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Chapitre 5 Differential effect of three ectomycorrhizal fungi on the physiological response of Picea glauca and Pinus banksiana seedlings exposed to a NaCl gradient

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Les resultats de l’expérience présentée dans ce chapitre ont été soumis pour publication dans un journal international avec comité de lecture (Bois, Bigras, Bertrand, Piché, Fung & Khasa 2005b). L’ensemble des mesures écophysiologiques fut réalisé avec la collaboration du Dr Francine Bigras (Ressources naturelles Canada, CFL, Québec). Les analyses biochimiques ont été réalisées en collaboration avec Annick Bertrand (Agriculture Canada, Québec). Les professeurs Damase Khasa et Yves Piché ont fourni l’encadrement scientifique. Martin Fung est intervenu pour la définition des besoins de l’industrie et fut la personne ressource au sein du bureau des affaires environnementales à Syncrude Canada Ltd. L’expérience 4, qui suit, a été conçue à partir des résultats obtenus dans les expériences 2 (Chapitre 3) et 3 (Chapitre 4). Ainsi, les travaux présentés dans ce chapitre portent sur les combinaisons des trois champignons basidiomycètes de l’expérience 2 avec les hôtes Pinus banksiana Lamb. et Picea glauca (Moench) Voss. Intégrant la combinaison Laccaria bicolor (Maire) Orton UAMH 8232 et P. banksiana, l’expérience 4 permet de rendre compte de la réponse physiologique de cette combinaison dans d’autres conditions d’inoculation et de croissance. À la différence de l’expérience 3, les semis ont été produits à Québec dans les serres du centre de foresterie des Laurentides (CFL). Ils ont ainsi été inoculés, fertilisés et irrigués afin d’obtenir les meilleurs résultats de mycorhization (Annexes II et III).

Une experience en serre a été réalisée pour évaluer l’effet de la mycorhization contrôlée de semis utilisés pour la végétalisation de rejets sableux sodiques issus de l’exploitation des sables bitumineux dans le Nord-Est de l’Alberta (Canada). Des semis d’épinette blanche (Picea glauca (Moench) Voss) et de pin gris (Pinus banksiana Lamb.) ont été inoculés avec trois champignons ECM, et la réponse physiologique de ces associations à un gradient de concentrations de NaCl (solutions de traitements de 0, 50, 100 et 200 mM de NaCl) a été déterminée au cours des quatre semaines de traitement. Les champignons ECM, Hebeloma crustuliniforme (Bull) Quél. UAMH 5247, Laccaria bicolor (Maire) Orton UAMH 8232 et un isolat de Suillus tomentosus (Kauff.) Sing., Snell and Dick provenant d’un terrain sodique ont été sélectionnés pour leur résistance in vitro à l’excès de Na+ et Cl-. La réponse des plantes à l’excès de NaCl a été caractérisée à l’aide de : (i.) l’accumulation et la répartition du Na+, (ii.) la fluorescence de la chlorophylle a, (iii.) la croissance du plant, (iv.) l’hydratation des tissus et (v.) l’accumulation d’osmolytes organiques. Les semis de pin gris se sont montrés plus sensibles que les semis d’épinette blanche à l’augmentation des concentrations en Na+ et en Cl-. Les deux espèces ont présenté une croissance réduite avec l’augmentation des concentrations de NaCl ainsi qu’une accumulation croissante d’osmolytes organiques et de Na+. Les semis d’épinette blanche inoculés avec l’isolat de S. tomentosus ont présenté la meilleure réponse de croissance à toutes les concentrations de NaCl testées. Bien que les semis de pin gris inoculés avec L. bicolor ou l’isolat de S. tomentosus aient montré la plus forte croissance avec les traitements de 50 et 100 mM de NaCl, les deux champignons ont augmenté le stress photochimique et la déshydratation des tissus de leur hôte à 200 mM de NaCl. À cette concentration, les semis de pin gris inoculés avec H. crustuliniforme ont montré la plus forte résistance.

A greenhouse experiment was set up to test the effect of ectomycorrhizal (ECM) inoculation on tree seedlings to be used in the revegetation of salt-affected tailing sands resulting from the exploitation of oil sand in northeastern Alberta (Canada). White spruce (Picea glauca (Moench) Voss) and jack pine (Pinus banksiana L.) seedlings were inoculated with three ECM fungi, and the physiological response of these associations to a gradient of NaCl concentration (0, 50, 100 and 200 mM) was assessed over four weeks. The ECM fungi Hebeloma crustuliniforme UAMH 5247, Laccaria bicolor UAMH 8232, and a Suillus tomentosus (Kauff.) Sing., Snell and Dick isolate from a sodic site, were selected for their in vitro resistance to excess Na+ and Cl-. The plant response to excess NaCl was characterized by: (i) Na+ accumulation and allocation, (ii) chlorophyll a fluorescence, (iii) growth, (iv) water content, and (v) organic osmolyte accumulation. Jack pine seedlings were more sensitive than white spruce seedlings to increasing Na+ and Cl- concentrations. Both species showed decreasing biomass accumulation, and increasing contents of organic osmotica and Na with increasing NaCl treatment. White spruce seedlings inoculated with the S. tomentosus isolate showed the best growth response over all NaCl concentrations assessed. Although jack pine seedlings inoculated with either L. bicolor or the S. tomentosus isolate exhibited the highest growth in the 50 and 100 mM treatments, both fungi increased photochemical stress and dehydration of their hosts in the 200 mM NaCl treatment. At this concentration, H. crustuliniforme inoculated jack pine seedlings showed a higher resistance.

The ability of a given plant to cope with fluctuating or permanent soil salinity or sodicity is dependent upon certain factors: the continued capacity to absorb water, the ability to cope with excess Na+, and the capacity to maintain ionic homeostasis with respect to essential ions (Levitt 1980, Cheeseman 1988, Munns 1993, 2005, Orcutt & Nilsen 2000, Kozlowski 1997, Hasegawa et al. 2000, Zhu 2001, 2002). Halophytes and adaptable glycophytes generally rely on osmotic adjustment and ionic compartmentalization to exclude and/or partition excess Na+ (Yeo 1998, Hasegawa et al. 2000). To withstand the physiological drought caused by high Na+ concentration within the soil solution, plants have to either counterbalance osmotic deficiencies with organic osmotica, or to accumulate Na+ or other ions in excess, as a low cost inorganic osmoticum (Yeo 1983, Niu et al. 1995, 1997, Bohnert & Shen 1999). In the latter case, the negative specific ion effects of Na+ and Cl- may take place once a critical concentration threshold is exceeded. Sodium and Cl- toxicity affects the plant’s phytohormonal balance, alters the enzymatic activity and the cell membrane integrity, causes protein and nucleic metabolism dysfunction, and reduces net photosynthesis and gaseous exchange (Munns 1993, 2005, Kozlowski 1997, Neumann 1997, Hasegawa et al. 2000, Mansour & Salama 2004).

Coniferous trees of the boreal forest generally form ectomycorrhizas with a range of fungi from the basidiomycetes and ascomycetes (Read et al. 2004). The resulting mutualistic symbiosis is essential for the completion of the life cycle of both partners in natural conditions. In return for enhanced mineral nutrition and survival (Smith & Read 1997), the host plant channels from 5 to 30% of its net photosynthetic production to its mycobionts (Söderstörm 1992). In the ectomycorrhizal (ECM) association, the fungal partner ensheathes the tree’s short roots and develops a Hartig net between the cortical cells; emanating hyphae explore the soil matrix for nutrients and water (Smith & Read 1997). The fungus being at the interface between the plant and the edaphic environment can reduce stresses such as nutrient deficiency (Marschner & Dell 1994, Smith et al. 1994, Read et al. 2004), drought (Dosskey et al. 1991, Lamhamedi et al. 1992), and heavy metal pollution (Kottke 1992, Jentschke & Goldbold 2000). However, little is known about the salt tolerance of the ECM association. In theory, the identification of salt tolerant strains of ECM fungi could help enhance tree survival and growth in saline and sodic environments. In an axenic experiment, Chen et al. (2001a) showed that, of 18 isolates of Pisolithus spp. from Australia tested, most were resistant to NaCl and Na2SO4 treatments of 200 mM. In a similar experiment, Kernaghan et al. (2002) showed that different ECM fungi isolated from the boreal forest, exhibited different resistance levels to a range of concentrations of various salts. The two most resistant isolates were the basidiomycetes Hebeloma crustuliniforme (Bull) Quel. UAMH 5247 and Laccaria bicolor Maire (Orton) UAMH 8232. Hebeloma crustuliniforme has also been shown to increase water conductance and to limit accumulation of Na+ in shoots of white spruce (Picea glauca (Moench) Voss) seedlings grown in the presence of 25 mM NaCl (Mushin & Zwiazek 2002). Furthermore, biomass accumulation and net photosynthesis of jack pine (Pinus banksiana Lamb.) colonized by L. bicolor was greater than that of non-inoculated controls exposed to excess NaCl (Bois et al. 2005c). In addition, a closely related species, Laccaria laccata (Scop.: Fr.) Cooke, has been shown to improve the salt stress tolerance of loblolly pine (Pinus taeda L.) (Dixon et al. 1993).

In the Athabasca region of northeastern Alberta (Canada), oil companies have cleared thousands of hectares of boreal forest, removed the overlying organic (‘topsoil’) and sedimentary layers (overburden), and excavated the oil sand ores. The emptied pits are filled with a mix of water and sand tailings issued from the oil extraction process and covered with the above mentioned spoil (Li & Fung 1998, Fung & Macyk 2000). The resulting reconstructed soils may be highly saline, alkaline and often sodic, and also contain oil residues (Li & Fung 1998, Fung & Macyk 2000). These substrates also lack certain components of the soil microbial community essential for natural reforestation (Danielson et al. 1983b, Bois et al. 2005d). The stabilized sand tailings amended with overburden and peat are revegetated with indigenous herbaceous, shrub and tree species (Fung & Macyk 2000). Jack pine and white spruce are the two main coniferous tree species used.

In a previous experiment, we assessed the influence of inoculation of L. bicolor UAMH 8232 on the growth of jack pine seedlings during the nursery phase, and its effect on salt resistance after exposure to excess NaCl as would occur after transplantation in a salt-affected reconstructed soil. Laccaria bicolor positively influenced the physiology of jack pine seedlings exposed to excess NaCl below a threshold concentration of toxicity lying between 100 and 300 mM NaCl (Bois et al. 2005c). However, the fungus increased Na and Cl content in shoot tissues and increased photochemical disturbance above this threshold. Moreover, using axenic culture conditions, we also showed that L. bicolor UAMH 8232 was more sensitive than isolates of Phialocephala sp., Hymenoscyphus sp., H. crustuliniforme, Suillus tomentosus (Kauff.) Sing., Snell and Dick to a range of NaCl concentrations (0 – 300 mM NaCl) (Bois et al. 2005a). To identify more reliable ECM fungal species, the present experiment sought to compare the physiological responses of jack pine and white spruce seedlings inoculated either with H. crustuliniforme, L. bicolor or a Suillus tomentosus (isolate from a sodic reconstructed soil (Bois et al. 2005a)), to a range of NaCl treatments. The combination of jack pine and L. bicolor was included in this experiment to assess the physiological response of the host in different growth and NaCl exposure conditions and to contrast the response of the other plant-fungus combinations. The first objective was to compare the growth response of inoculated seedlings since that parameter is used to assess nursery stock quality. The growth parameters are also good indicators of short-term stress resistance/tolerance after transplantation. As certain ECM fungi show a degree of host specificity (Molina et al. 1992), the response of different host plants inoculated with the same fungus may differ. Depending on the host species, the mycobiont conveying the greatest degree of resistance to an excess of NaCl may differ and the strategies of resistance may not be the same. The second objective was to investigate osmotic adjustment patterns and allocations of some resources, and to assess the efficiency of the photochemical apparatus. This will help to unravel the effect of each mycobiont on it’s host resistance mechanisms. Finally, soil salinity (electrical conductivity (EC)) and sodicity (sodium absorption ratio (SAR)) indicators were used to relate the physiological response to in situ chemical conditions -Na+ dispersion effects on soil colloids set apart-.

The L. bicolor UAMH 8232 inoculum was produced in batch culture in 1 l Erlenmeyer flasks containing 100 ml of modified Melin-Nokran (MMN) medium (Marx 1969). The H. crustuliniforme UAMH 5247 and S. tomentosus solid inocula were grown in 500 ml Erlenmeyer flasks containing 250 ml sterile Peat:Vermiculite (P:V) (1:28, v:v) amended with 125 ml of MMN. Cultures were incubated for 28 d in the dark at 23ºC. Prior to the present experiment, identification of the three fungal species was performed by sequencing the ITS region of the rDNA (obtained from pure mycelium) following the method outlined by Bois et al. (2005d). The sequences were then subjected to the GenBank (http://www.ncbi.nlm.nih.gov) sequence homologies search engine.

Seeds of jack pine and white spruce were pre-germinated under greenhouse conditions (30-40% RH, 8/16 h night/day, 18/23ºC night/day, photon flux density (PFD) of 100-150 μmol m-2 s-1) in trays containing Turface® MVP (ProfileTM, Buffalo Grove, IL, USA). To favor short root development and ectomycotrophy, seedlings of both species were grown in the same trays for six weeks without fertilization prior to inoculation (Guérin-Laguette personal communication). The seedlings were subsequently transferred to IPL 15-320 plastic containers (IPL, St-Damien, Québec, Canada). Prior to transplantation, each cavity was half filled with a growth substrate consisting of a Sand:Turface®:Peat:Perlite (S:T:P:Pe) (16:16:1.2:1.75, v:v:v:v) mix. For the L. bicolor treatment, roots of seedlings were dipped into a suspension (in sterile distilled H2O) of rinsed (to remove any trace of the MMN medium) and chopped hyphae. Seedlings were placed singly in the cavities and the roots covered with 20 ml of sterile P:V (see above) amended with 5 ml of the suspension before filling the cavity with further growth substrate (Guérin-Laguette personal communication). For seedlings inoculated with H. crustuliniforme and S. tomentosus, the sterile P:V (see above) was replaced by 20 ml of solid inoculum. Control seedlings received 20 ml of sterile P:V (see above). The seedlings were grown for 21 weeks under the greenhouse conditions described previously. The seedlings were watered every two days and fertilized weekly with 20-8-20r (Plant Prod Québec, Laval, Québec, Canada) (total N applied: 46 mg per cavity). At the end of this period, fertilization was stopped and dormancy was induced over 17 d by reducing the photoperiod (16/8 h night/day). During the last seven days, seedlings were placed in the dark in a cold room at 4ºC for the night periods. Seedlings were held dormant for six weeks in the dark at 4ºC prior to the NaCl treatment application.

The seedling height was measured, and individuals lying between the second and third quartiles were selected for the experiment and transplanted into 1 l pots containing the S:T:P:Pe substrate mix. To preserve the integrity of the mycelial network and limit transplantation shock, care was taken to keep the root plugs intact. To break dormancy, seedlings were returned to the greenhouse and exposed to a long photoperiod (30-40% RH, 6/18 h night/day, 18/23ºC night/day, PFD of 100-150 μmol m-2 s-1). During the first two weeks following transplantation, seedlings were fertigated weekly by immersion of the pots (15 min) in a solution of 20-8-20r (0.5 g l-1). During the subsequent four weeks, NaCl was applied weekly with the fertigating solution to obtain and maintain concentrations in the growth substrate of either 0, 50, 100 or 200 mM. To limit fluctuations in NaCl concentration, direct evaporation from the growth substrate was reduced by a plastic mulch. The choice for short-term exposure to these levels of NaCl application was determined based on Lichtenthaler’s (1996) views of the plant stress concept: when the threshold of stress-tolerance or stress-resistance has been passed, a short-term high-level stress can induce the same damage as a long-term low-level stress. Glycophytes tend to be sensitive to salt stress in NaCl concentration close to 50 mM (Orcutt & Nilsen 2000) and, as described above, jack pine seedlings were shown to reach a threshold of resistance between 100 and 300 mM NaCl (Bois et al. 2005c).

At the time of harvest, measurements of the chlorophyll a fluorescence (ChFl) of photosystem II (PSII) were performed on five needles (still attached) from growing (produced after dormancy) and mature (produced prior to dormancy) sections of stem, using a portable fluorometer (model PAM-2000, Heinz Walz GmbH, Effeltrich, Germany). The effective quantum yield (ΦPSII = (F m’-F t) / F m') was evaluated on light-adapted (steady state) needles at 23ºC in a ventilated room. The actual fluorescence (F t) induced by an actinic red light illumination (photosynthetic active radiation (PAR) of 340-350 μmol m-2 s-1) and the light-adapted maximal fluorescence (F m') induced by a saturating pulse (0.8 s) of light (PAR of 2000 μmol m-2 s-1) were recorded. Seedlings were subsequently held in a dark ventilated room for 1 hour prior to measuring dark-adapted ChFl on growing and mature needles. The dark-adapted minimal fluorescence (F 0) was obtained using a modulated light of low intensity (PAR less than 1 μmol m-2 s-1). A saturating pulse (0.8 s) of light was applied to obtain the dark-adapted maximal fluorescence (F m). The dark-adapted variable fluorescence (F v = F m - F 0) was calculated and used to obtain the F v/F m ratio.

Following ChFl measurements, shoots were harvested and the fresh mass (FM) determined. Roots were rinsed under running water to remove any adhering growth substrate and the FM determined. Weighed sub-samples of at least 300 viable root tips were collected to evaluate the level of ECM fungal colonization for each seedling. Percentage colonization was determined as the proportion of mycorrhizal root tips to the total number of short root tips. Mycorrhizas were sorted into different morphotypes using criteria described by Agerer (1999). To confirm species identification, molecular typing (ITS-RFLP) was done using the method outlined in Bois et al. (2005d). A 0.3 g subsample of growing shoot and root tissues were collected and stored at -20ºC prior to biochemical analyses (see below). Shoots and the remaining roots were oven dried (65ºC ± 2, 72 h) and the dry mass (DM) determined. The percentage of water in shoot and root tissues at harvest was calculated as (FM – DM) / FM.

After harvest, the electrical conductivity (EC), the sodium absorption ratio (SAR) and the pHH2O of the growth medium were measured to insure the reliability of the NaCl treatments. The EC and SAR ( ) were determined as indicators of the soil solution characteristics and were used in this experiment to relate artificial conditions to revegetation site characteristics. These parameters were measured on a 1:5 soil:water (w:v) extract (EC1:5 and SAR1:5) which is a more convenient method than on saturated paste extract (ECe and SARe). As only the chemical conditions of saline or sodic soils were intended to be reproduced, this experiment used an almost inert artificial substrate comparable to that of semi-hydroponic systems.

The shoot and root tissues (0.3 g) were ground separately in liquid N and compatible osmolytes extracted by adding a 6 ml aliquot of a methanol:chloroform:water (12:5:3, v:v:v) solution. The samples were incubated at 65ºC (30 min) to halt enzymatic activity. Tubes were centrifuged (13 000 x g) for 10 min and 1 ml of supernatant was collected. To induce phase separation, a 0.250 ml aliquot of water was added to the extract. After shaking, the tubes were centrifuged (13 000 x g) for 10 min and the aqueous phase collected. A 1 ml sub-sample was used for proline determination and 1 ml was evaporated to dryness on a rotary evaporator, re-solubilized in 1 ml of water, and centrifuged (13 000 x g) for 3 min prior to HPLC analysis. The HPLC analytic system was controlled by WATERS Millennium32 software (WATERS, Milford, MA, USA) and was composed of a Model 515 pump and a Model 717plus autosampler. Stachyose, raffinose, sucrose and fructose were separated on a WATERS Sugar-Pak column (6.5 × 300 mm) eluted isocratically with EDTA (Na+, Ca2+, 50 mg l-1) at 85ºC (flow rate of 0.5 ml min-1) and detected on a refractive index detector (Waters, Model 2410). Pinitol, glucose, glycerol and mannitol were separated on a Bio-Rad Aminex HPX-87P column (7.8 × 300 mm) eluted isocratically with water at 85ºC and detected on a refractive index detector (Waters, Model 2410). Carbohydrate peaks were identified and concentrations determined by comparison with standards.

For proline determination, a 500 μl aliquot of the 1 ml subsample was mixed with 300 μl of a ninhydrine solution (0.125 g ninhydrine in 5 ml of a solution of 6 M H3PO4:glacial CH3COOH (2:3, v:v)) and 200 μl glacial CH3COOH (Paquin & Lechasseur 1979). Each sample was thoroughly mixed, incubated at 100ºC for 45 min, cooled, and a 800 μl aliquot of toluene added. After 45 min the optical density (OD) of the upper phase (toluene) was assessed by spectrophotometry at 515 nm. The proline content was calculated from the regression curve of the OD obtained for a range (0-10 μg) of standard solutions of pure proline.

All dried shoot and root tissues were ground and the K, Ca and Na content analyzed following the methods outlined by Kalra & Maynard (1992).

The experiment consisted of a factorial design comprising three completely randomized blocks. Each block comprised 32 experimental units (a pot) representing three factors in combination: (i) the host plant species (two levels), (ii) the inoculation treatments (four levels), and (iii) the NaCl treatments (four levels). A three-way ANOVA was done to analyze the physiological responses of the seedlings using the procedure PROC GLM (SAS system, The SAS Institute, Cary, NC, USA). Main effects of the inoculation and the NaCl treatments and their interaction were assessed using the following model: yijkl = µ + τi + βj + αk + (τβ) ij + (τα) ik + (βα) jk + (τβα) ijk + δl + εijkl (where yijkl is the response, µ is the mean value of the response, τi is the effect of the ith level of the host species, βj is the effect of the jth level of the inoculation treatment, αk is the effect of the kth level of the NaCl treatment, (τβ) ij , (τα) ik , (βα) jk and (τβα) ijk are interactions between the three controlled factors, δl is the effect of the lth block, and εijkl is the error term ((τδ) il + (βδ) jl + (αδ) kl + (τβδ) ijl + (ταδ) ikl + (βαδ) jkl + (τβαδ) ijkl ) (Steel et al. 1997)). In a preliminary in vitro study, we observed that, under non-stressed conditions, H. crustuliniforme UAMH 5247 was a slow growing isolate, while L. bicolor UAMH 8232 and the S. tomentosus isolate grew faster. Therefore, the following contrasts were used: (i) control plants vs inoculated plants (C1), (ii) the H. crustuliniforme treatment vs the L. bicolor and the S. tomentosus isolate treatments (C2), (iii) and the L. bicolor treatment vs the S. tomentosus isolate treatment (C3). The NaCl gradient effect was analyzed using polynomial contrasts.

For comparative purposes, a naturally saline soil has an ECe higher than 4 dS m-1 (approximately equivalent to an EC1:5 > 0.2 dS m-1 for a sandy-like substrate (Slavich & Petterson 1993)) and an exchangeable sodium percentage higher than 15% (Sumner 1993) (approximately equivalent to an SAR1:5 > 10 (Sumner et al. 1998)). In the NaCl control treatment, the growth substrate was lightly saline (possibly from fertilisation salts) but did not show sodic soil chemical conditions. Addition of increasing amount of NaCl increased substrate EC1:5 and SAR1:5 and all substrates reached levels comparable to that of saline and sodic soil, in all NaCl treatments (Table 5.1).

Non-inoculated seedlings were not contaminated by fungi from the other inoculation treatments; however, certain seedlings exhibited low levels of contamination by Thelephora americana Lloyd, an ECM fungus commonly encountered in tree nurseries (Marx 1991). Of the inoculated seedlings, all root systems were colonized by the ECM species chosen for the treatments. The NaCl treatments had little influence on percentage colonization, which ranged between 80 and 100% (data not shown).

Increasing the amount of NaCl applied increased the Na content of shoot (Figure 5.1, Table 5.2) and root tissues of both inoculated and non-inoculated seedlings. Jack pine seedlings showed significantly higher Na+ concentration in their tissues than white spruce seedlings (P < 0.001). In white spruce inoculated with H. crustuliniforme, higher amounts of Na+ were allocated to shoots than to roots compared to the other inoculated seedlings, in all but the 200 mM treatment. By contrast, jack pine seedlings inoculated with H. crustuliniforme had significantly lower amounts of Na+ in shoot tissues than in roots when compared to the other inoculation treatments (P < 0.05) (Figure 5.1, Table 5.2). The K/Na and the Ca/Na ratios significantly (P < 0.001) decreased with increasing NaCl concentrations (data not shown) and were significantly (P < 0.001) lower in jack pine seedlings than in white spruce seedlings.

Open symbols represent white spruce seedlings and solid symbols represent jack pine seedlings.

The F v/F m ratio decreased in the 100 mM and 200 mM treatments (Figure 5.2). White spruce seedlings showed significantly (P < 0.001) less photochemical stress than jack pine seedlings and the difference was greatest in the 200 mM treatment (Figure 5.2, Table 5.2). Growing parts of jack pine seedlings showed significantly higher F v/F m ratio values in the 100 and 200 mM NaCl treatments than did mature parts (P < 0.01); white spruce seedlings did not exhibit such a difference. Most of the differences between inoculation treatments were observed in jack pine seedlings. In the 200 mM treatment, jack pine seedlings inoculated with L. bicolor and the S. tomentosus isolate showed the lowest F v/F m ratio of growing and mature needles, respectively. The H. crustuliniforme treatment reduced the F v/F m ratio of mature needles of white spruce seedlings in the 200 mM NaCl treatment but increased it in mature needles of jack pine seedlings in all NaCl treatments. The ΦPSII parameter exhibited a linear decrease with increasing NaCl concentrations in the growth substrate (data not shown). This parameter showed a similar response to that of the F v/F m ratio. The inoculated seedling response was similar and showed significantly (P < 0.05) lower yields with increasing NaCl treatments than did non-inoculated seedlings.

Open symbols represent white spruce seedlings and solid symbols represent jack pine seedlings.

In general, seedlings exhibited a significant (P < 0.001) linear decrease in shoot and root biomass with increasing NaCl concentration (Figure 5.3, Table 5.2). Non-inoculated controls were significantly (P < 0.001) smaller (data not shown), and had lower shoot and root DM than inoculated seedlings (P < 0.001). Seedlings inoculated with the S. tomentosus isolate showed the highest biomass, with L. bicolor-inoculated seedlings ranking second and those inoculated with H. crustuliniforme ranking third. Although, white spruce seedlings inoculated with H. crustuliniforme exhibited the lowest shoot DM compared to the other inoculation treatments, those inoculated with L. bicolor showed the greatest decrease with increasing NaCl concentration. The shoot:root ratio of both host species increased with increasing NaCl concentration (data not shown). The shoot:root ratio of white spruce seedlings (1.5) was significantly (P < 0.001) higher than that of jack pine seedlings (1.1). Moreover, the shoot:root ratio of white spruce seedlings increased to a greater extent with NaCl increase than did that of jack pine seedlings (significant linear interaction, P < 0.05). Inoculation treatments did not affect the shoot:root ratio response.

Open symbols represent white spruce seedlings and solid symbols represent jack pine seedlings.

Jack pine seedlings exhibited the greatest decrease in shoot water content with increasing NaCl, dropping to 65% in the 200 mM NaCl treatment when inoculated with either L. bicolor or the S. tomentosus isolate (Figure 5.4, Table 5.2). White spruce seedlings either non-inoculated or inoculated with L. bicolor or the S. tomentosus isolate showed a decrease in root water content with increasing NaCl treatments. By contrast, seedlings of both species inoculated with H. crustuliniforme had a significantly (< 0.01) higher root water content in all NaCl treatments, and it increased with increasing NaCl treatments.

Open symbols represent white spruce seedlings and solid symbols represent jack pine seedlings.

Host response - Inoculation significantly affected concentration of proline and sugars (except raffinose) between shoot and/or root tissues (Figure 5.5 to Figure 5.9). Proline (Figure 5.5, Table 5.2) and total sugar content (the sum of all sugar assessed, data not shown), increased in shoots of both host species with increasing NaCl concentration (P < 0.001). However, these osmolytes tended to remain constant or to increase slightly in root tissues. This response was most evident in the 200 mM NaCl treatment, which induced the highest host tissue content of proline and total sugar content. White spruce and jack pine seedlings showed significantly (P < 0.001) higher amounts of fructose, glucose (Figure 5.6, Table 5.2), pinitol (Figure 5.7, Table 5.2), mannitol (negligible in roots), and glycerol in shoots than in roots. Both host species also showed significantly (P < 0.001) higher sucrose content in roots (Figure 5.8, Table 5.2) than in shoots (data not shown). The predominant sugars of the total sugar content were glucose, fructose and pinitol, and to a lesser extent sucrose and glycerol. White spruce seedlings tended to maintain higher level of glucose and fructose, while jack pine seedlings showed a higher accumulation of pinitol.

Open symbols represent white spruce seedlings and solid symbols represent jack pine seedlings.

Open symbols represent white spruce seedlings and solid symbols represent jack pine seedlings.

Open symbols represent white spruce seedlings and solid symbols represent jack pine seedlings.

Mycobiont influence - With increasing NaCl concentration, significantly (P < 0.001) higher amounts of proline were accumulated in non-inoculated white spruce seedlings and in jack pine seedlings inoculated with L. bicolor and S. tomentosus (Figure 5.5, Table 5.2). The pinitol content of shoots was significantly (P < 0.05) higher in seedlings of both species inoculated with S. tomentosus. In addition, the sucrose content in roots of white spruce seedlings inoculated with S. tomentosus exhibited a significantly (P < 0.05) higher increase with increasing NaCl concentrations (Figure 5.8). Increases in glycerol, mannitol and stachyose with increased NaCl treatments were specifically linked to certain host-fungus combinations: H. crustuliniforme inoculated white spruce seedlings showed significantly (P < 0.05) higher levels of stachyose in root tissues in the 100 mM NaCl treatment (data not shown) and showed increased glycerol in shoot tissues in all NaCl treatments (Figure 5.9, Table 5.2); with increasing NaCl concentration, the shoot mannitol content was significantly (P < 0.05) higher in shoots of inoculated jack pine seedlings than in shoots of non-inoculated seedlings (data not shown).

Open symbols represent white spruce seedlings and solid symbols represent jack pine seedlings.

Inoculation improved the growth and/or reduced NaCl stress in white spruce and jack pine seedlings. This result is in accordance with the results obtained by Dixon et al. (1993) with loblolly pine inoculated with three selected ECM fungi. White spruce seedlings inoculated with S. tomentosus exhibited the highest biomass and low photochemical disturbance over the range of NaCl concentrations tested. We can infer from this result that, under saline and/or sodic site conditions, white spruce seedlings inoculated with the S. tomentosus isolate would probably show the highest biomass accumulation and should be considered in future revegetation work. The S. tomentosus isolate originated from a sodic reconstructed soil prepared for revegetation at Syncrude Canada Ltd. (AB, Canada). Therefore, this fungus is likely to be more suitable for this type of reclamation work and should be more resistant to other site-specific stresses (e.g., toxic oil residues). This in turn would make it more competitive and persistent than species isolated from undisturbed boreal forest soil (Kropp & Langlois 1990). Furthermore, Suillus species exhibit a high degree of host specificity to conifer trees and are easy to culture axenically for inoculum production (Dahlberg & Finlay 1999). In field situation where the excess of Na+ and salinity are less than in the 200 mM NaCl treatment (approx. SAR1:5 = 32 and EC1:5 = 2 dS m-1), high biomass production may be obtained with L. bicolor which could be another good candidate for inoculation of white spruce seedlings produced for revegetation of saline and/or sodic sites.

The ChFl, water content and Na+ accumulation/allocation results suggest that two ranges of NaCl concentrations should be distinguished for jack pine seedlings: (i) from 0 to 100 mM and (ii) from 100 to 200 mM NaCl. Sodic sites in the first range of NaCl concentrations may be revegetated with jack pine seedlings inoculated with either the L. bicolor or the S. tomentosus isolates. Danielson & Visser (1989) observed that species of Suillus were the major indigenous fungi colonizing jack pine seedlings used for revegetation of oil sand tailings; Visser (1995) found species of Suillus associated with jack pine over a wide range of stand ages following wildfire disturbance in Alberta. Furthermore, Danielson (1991) observed that Suillus species may colonize container-grown jack pine seedlings in tree nurseries. This suggests that the S. tomentosus isolate used in this study may also be a good candidate for mass inoculation of jack pine seedlings in tree nurseries.

Under high NaCl stress (200 mM), H. crustuliniforme was the most beneficial fungus for jack pine seedlings, whereas the two other fungi induced higher photochemical stress. Seedlings inoculated with H. crustuliniforme grew at a slower rate than the other inoculated plants and this may be an advantage in highly stressful conditions (Orcutt & Nilsen 2000, Zhu 2001). White spruce seedlings inoculated with H. crustuliniforme did not show any resource limitations (similar biomass accumulation to that of controls) and the fungus developed an extensive extraradical mycelium (visual estimation at harvest on undisturbed substrate). This indicates a net advantage in terms of resource exchange toward the mycobiont (Colpaert et al. 1992) as ECM growth did not benefit plant growth (Tinker et al. 1994). Moreover, jack pine seedlings showed a lower root DM when colonized by H. crustuliniforme, indicating a probable strong fungal sink for resources. In given nutritional conditions, a special phytohormonal balance and resource exchange dynamic may be set up between each partner to maintain the symbiosis, while favoring the growth of either the host or the mycobiont (Nylund & Wallander 1989, Colpaert & Verstuyft 1999). Miller et al. (1989) reported that H. crustuliniforme was a strong sink for C when it was associated with Pinus sylvestris L. in substrates with low biotic activity (e.g., little existing mycelia or pathogenic organisms), and as such, with little competition. By contrast, the increased energy demand for the development of the extraradical mycelium resulted in a reduction of host growth (Miller et al. 1989, Tinker et al. 1994). In vitro, the L. bicolor and S. tomentosus isolates grew faster than the H. crustuliniforme isolate even with excess NaCl in the growth medium (Bois et al. 2005a). In the present study, the former two species may have been less dependent on host C for substrate colonization and may have been weaker sinks for photosynthate.

Although both species accumulated increasing amounts of Na with increased NaCl concentrations, in the 50 and 100 mM treatments neither host species exhibited photochemical stress. In these NaCl treatments, toxic effects were likely avoided by vacuolization of excess ions and by a higher concentration of organic osmolytes in shoot tissues (Niu et al. 1995, Hasegawa et al. 2000). While white spruce seedlings did not show a higher accumulation of the compatible osmolytes measured, they showed a higher capacity to exclude Na+ from its tissues. This confirms the results obtained by Renault et al. (1999) that reported a lower accumulation of Na+ in shoots of white spruce seedlings exposed to saline conditions than in shoots of jack pine seedlings. In white spruce, the higher retention of excess ions (or control of Na+ allocation) at the root level, compared to jack pine seedlings, also helped to avoid toxic effects on essential shoot mechanisms (e.g., photochemistry) compared to jack pine seedlings.

In jack pine seedlings, the S. tomentosus isolate induced the greatest changes in the quantity and diversity of accumulated organic osmotica. Similarly, white spruce seedlings were more responsive to colonization by H. crustuliniforme. In both cases, these ECM fungi caused an increased accumulation of the major sugars by their hosts (i.e., glucose and fructose for white spruce and pinitol for jack pine seedlings) with increasing NaCl concentration. There is a certain degree of specificity between host and mycobionts (Molina et al. 1992) that may be reflected at both the phyto-hormonal and biochemical level, i.e., each of the two symbionts may influence carbohydrate metabolism of the other (Wedding & Harley 1976, Pfeffer et al. 2001). A specific osmolyte balance could be set up for each symbiotic combination which would result in a specific potential of resistance to NaCl stress. It is well known that a particular solute may have more than one function and that different compatible solutes may have different functions (Bohnert & Shen 1999). In general, the multitude of compatible solutes which accumulate in response to osmotic stress are considered to play an osmoregulatory function (Bohnert & Shen 1999). In the present study, proline, glucose, fructose and pinitol were the major shoot organic osmoregulators. Sucrose is both a stress signal and the principal form in which C is transported. This molecule can be broken down by invertase to give glucose and fructose (Kleinschmidt et al. 1998, Koch 2004) which could be used as the substrate from which other sugars are synthesized (Bohnert et al. 1995). Proline, pinitol, mannitol, glycerol and stachyose are all known to have osmoprotective functions (Thomas et al. 1992, Shen et al. 1997, 1999, Kleinschmidt et al. 1998, Bohnert & Shen 1999, Sundblad et al. 2001). Some of these compounds could exert osmoprotective functions even at low osmotically insignificant concentrations by scavenging radical oxygen or by maintaining membrane integrity (Bohnert & Shen 1999). Moreover, the accumulation of such compatible osmolytes could prevent the inhibitory effects of potentially toxic ions on enzyme activity and the dissociation of the oxygen evolving system of photosystem II (Papageorgiou & Murata 1995). Sucrose and stachyose accumulated specifically in white spruce roots, and the accumulation of these sugars was enhanced by the S. tomentosus and the H. crustuliniforme isolates, respectively. Sucrose concentration was significantly higher in roots of white spruce seedlings than in those of jack pine seedlings and as such, may play a role in the exclusion of Na+ from roots (and subsequently shoot) of white spruce seedlings. For both conifer species, the overall strategy in the presence of excess Na+ in the soil solution was to allow the accumulation of a certain amount of Na+ in roots, while protecting cell membranes, organelles, and general metabolism by the accumulation of osmoprotectant solutes. The osmotic potential of shoots was lowered by accumulation of compatible osmoregulatory solutes, along with the accumulation of non-toxic levels of Na+. The resulting lower osmotic potential in shoots compared to roots may facilitate water flux through the plant.

In our study, the accumulation of Na+ was greater in jack pine than in white spruce seedlings indicating a higher resistance of white spruce to high NaCl stress (Renault et al. 1999). Low accumulation of Na+ in shoots and high K/Na and Ca/Na ratio have been shown to be good indicators of resistance to sodic stress in many plant species (Niu et al. 1995, Orcutt & Nilsen 2000, Tyerman & Skerrett 1999). The negative effect of Na+ accumulation was most evident in the 200 mM NaCl treatment in which jack pine seedlings inoculated with either the L. bicolor or the S. tomentosus isolates exhibited the highest Na+ accumulation in shoots and the highest photochemical disturbance and the lowest shoot water content. Interestingly, toxic effects occurred at a level of Na+ in shoot tissues lying between 1 and 1.5% DM which was also observed at higher NaCl treatments in a previous experiment (Bois et al. 2005c) on similar sized jack pine seedlings inoculated with L. bicolor. Furthermore, beyond that threshold (above 100 mM NaCl) jack pine seedlings inoculated with the L. bicolor or the S. tomentosus isolates switched partitioning of excess toxic ions towards the shoot, which accumulated 1.5-time more Na+ than the roots. Marcum & Murdoch (1992) reported a similar pattern of increasing Na+-regulated osmotic adjustment in the shoot in function of the NaCl concentration of the substrate for the perennial halophytic grass Sporobolus virginicus L. Kunth. This species exhibited a K+-regulated shoot osmotic adjustment under 1 mM NaCl concentrations; however, this was progressively replaced by a Na+-regulated shoot osmotic adjustment with increasing NaCl concentration, up to 450 mM NaCl. Proline accumulation was another good indicator of the higher stress level in jack pine seedlings inoculated with the L. bicolor and the S. tomentosus isolates in the 200 mM NaCl treatment. The proline amino acid is one of the most widely distributed compatible osmolyte and it accumulates in a wide range of organisms from bacteria to higher plants as a resistance mechanism against water deficit and salinity (Marcum & Murdoch 1992, Thomas et al. 1992, Taylor 1996). Proline acts as an organic N reserve (for recovery after stress period), as an osmoprotectant for membranes and proteins (against accumulation of inorganic ions), as a cryoprotectant and as a radical scavenger (Santarius 1992, Thomas et al. 1992, Taylor 1996, Bohnert & Shen 1999). As a consequence, accumulation of proline indicates either more efficient protection mechanisms (damage prevention) or higher stress effects (damage salvation) (Shah et al. 1990, Kohl 1997). In jack pine seedlings inoculated with either the L. bicolor or the S. tomentosus isolates, proline content increased markedly in shoots in response to high stress but did not deter damage to the light harvesting antenna (Santarius 1992).

The ChFl results suggest that the inner threshold of toxicity may be specific to a plant-fungus combination with a specific osmotic stress tolerance. The threshold for white spruce seedlings or for H. crustuliniforme inoculated jack pine seedlings was not reached in the range of NaCl concentrations assessed in the present study. White spruce seedlings or H. crustuliniforme inoculated jack pine seedlings exhibited shoot:root ratios of Na+ lower than one and may have been more efficient in resisting osmotic stress as they avoided an Na+-dominant osmotic adjustment mechanism. White spruce seedlings exhibited only slight photochemical disturbance in the 200 mM NaCl treatment but showed lower inorganic osmolyte accumulation (e.g., shoot Na+), and accumulated only certain organic osmolytes (i.e., sucrose, glycerol and stachyose) to a greater extent than in jack pine seedlings. This indicates a more efficient osmotic adjustment strategy for white spruce than for jack pine seedlings at high NaCl concentrations. Bledsoe & Rygiewicz (1986) reported that H. crustuliniforme inoculated Douglas fir exhibited reduced Na+ uptake compared to non-mycorrhizal seedlings. Furthermore, in a previous in vitro experiment (Bois et al. 2005a), H. crustuliniforme was shown to be resistant to absorption of high concentrations of Na+ and Cl- (above 4% and 20% DM, respectively), and to produce high amounts of mannitol and trehalose in response. Therefore, when inoculated on jack pine, this fungus could have increased the root’s capacity to accumulate Na+ and so limited the release of excess Na+ toward the shoot in the 200 mM NaCl treatment. Seedlings of both species inoculated with H. crustuliniforme showed high shoot and root water content in all NaCl treatments. This fungus, which was also shown to improve root hydraulic conductance (Mushin & Zwiazek 2002), may favor osmotic adjustment and improve osmotic stress resistance of its host in highly sodic conditions.

Overall, it is important to bear in mind that in terms of growth S. tomentosus would be the best ECM fungal candidate for seedlings to be used for the revegetation of saline and/or sodic reconstructed soils. Nonetheless, although, jack pine seedlings inoculated with either the L. bicolor or the S. tomentosus isolates showed higher biomass yield, shoot metabolism protection by compatible osmolytes was possibly overloaded in the 200 mM NaCl treatment. By contrast, jack pine seedlings inoculated with H. crustuliniforme showed lower biomass yield but were more resistant to high osmotic and ionic stress. Thus, H. crustuliniforme would be the best candidate in terms of survival for jack pine seedlings to be used for revegetation of highly saline and/or sodic sites. To confirm conclusions drawn from this greenhouse experiment, it will be necessary to monitor seedling response to long-term exposure to saline and/or sodic conditions in situ.

This research was funded by Syncrude Canada Ltd. and NSERC (CRDPJ 250448-01 to D.P.Khasa). The authors wish to thank Alexis Guérin-Laguette for his help with fungal inoculation, Yves Dubuc for all his help with plant physiological measurements, Lucette Chouinard and Pierre Lechasseur for biochemical analyses, Alain Brousseau for elemental analyses, and Michèle Bernier-Cardou for advices in statistical analyses. The authors also thank Andrew Coughlan and Jean-Luc Jany for help in solving scientific problems and preparing the manuscript. The authors are grateful to Erin Bergrand and to Claude Fortin for technical help and advice.

© Gregory Bois, 2005