al. 2004), other studies reported no effect (Maestre et al. 2002; Teste et al. 2004). Some of these discrepancies may be due to differences in root colonization and (or) competition from native mycorrhizal fungi as well as to the genetic intraspecific variability (Teste et al. 2004; Campagnac and Khasa 2013). Since the inoculation of container tree seedlings with mycorrhizal fungi increases production costs, more research is needed to demonstrate the benefits of seedling mycorrhization for their postplanting survival and growth. The present study was carried out with jack pine (Pinus banksiana) and white spruce (Picea glauca) seedlings, which are commonly used for the oil sands reclamation. Noninoculated seedlings were compared with seedlings inoculated with three ECM fungi individually or in all possible combinations. The fungi in this study included Hebeloma crustuliniforme, Laccaria bicolor, and Suillus tomentosus since they had been previously reported to increase growth and stress resistance of conifer seedlings under laboratory conditions (Nguyen et al. 2006; Yi et al. 2008; Calvo-Polanco and Zwiazek 2011). However, the effectiveness of seedling inoculation with single cultures and combinations of ECM fungi in improving the establishment and subsequent growth of seedlings following planting in harsh reforestation sites needs to be determined. The aim of this study was to examine the effect of seedling inoculation with ECM fungi at the tree seedling production stage on the survival and growth of seedlings following planting in oil sands reclamation sites. We hypothesized that, since mycorrhizal

fungi help protect plants against the effects of environmental stresses, including salinity, drought, and high soil pH conditions, which characterize oil sands reclamation sites, the inoculation of seedlings prior to planting would improve seedling growth and survival. We also tested the hypothesis that the effectiveness of mycorrhizal fungi would be different for jack pine and white spruce due to the differences in persistence of the mycorrhizal fungi in the root system.

Materials and methods Seedling production and inoculation Jack pine (P. banksiana Lamb.) and white spruce [P. glauca Moench (Voss)] seedlings were grown in commercial tree seedling nursery (Bonnyville Forest Nursery, Bonnyville, AB, Canada) between April and September, 2008 in preparation for planting in June, 2009. The seedlings were grown in 60-cavity Styroblock containers (Beaver Plastics, Acheson, AB, Canada) of 220 ml capacity. The seeds were planted in non-sterile peat: vermiculite growing media (4:1, by volume) and seedlings grown under standard tree nursery production conditions. H. crustuliniforme (Bull.) Quel. (strain # UAMH 5247) (HB), L. bicolor (R. Maire) Orton (strain # UAMH 8232) (LB) and S. tomentosus (Kauffman) Singer, Snell and Dick (strain # UAMH 6252) (ST) were obtained from the University of Alberta Microfungus Collection and Herbarium and grown in modified Melin Norkrans liquid medium (Kernaghan et al. 2002). Before inoculating, liquid culture was homogenized and diluted to produce a concentration of 5 × 103 viable propagules ml−1 or 0.46 mg dry mycelia ml−1 for H. crustuliniforme, 5 × 103 viable propagules ml−1 or 0.39 mg dry mycelia ml−1 for L. bicolor, and 5 × 102 viable propagules ml −1 or 0.34 mg dry mycelia ml −1 for S. tomentosus. The mycelial slurry was mixed into the growing medium of peat/vermiculite, in a 4:1 mix by volume, which was used to fill the Styroblock cavities. Each container cavity (220 ml) received 15 ml of the diluted mycelial slurry. The inoculation was carried out with each fungal species individually or in combinations with four replicates as follows: HC (H. crustuliniforme), LB (L. bicolor), ST (S. tomentosus), HC + LB, HC + ST, ST + LB, HC + LB + ST, plus non-inoculated control (CTRL). A total of eight inoculation treatments with four replicate blocks were established for both jack pine and white spruce plots. Three to four seeds of jack pine (P. banksiana Lamb.) and white spruce [P. glauca (Moench) Voss] were sown in each container cavity and misted daily during the germination period. Two weeks after germination, seedlings were thinned and transplanted to empty cavities so that each cavity contained one seedling. The seedlings were fertilized with nursery standard starter feed solution (80N-50P-185K) plus micronutrients

Mycorrhiza

approximately every week from May through September, 2008. The seedlings were grown for 6 months, hardened off for 6 weeks, and stored at −2 °C for 5 months prior to planting in spring 2009. The experimental design consisted of a randomized complete block design for each plant species with four replicates blocks. The seedling inoculation was carried out with three different ectomycorrhizal species and a consortium of the three-ectomycorrhizal fungi. Seedling status before planting Pre-planting seedling status in both studied tree species was evaluated by measuring heights and root collar diameters in ten randomly selected seedlings per species per treatment. For mycorrhizal assessment, root samples of both tree species were collected from randomly selected 2–3 root plugs from each treatment, and several roots from each plug were pooled for the total of 48–72 roots per treatment. The roots were examined by morpho- and molecular typing (Gagné et al. 2006) and their colonization rates were recorded. The inoculation success was also assessed by examining the numbers of short roots colonized by the fungi (fungus) based on the hyphal morphology and Hartig net formation. Study site and seedling establishment The study was conducted at Suncor Energy Inc. mine reclamation area in Alberta, Canada. The landform, reconstructed in spring 2009, is referred to as Mine Dump 5 (MD5) and is located at 56°58′ 55′′ N, 111° 19′ 97′′W. The study site located within MD5 was approximately 0.3 ha and was constructed in accordance with the oil sand’s plant and mine approval (Government of Alberta 2009). The site construction involved placing the top soil, which had been removed prior to mining, on the benches (clean overburden) and peat/mineral mix (60:40 by volume). The total average rainfall precipitation in the area is 342 mm and a total average snowfall of 155.8 mm per year while the average annual temperature is 0.7 °C (Environment Canada, Fort McMurray Weather Station, 30-year average data for 1971–2000). Soil samples of approximately 500 g were collected from the 10 to 25 cm depth along a 10 m transect for the total of 24 samples and their physical and chemical properties analyzed by the University of Alberta Analytical Laboratory. Texture was determined using the hydrometer method as outlined in (Sheldrick and Wang 1993). Total carbon and nitrogen contents were measured using a Costech 4010 Elemental Analyzer System (Costech Analytical Technologies Inc., Valencia, CA, USA) fitted with a thermal conductivity detector. Cation analyses were carried out using a Varian 880 Atomic Absorption Spectrometer and microbial biomass C and was analyzed as explained by Voroney et al. (2008) using the Shimadzu TOC-V/TN instrument. The

physical and chemical characteristics of the soil are summarized in Table 1. Study design The experiment was a randomized complete block design with each plot of 56 × 30 m for each plant species and four replicated blocks. Each treatment block (5 by 4 m) was planted with 30 seedlings at a distance of 1 m and block distance of 2 m. A total of 960 seedlings per plant species were planted in each experimental plot for the total of 1920 seedlings planted in a 6720 m2 study area. Measurements and statistical analyses Measurements were taken after 6 and 12 months of planting, i.e., in September 2009 and 2010 with one destructive sampling in 2010. Five randomly selected seedlings per plant species from each of the four blocks were used for the measurements which included shoot height, stem diameter at the root collar and dry biomass. The data were used to calculate the stem-volume index (SVI) where SVI = (root collar diameter)2 × total terminal shoot height (Pontailler et al. 1997) and the plot-volume index (PVI) where PVI = stem-volume index × number of surviving plants per treatment (Marx et al. 1991). Relative height growth rate (RHGR) was calculated for both growing seasons as (hf − hi)/hi × 1/tf × ti × 100, where hf is the final height (centimeter) at the measurement time (tf ) (September 2009 and 2010) and hi is the initial height (centimeter) at the planting time (ti) (Pera et al. 1999). Seedling survival was determined for all treatments at the end of the second growth season. Table 1 Physical and chemical soil properties in MD5 reclamation site. Values are means±SE (n = 8) Soil property

Value

Mg (mg kg−1) Na (mg kg−1) K (mg kg−1) P (mg kg−1) Ca (mg kg−1) Total N (mg kg−1) Organic S (mg kg−1) Organic C (mg kg−1) pH Electrical conductivity (μS m−1) Microbial biomass C Silt (%) Clay (%) Sand (%)

583.4±41.7 36.2±3.5 102.1±14.1 0.8±0.1 7,7000±283.9 6.7±0.8 1000±0.0 56,000±0.64 8.0 499.5 83.9 35.0 14.0 51.0

Mycorrhiza

To determine the effect of mycorrhizal inoculum on dry biomass, field mycorrhizal inoculum dependency (FMID) was calculated as (Bi − Bc)/Bi × 100, where Bi is the dry biomass of inoculated and Bc dry biomass of non-inoculated seedlings (Plenchette et al. 1983). The design of the experiment was a randomized block with the block and treatment effects being the random and fixed factors, respectively. All data were checked for normal distribution and homoscedasticity. The homogeneity of variance was tested using the residual plots in which no particular patterns were observed, suggesting that the assumption was met. Because the model has both random and fixed factors, all growth data were analyzed by one-way analysis of variance (ANOVA) PROC MIXED model to determine the effects of inoculation. Following a significant treatment effect (p ≤ 0.05), protected LSD multiple comparisons were carried out to determine the differences between treatments. Some contrasts were also done to specify the block effect and to compare the single fungus treatments (HC, LB, ST) with the inoculation with two (HC + LB, HC + ST, LB + ST) and three fungi (HC + LB + ST). For the survival data, a binomial randomized complete block ANOVA was applied to the data. The data were subjected to the ANOVA using SAS 9.2 software (SAS Institute Inc 2008). Morphology description of mycorrhizas Root samples, each containing approximately 1.5 g of roots, were collected during the destructive sampling in 2010 from four equidistant points around a tree in equal proportions. After washing the roots under running water, fine roots were incised into 5- to 7-cm-long segments which were spread in water in a glass dish and examined under the stereomicroscope. ECM types were grouped using branching patterns, color, tip shape, texture, emanating hyphae, and outer mantle (Agerer 1987). Root tips and segments were obtained from all treatments and stored in cetyl trimethylammonium bromide (CTAB) buffer (Henrion et al. 1994) at 20 °C for molecular analyses. Representative root samples were stained with trypan blue to confirm the presence of the Hartig net (Brundrett et al. 1996). After the microscopic examination, the percentage of colonized root tips was calculated in 20 seedlings per treatment as the number of ECM root tips divided by the total number of root tips and multiplying by 100 (Gehring and Whitham 1994). DNA extraction and amplification Between two and five representative root tips of each morphotype were selected for DNA extraction for each sampled plot. Total genomic DNA was extracted using Sigma Extract-N-Amp Tissue Kit following the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO, USA). To identify the ECM fungi present in the roots, a total of 150 DNA extracts were obtained from root tips sampled in 2010. The internal

transcribed spacer (ITS) of the nuclear ribosomal DNA (rDNA) was amplified using ITS1F and ITS4 primers (Gardes and Bruns 1993; White et al. 1990) as listed in Table 2. Amplifications were performed in a vapo-protect thermocycler (Eppendorf, Mississauga, ON, Canada) with an initial denaturation cycle at 95 °C for 2 min, followed by 15 cycles of 95 °C for 2 min, 55 °C for 2 min and 72 °C for 2 min; then 25 cycles of 94 °C for 35 s, 53 °C for 55 s and 72 °C for 3 min, with a final extension of 72 °C for 5 min. All PCR products were visualized on a 1 % agarose gel. PCR products were purified using ExoSAP-IT (USB, Cleveland, OH, USA) and, when necessary, by gel incision following the manufacturer’s protocol (Qiagen, Toronto, ON, Canada). Sanger sequencing was carried out in one direction using the Big Dye Terminator Sequencing Mix (v. 3.1, Life Technologies Corporation, Carlsbad, CA, USA) with the same PCR forward primer at a final concentration of 0.1 μM. The resulting products were precipitated using EDTA/ethanol following the manufacturer’s instructions. Persistence of inoculated species and primer design Specific primers were designed for each fungus that was inoculated onto seedlings. DNA extraction–PCR was conducted from fungal plate cultures following the same procedure as above but with the use of NSI1 and NLB4 primer pair because of their success in targeting basidiomycetes fungi (Martin and Rygiewicz 2005). The PCR products were Table 2 List of primers used in PCR amplification of fungal DNA extracts Primers Sequence (5′——————3′)

Melting temperature (°C)

Expected product size (∼bp)

ITS1F ITS4

55

550–750

NSI1 NLB4 HCFa HCRa LBFa LBRa STFa STRa

a

CTTGGTCATTTAGAGGAA GTAA TCCTCCGCTTATTGATAT GC GATTGAATGGCTTAGTGA GG GGATTCTCACCCTCTATG AC TGGTTGTTGCTGGCTCTT TCGAGG TGCAGATGTCCACGGC GTAGAT CGGATTTGAGGATCGC CGTGCTGT TTGTACACGGTCCAGC GCGGAT CGTGCACGCCCTCTTTCT CGA AACAGGTCTCCGGCAG CCTC

Designed primers

53 50.8

550–800

51.7 61.7

400–480

60.7 63.5

500–520

63.3 62.1 62.1

350–430

Mycorrhiza

purified and sequenced in both forward and reverse directions to confirm identity and similarity to the sequences submitted to the National Centre for Biotechnology Information (NCBI) database. Having obtained 99 % identity to isolates with accession numbers EU379675, FJ845417, FJ845541, for H. crustuliniforme, L. bicolor and S. tomentosus, respectively; the sequences were then used to design primers for each organism. The sequences were aligned with Bioedit software (Hall 1999) while primers were designed using the NCBI primer-blast tool (http://www.ncbi.nlm.nih.gov/tools/primerblast/). The primers were synthesized by Integrated DNA Technologies considering melting temperature, noncomplementarity of the 3′ ends, the G + C contents between 40 and 60 %, absence of secondary structures, absence of cross hybridization and the length between 18 and 28 nucleotides of the oligonucleotides (van Tuinen et al. 1998). Primer specificity and efficiency The designed primers were tested on pure cultures of H. crustuliniforme, L. bicolor, and S. tomentosus to confirm their efficiency. The PCR products were separated electrophoretically and visualized on Sybersafe-stained 1 % agarose gels. Sizes of the PCR products were ∼500 to 750 bp. The designed primers were tested for specificity on a variety of fungal pure cultures obtained from UAMH, including Amphinema byssoides, Cenococcum geophilum, Hebeloma longicaudum, Lactarius affinis, Paxillus involutus, Tricholoma flavovirens, and Wilcoxina mikolae. To enhance their specificity, a touchdown PCR in a final volume of 10 μl with 0.1 μM of appropriate primers was optimized (Hecker and Roux 1996). The PCR protocol consisted of 95 °C for 2 min, 10 cycles of 94 °C for 1 min, 65 °C for 1 min, 72 °C for 1 min; 10 cycles of 94 °C for 1 min, 63 °C for 1 min, 72 °C for 1 min; 15 cycles of 94 °C for 35 s, 60 °C for 1 min, 72 °C for 2 min and a final elongation of 72 °C. The PCR products were separated electrophoretically on 1 % agarose gel and stained using a Sybersafe DNA stain at a rate of 1 μl/10 ml gel. Therefore, to determine the persistence of each inoculated ECM fungi, PCR products obtained using IT1F and ITS4 primers were used as templates. This is referred to as a nested PCR as this method helps to amplify specific sequences of DNA from a large complex mixture of DNA using a specific primer pair (Martin and Rygiewicz 2005).

Results Seedling pre-planting status Fungal inoculation treatments did not significantly affect seedling heights and root collar diameters (data not shown). In both tree species, the inoculated seedlings were ectomycorrhizal

with all of the tested fungi as confirmed by the morpho- and molecular typing. In different treatments, root colonization rates ranged from 61 to 84 % including 3–6 % of roots that were also colonized by the native, nursery-adapted, fungi identified as Thelephora americana and A. byssoides. These two fungi were also identified in 17–28 % of roots in noninoculated control seedlings.

Growth parameters Colonization rates presented in Table 3 refer to all ectomycorrhizal fungi colonizing the roots including native soil fungi. The ectomycorrhizal colonization varied from approximately 20 % in non-inoculated seedlings of both plant species to 76.6 and 87.7 % in white spruce and jack pine seedlings, respectively, inoculated with HC + LB (Table 3). The effect of inoculation on plant height was greater in jack pine compared with white spruce (P = 0.023) (Fig. 1, Table 3). In both plant species, the HC + LB treatment had the greatest effect on heights (Fig. 1). In jack pine, there was also a positive effect of the inoculation treatments on seedling RHGR, when Table 3 Growth parameters and root colonization of jack pine and white spruce seedlings at the end of the second growth season in 2010 Treatment

Jack pine CTRL HC LB ST HCLB HCST LBST HCLBST White spruce CTRL HC LB ST HCLB HCST LBST HCLBST

Height (cm)

Dry Root/shoot FMID Colonization biomass (g) dry biomass (%)

50.8a 59.5b 55.1ab 53.9 ac 59.9b 57.0b 58.4b 57.0b

102.3a 136.7b 135.8b 134.7b 137.4b 136.1b 132.b 118.3ab

0.31a 0.33a 0.32a 0.34a 0.34a 0.30a 0.31a 0.30a

0.00 25.2 24.7 24.1 25.6 24.8 22.6 13.6

46.5a

107.3ab

0.29a

0.0

20.1a

51.4bc 49.1 ac 50.6 ac 51.7bc 49.7 ac 50.3 ac 51.4bc

117.9ab 101.2a 109.5ab 122.5ab 117.1ab 110.2ab 127.8b

0.30a 0.24a 0.26a 0.30a 0.29a 0.28a 0.24a

11.5 −7.5 2.5 16.1 10.8 3.3 19.9

68.6b 67.7b 52.2c 76.6d 64.5b 59.5e 63.7be

21.5a 64.1b 76.3c 61.8b 87.7d 67.5b 68.7b 75.8b

FMID field mycorrhizal inoculation dependency (dry biomass of inoculated−dry biomass of non-inoculated/dry biomass of inoculated seedlings×100). Treatments are CTRL control-non-inoculated; LB L. bicolor; HC H. crustuliniforme; ST S. tomentosus; HCLB H. crustuliniforme and L. bicolor; HCST H. crustuliniforme and S. tomentosus; LBST L. bicolor and S. tomentosus; HCLBST H. crustuliniforme, L. bicolor, and S. tomentosus. Colonization refers to all ectomycorrhizal fungi colonizing the roots including native soil fungi

Mycorrhiza

Fig. 1 Changes in heights of inoculated and non-inoculated jack pine (PJ) and white spruce (SW) seedlings after one and two growing seasons. Means±SE (n = 20) are shown. Treatments are CTRL control-non-inoculated; LB L. bicolor; HC H. crustuliniforme; ST S. tomentosus; HCLB H.

crustuliniforme and L. bicolor; HCST H. crustuliniforme and S. tomentosus; LBST L. bicolor and S. tomentosus; HCLBST H. crustuliniforme, L. bicolor, and S. tomentosus

calculated for 2009 and 2010 (Fig. 2). In 2010, jack pine seedlings inoculated with the ECM fungi showed about 50 % to over 100 % higher RGHR than non-inoculated seedlings (CTRL) depending on the inoculation treatment. The least effective treatments in terms of RHGR increase were those of ST and HC + LB + ST (Fig. 2). With the exception of HC + LB + ST, all of the inoculation treatments significantly increased seedling dry biomass compared with control in jack pine when measured at the end of

the second growing season, resulting in FMID values of about 25 % (Table 3). There was no significant effect of inoculation on seedling dry biomass in white spruce (Table 3). There was also no significant effect of inoculation on root to shoot dry biomass ratios in jack pine and in white spruce (Table 3). In white spruce, the SVI was not significantly affected by any of the inoculation treatments (Fig. 3A). There was a general increasing trend in PVI of white spruce seedlings inoculated with mycorrhizal fungi; however, only the

Fig. 2 Relative height growth rates (RHGR) of inoculated and noninoculated jack pine (PJ) and white spruce (SW) seedlings over two growth seasons. Means (n = 20) are shown. Treatments are CTRL control-non-inoculated; LB L. bicolor; HC H. crustuliniforme; ST S.

tomentosus; HCLB H. crustuliniforme and L. bicolor; HCST H. crustuliniforme and S. tomentosus; LBST L. bicolor and S. tomentosus; HCLBST H. crustuliniforme, L. bicolor, and S. tomentosus

Mycorrhiza

White Spruce 60

1000

a

b a

a

a

800

a

a

a

3

Stem volume (cm )

50

a

a

a

ab

ab

ab

ab

PVI (cm3)

40

30

ab

600 b

b 400

20 200

10

0

0 CTRL

HC

LB

ST

HCLB

HCST

LBST HCLBST

CTRL

HC

LB

Treatment

ST

HCLB

HCST

LBST HCLBST

Treatment

Jack Pine 80

800

d

b b

60

ab

b

b

a

a

a

a

a

a

b 600

a

a

PVI (cm3)

3

Stem volume (cm )

c

a 40

20

a

400

200

0

0 CTRL

HC

LB

ST

HCLB

HCST

LBST HCLBST

CTRL

Treatment

HC

LB

ST

HCLB

HCST

LBST HCLBST

Treatment

Fig. 3 Effect of mycorrhizal inoculation on stem-volume index (SVI) (a and c) and plot-volume index (PVI) (b and d) of field-planted jack pine (a and b) and white spruce (c and d) seedlings at the end of the second growth season following planting. Means±SE (n = 20) are shown. Different letters indicate significant differences at p≤0.05 (Fisher LSD).

Treatments are CTRL control-non-inoculated; LB L. bicolor; HC H. crustuliniforme; ST S. tomentosus; HCLB H. crustuliniforme and Laccaria; HCST H. crustuliniforme and Suillus; LBST L. bicolor and S. tomentosus; HCLBST H. crustuliniforme, L. bicolor, and S. tomentosus

inoculation with HC + LB resulted in an over 100 % increase of PVI compared with CTRL (Fig. 3B). In contrast to white spruce, the SVI in jack pine increased by the inoculation treatments with the exception of ST and HC + LB + ST in which the increase was not significant at p ≤ 0.05 (Fig. 3C). The highest, over 50 %, increase over CTRL value was measured in HC seedlings (Fig. 3C). For PVI, the effect of inoculation produced the same increasing trend in jack pine as in white spruce (Fig. 3D). However, none of the inoculation treatments in jack pine produced statistically significant increases in PVI compared with CTRL (Fig. 3D).

Survival There was no overall significant effect of inoculation on seedling survival in jack pine when compared with CTRL (Fig. 4). The survival of white spruce seedlings was significantly higher (over 50 %) in ST, HC + LB, HC + ST, and HC + LB + ST inoculation treatments than in non-inoculated seedlings. The increases in survival rates for the remaining inoculation were not significantly different from CTRL at p ≤ 0.05 level (Fig. 4). Survival of white spruce was higher in doubleand triple-inoculated seedlings compared with CTRL or seedlings inoculated with a single fungus (Fig. 4).

Mycorrhiza Fig. 4 Survival rates of jack pine (PJ) and white spruce (SW) seedlings at the end of the second growth season. Vertical bars represent means (n = 20)±SE. Different letters indicate statistically significant differences between treatments at p≤0.05 (Fisher LSD). Treatments are CTRL control-non-inoculated; LB L. bicolor; HC H. crustuliniforme; ST S. tomentosus; HCLB H. crustuliniforme and L. bicolor; HCST H. crustuliniforme and S. tomentosus; LBST L. bicolor and S. tomentosus; HCLBST H. crustuliniforme, L. bicolor, and S. tomentosus

Morphology and molecular typing For both plant species, about 20 % of CTRL roots were colonized by fungi, compared with 52–87 % of roots in inoculated seedlings (Table 3). A gross morphology of 17 morphotypes was distinguished. In both plant species, mantle and Hartig net were observed. Roots were also observed to have septate runner hyphae, zoospores, and occasional vesiclelike structures. A total of 70 successful amplifications out of 150 tested roots were obtained using general fungal primers (ITS1F and ITS4). Multiple PCR fragments that barred further sequencing accounted for 15 % of amplified samples. Only the 27 samples with high DNA concentration were further sequenced to ascertain species identity. ITS sequencing produced a total of eight taxa identified to family (one), genus (two), and species (five) levels (Table 4). Five samples could not match any known sequences in the GenBank. In the examined samples, A. byssoides and T. americana were the most frequent fungal species identified (Table 4). Other identified fungi included one Ascomycetous mycorrhizal fungus, Wilcoxina sp., and the pathogenic fungus Olpidium brassicae belonging to the class Chytridiomycetes (Table 4).

Discussion Seedling inoculation resulted in higher ectomycorrhizal colonization rates compared with non-inoculated control, which had also a relatively small proportion of roots colonized by the nursery contaminant fungi identified as A. byssoides and T. americana. The effects of inoculation on height and dry biomass were greater in jack pine compared with white

spruce. The effectiveness in conferring different physiological attributes to plants by different species of ECM fungi has been recognized and attributed to plant-fungus compatibility and (or) site characteristics (Quoreshi et al. 2008). In earlier studies, L. bicolor and H. crustuliniforme were more effective in conferring salt resistance to jack pine compared with white spruce (Nguyen et al. 2006). However, S. tomentosus was reported to be more effective in conferring salt and fluoride tolerance to white spruce compared with jack pine (CalvoPolanco et al. 2009).

Table 4 Fungal taxa identified in root samples of planted seedlings Fungal taxon

Numbers of Maximum Corresponding Inoculated/ nonmatching homology E valuea inoculated sequences (%)

Thelephora americana Amphinema byssoides Sebacina sp. Suillus tomentosus Wilcoxina sp. Laccaria bicolor Olpidium brassicae Pyronemataceae Unidentified

7

99

0.0

Both

9

99

0.0

Both

1 2

90 84

0.0 3e-151

Inoculated Inoculated

1 2

99 99

0.0 0.0

Inoculated Both

1

86

0.0

Inoculated

1 5

92 -

0.0 -

Inoculated Both

a

E (expect value) of blast is a parameter that describes the number of hits one can “expect” to see by chance when searching a database of a particular size

Mycorrhiza

Mycorrhizal colonization under field conditions is influenced by factors such as soil characteristics, abundance of fungal inocula and plant diversity (Hedlund and Gormsen 2002). According to Grossnickle and Reid (1982) and Kerngahan et al. (2003), conifer seedlings could be easily colonized by a variety of fungal and bacterial endophytes as well as ectomycorrhizal fungi present in the greenhouse environment. These endophytes are endosymbionts, often bacteria or fungi, which live in the inner tissues of plants without causing any apparent disease (Wilson 1995). Therefore, in the absence of fungal-free controls, it is difficult to speculate what effect these endophytes may have on seedling physiology. This may help explain why some studies found no beneficial effects of mycorrhization (Maestre et al. 2002; Teste et al. 2004). Fungal compatibility with plants is a complex issue involving the ability of the fungus to colonize and persist in roots as well as different physiological responses that it may produce in a plant. In Populus balsamifera, mycorrhizal fungi W. mikolae and H. crustuliniforme triggered different responses in plants: W. mikolae promoted growth while H. crustuliniforme stimulated root hydraulic conductivity (Siemens and Zwiazek 2008). Furthermore, in vitro studies reported the ability of H. crustuliniforme and L. bicolor to tolerate saline-alkaline conditions found in composite tailings that are generated during oil sand extraction (Kernaghan et al. 2002). A more recent study has shown fungal genetic variability in tolerance to saline conditions (Campagnac and Khasa 2013). Due to the direct impact on the applications of mycorrhizas, a variety of plant species have been tested with different ECM fungi. Another approach involves combining several fungal species in the inoculum applied to plants. Parlade and Alvarez (1993) studied the co-inoculation of Douglas fir with four pairs of ECM fungi. Their study revealed L. bicolor to be an aggressive root colonizer and the ability of all three types of fungi to significantly increase total plant biomass. Similarly, our study showed significant differences in biomass of jack pine between control and plants inoculated with ECM fungi with the exception of HC + LB + ST treatment. Since HC + LB + ST treatment was at least as effective in height growth stimulation in jack pine as other mycorrhizal treatments, the apparent lack of a significant effect on dry biomass of jack pine seedlings may suggest greater energy demands for the maintenance of fungal hyphae in HC + LB + ST treatment. The results of our study did not show a clear and consistent advantage of combining several species in one inoculum. In fact, for some of the measured parameters, fungal combinations were less effective than a single species. Although little understood, the combination of fungal species may result in synergistic plant fitness responses (Bois et al. 2006). Clearly, the issue of single versus multiple fungal inocula applications warrants further investigation.

In the present study, inoculated white spruce showed generally higher survival rates compared to jack pine and contrary to jack pine, survival of white spruce seedlings increased significantly by inoculation with HC + LB, HC + ST and HC + LB + ST, enforcing the point that the effects of mycorhizal fungi on plant responses which affect survival will vary between the different fungal and plant species (Nguyen et al. 2006; Calvo-Polanco et al. 2009; b). Additionally, the benefits of a particular mycorrhizal fungus may be site-dependent and vary with environmental factors. Inoculation treatments increased stem volume in jack pine, but not in white spruce. There also appeared to be some effect of inoculation on PVI in both tree species, but only HC + LB inoculation in white spruce produced a statistically significant increase (Fig. 3). Other studies have also reported increased PVI of mycorrhizal plants (Gagné et al. 2006; Quoreshi et al. 2008) including white spruce seedlings (Gagné et al. 2006). The results confirm potential beneficial effect of mycorrhizas on postplanting performance of seedlings. In the present study, we found 17 types of morphotypes in both plant species. Similar numbers of morphotypes were also reported in other studies (Visser 1995; Wurzburger et al. 2001; Teste et al. 2012). Different ECM fungi colonize host root tips at various plant stages, leading to the categories of “early"-, “multi”- and “late”-stage fungi (Visser 1995). The fungi identified in the present study belong to the early- and multi-stage fungal species and were found on young and mature root tips. A. byssoides and T. americana were the most frequent fungal species present on the site. One Ascomycetous mycorrhizal fungus, Wilcoxina sp., and a pathogenic fungus O. brassicae were also detected. These results are congruent with the previous studies reporting dominance of A. byssoides, C. geophilum, and Thelephora sp. in outplanted seedlings (Gagné et al. 2006; Quoreshi et al. 2008; Teste et al. 2012) and suggest the aggressive colonizing nature of these fungal species. It is still uncertain as to whether these fungi are field or tree nursery root colonizers (Flykt et al. 2008; Teste et al. 2012). Unfortunately, we did not systematically collect the data through morpho- and molecular typing that would allow determining the frequency or amount of root colonization by different fungal species in inoculated seedlings and noninoculated controls. Such data would help understand better the physiological responses of plants to each fungal strain following planting. The benefits of mycorrhizal inoculation are affected by persistence of inoculated strains in plants (Grove and Le Tacon 1993). Of the three inoculated fungal species, H. crustuliniforme was the only one that was not detected on plant roots after two growing seasons. The colonization of the host root system by resident ECM fungi after planting is an expected phenomenon and H. crustuliniforme was previously reported to be replaced over time by other mycorrhizal fungi (Bledsoe et al. 1982; Menkis et al. 2007). However, it is

Mycorrhiza

interesting that we found growth responses in plants despite the absence of H. crustuliniforme in the roots when examined after two growing seasons. This means that early advantage of inoculation observed in the greenhouse (data not shown) was maintained in the field. Repeated examination of roots and identification of mycorrhizal fungi over time following planting would be required to determine the mycorrhizal dynamics in both conifer species.

Conclusions In summary, we demonstrated increased growth in jack pine and increased postplanting survival in white spruce seedlings inoculated with ECM fungi by a tree nursery and planted in oil sands reclamation areas. We suggest that ECM inoculation of conifer seedlings by tree nurseries should be considered to improve postplanting performance in oil sands reclamation sites. However, a selection of mycorrhizal fungal species or their combinations may be required to achieve specific objectives. More studies are required to gain understanding of the changes in mycorrhizal dynamics that take place in roots of outplanted seedling inoculated with mycorrhizal fungi as well as their interactions with the native soil microbial community. Acknowledgments Financial supports for this project came from Suncor Energy Inc. and Symbiotech Research Inc. We express our gratitude to Gataen Daigle of Department of Mathematics and Statistics, Université Laval for statistical consultation as well as Francis Salifu of Suncor Energy and Charles Greer of the National Research Council of Canada for support and advice. We also thank the anonymous reviewers for their valuable comments.

References Agerer R (1987–2001) Descriptions of ectomycorrhizae. Einhorn-Verlag Eduard Dietenberger GmbH., Schwäbisch Gmund, Munich. Bledsoe CC, TennysonK, Lopushinsky W (1982) Survival and growth of outplanted Douglas fir seedlings inoculated with mycorrhizal fungi. Can J For Res 12:720–723 Bois G, Piché Y, Fung MYP, Khasa DP (2005) Mycorrhizal inoculum potentials of pure reclamation materials and revegetated tailing sands from the Canadian oil sand industry. Mycorrhiza 15:149–158 Bois G, Bigras FJ, Bertrand J, Piché Y, Fung MYP, Khasa DP (2006) Ectomycorrhizal fungi affect the physiological responses of Picea glauca and Pinus banksiana seedlings exposed to an NaCl gradient. Tree Physiol 26:1185–1196 Brundrett MC (1991) Mycorrhizas in natural ecosystems. In: Macfayden A, Begon M, Fitter AH (eds) Advances in ecological research. Academic, London, UK, pp 171–313 Brundrett M, Bougher N, Dell B, Grove T, Malajczuk N (1996) Working with mycorrhizas in forestry and agriculture. ACIAR Monograph 32, Canberra, Australia Calvo Polanco M, Zwiazek JJ, Jones MD, MacKinnon MD (2009) Effects of NaCl on responses of ectomycorrhizal black spruce (Picea mariana), white spruce (Picea glauca) and jack pine (Pinus banksiana) to fluoride. Physiol Plant 135:51–61

Calvo-Polanco M, Zwiazek JJ (2011) Role of osmotic stress in ion accumulation and physiological responses of mycorrhizal white spruce (Picea mariana) and jack pine (Pinus banksiana) seedlings exposed to soil fluoride and NaCl. Acta Physiol Plant 33: 1365–1373 Calvo-Polanco M, Jones MD, Zwiazek JJ (2009) Effects of pH on NaCl resistance of American elm (Ulmus americana) seedlings inoculated with Hebeloma crustuliniforme and Laccaria bicolor. Acta Physiol Plant 31:515–522 Campagnac E, Khasa DP (2013) Relationship between genetic variability in Rhizophagus irregularis and tolerance to saline conditions. Mycorrhiza. doi:10.1007/s00572-013-0517-8 Danielson RM, Visser S (1989) Host response to inoculation and behavior of induced and indigenous ectomycorrhizal fungi of jack pine grown on oil-sands tailings. Can J For Res 19:1412–1421 Flykt E, Timonen S, Pennanen T (2008) Variation of ectomycorrhizal colonisation in Norway spruce seedlings in Finnish forest nurseries. Silva Fenn 42:571–585 Franklin JA, Renault S, Croser C, Zwiazek JJ, MacKinnon MD (2002) Jack pine growth and elemental composition are affected by saline tailings water. J Environ Qual 31:648–653 Gagné A, Jany JL, Bousquet J, Khasa DP (2006) Ectomycorrhizal fungal communities of nursery inoculated seedlings outplanted on clearcut sites in northern Alberta. Can J For Res 36:1684–1694 Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for Basidiomycetes-application to the identification of mycorrhizas and rusts. Mol Ecol 2:113–118 Gehring CA, Whitham TG (1994) Comparisons of ectomycorrhizae on pinyon pines (Pinus edulis; Pinaceae) across extremes of soil type and herbivory. Amer J Bot 81:1509–1516 Government of Alberta (2009) Environmental management of Alberta’s oil sands. Alberta Environment, Edmonton, Alberta, Canada, ISBN: 978-07785-7677-8 Grossnickle SC, Reid CP (1982) The use of ectomycorrhizal conifer seedlings in the revegetation of a high-elevation mine site. Can J For Res 12:354–361 Grove TS, Le Tacon F (1993) Mycorrhiza in plantation forestry. Adv Plant Pathol 23:191–227 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Series 41:95–98 Hecker KH, Roux KH (1996) High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. BioTechniques 20:478–485 Hedlund K, Gormsen D (2002) Mycorrhizal colonization of plants in setaside agricultural land. Appl Soil Ecol 19:71–78 Henrion B, Di Battista C, Bouchard D, Vairelles D, Thompson BD, Le Tacon F, Martin F (1994) Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery-grown Douglasfir by PCR of the rDNA intergenic spacer. Mol Ecol 3:571–580 Kamaluddin M, Zwiazek JJ (2002) Naphthenic acids inhibit root water transport, gas exchange and leaf growth in aspen (Populus tremuloides) seedlings. Tree Physiol 22:1265–1270 Kernaghan G, Hambling B, Fung M, Khasa DP (2002) In vitro selection of boreal ectomycorrhizal fungi for use in reclamation of salinealkaline habitats. Restoration Ecol 10:43–51 Kernaghan G, Sigler L, Khasa DP (2003) Mycorrhizal and root endophytic fungi of containerized Picea glauca seedlings assessed by rDNA sequence analysis. Microbial Ecol 45:128–136 Maestre FT, Bautista S, Cortina J, Diaz G, Honrubia M, Vallejo R (2002) Microsite and mycorrhizal inoculum effects on the establishment of Quercus coccifera in semi-arid degraded steppe. Ecol Eng 19:289– 295 Martin KJ, Rygiewicz PT (2005) Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiol 5:28–39

Mycorrhiza Marx D (1990) Soil-pH and nitrogen influence isolithus ectomycorrhizal development and growth of loblolly pine seedlings. For Sci 36: 224–245 Marx DH, Ruehle JL, Cordell CE (1991) Methods for studying nursery and field response of trees to specific ectomycorrhiza. In: Norris JR, Read D, Varma AK (eds) Techniques for mycorrhizal research. Academic, San Diego, pp 384–411 Menkis A, Vasiliauskas R, Taylor AFS, Stenlid J, Finlay R (2007) Afforestation of abandoned farmland with conifer seedlings with three ectomycorrhizal fungi-impact on plant performance and ectomycorrhizal community. Mycorrhiza 17:337–348 Molina R, Massicotte H, Trappe JM (1992) Specificity phenomena in mycorrhizal symbiosis: community-ecological consequences and practical implications. Chapman and Hall, New York, USA, pp 357–423 Muhsin TM, Zwiazek JJ (2002) Colonization with Hebeloma crustuliniforme increases water conductance and limits shoot sodium uptake in white spruce (Picea glauca) seedlings. Plant Soil 238:217–225 Nguyen H, Calvo Polanco M, Zwiazek JJ (2006) Gas exchange and growth responses of ectomycorrhizal Picea mariana, Picea glauca, and Pinus banksiana seedlings to NaCl and Na2SO4. Plant Biol 8: 646–652 Ortega U, Dunabeitia M, Menendez S, Gonzalez-Murua C, Majada J (2004) Effectiveness of mycorrhizal inoculation in the nursery on growth and water relation of Pinus radiata in different water regimes. Tree Physiol 24:64–73 OSVRC (1998) Guidelines for reclamation to forest vegetation in the Athabasca oil sands region. Report #ESD/LM/99-1. Oil Sands Vegetation Reclamation Committee. Alberta Environmental Protection, Edmonton, Alberta, Canada. Parlade J, Alvarez IF (1993) Coinoculation of aseptically grown Douglas fir with pairs of ectomycorrhizal fungi. Mycorrhiza 3:93–96 Parlade J, Luque J, Pera J, Ricon AM (2004) Field performance of Pinus pinea and P. halepensis seedlings inoculated with Rhizopogon spp. and outplanted in formerly arable land. Ann For Sc 61:507–514 Pera J, Alvarez IF, Rincon A, Parlardé J (1999) Field performance in northern Spain of Douglas-fir seedlings inoculated with ectomycorrhizal fungi. Mycorrhiza 9:77–84 Plenchette C, Fortin JA, Furlan V (1983) Growth responses of several plant species to mycorrhizae in a soil of moderate P-fertility. I. Mycorrhizal dependency under field conditions. Plant Soil 70:199–209 Pontailler JY, Ceulemans R, Guittet J, Mau F (1997) Linear and nonlinear functions of volume index to estimate woody biomass in high density young poplar stands. Ann Sci For 54:335–345 Quoreshi A, Piché Y, Khasa D (2008) Field performance of conifer and hardwood species 5 years after nursery inoculation in the Canadian Prairie provinces. New For 35:235–253

Renault S, Zwiazek JJ, Fung M, Tuttle S (2000) Germination, growth and gas exchange of selected boreal forest seedlings in soil containing oil sands tailings. Environ Pollut 107:357–365 SAS Institute Inc. (2008) SAS/STAT® 9.2 Users Guide. Cary, NC: SAS Institute Inc., Cary, NC, USA. Sheldrick BH, Wang C (1993) Particle-size distribution. In: Carter MR (ed) Soil sampling and methods of analysis. Canadian Society of Soil Science, Lewis, Ann Arbor, MI, pp 499–511 Siemens JA, Zwiazek JJ (2008) Root hydraulic properties and growth of balsam poplar (Populus balsamifera) mycorrhizal with Hebeloma crustuliniforme and Wilcoxina mikolae var. mikolae. Mycorrhiza 18: 393–401 Siemens JA, Zwiazek JJ (2011) Hebeloma crustuliniforme modifies root hydraulic responses of trembling aspen (Populus tremuloides) seedlings to changes in external pH. Plant Soil 345:247–256 Teste FP, Schmidt MG, Berch SM, Bulmer C, Egger K (2004) Effects of ectomycorrhizal inoculation on survival and growth of interior Douglas-fir seedlings on reforestation sites and partially rehabilitated landings. Can J For Res 34:2074–2088 Teste FP, Lieffers VJ, Strelkov SE (2012) Ectomycorrhizal community responses to intensive forest management: thinning alters impact of fertilization. Plant Soil 360:333–347 van Tuinen D, Jacquot E, Zhao B, Gollotte A, Gianinazzi-Pearson V (1998) Characterization of root colonization profiles by a microcosm community of arbuscular mycorrhizal fungi using 25S rDNAtargeted nested PCR. Mol Ecol 7:879–887 Visser S (1995) Ectomycorrhizal fungal succession in jack pine stands following wildfire. New Phytol 129:389–401 Voroney PC, Brookes P, Beyaert RP (2008) Soil microbial biomass C, N, P, and S. In: Carter MR, Gregorich EG (eds) Soil sampling and methods of analysis, 2nd edn. CRC, Boca Raton, FL, pp 637– 651 White TJ, Bruns TD, Lee S, Taylor DJ (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Snisky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic, New York, pp 315– 322 Wilson D (1995) Endophyte—the evolution of a term and clarification of its use and definition. Oikos 73:274–276 Wurzburger N, Bidartondo MI, Bledsoe CS (2001) Characterization of Pinus ectomycorrhizas from mixed conifer and pygmy forests using morphotyping and molecular methods. Can J Bot 78:1211– 1216 Yi H, Calvo Polanco M, MacKinnon MD, Zwiazek JJ (2008) Responses of ectomycorrhizal Populus tremuloides and Betula papyrifera seedlings to salinity. Environ Exp Bot 62:603–622