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Chapitre 4 Physiological response to NaCl of nursery-grown Pinus banksiana inoculated with Laccaria bicolor

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Les résultats présentés dans ce chapitre ont été exploités pour la publication d’un chapitre de livre (Bois, Bigras, Piché, Fung, Khasa 2005c) et ont été présentés (affiche) à la conférence internationale sur les mycorhizes en août 2003 (ICOM 4, Montreal, QC, Canada). L’ensemble des mesures écophysiologiques fut réalisé avec la collaboration du Dr Francine Bigras (Ressources naturelles Canada, CFL, 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 3, qui suit, porte sur l’association Pinus banksiana Lamb. et Laccaria bicolor (Maire) Orton UANH 8232. Elle fut réalisée sur des semis inoculés produits pour l’expérience 6 (expérience B, Chapitre 6). Ces deux expériences furent mises en place dans un souci d’application immédiate pour obtenir des résultats sur le potentiel d’inoculation d’un champignon ECM dans des conditions courantes de végétalisation (pépinière et plantation). La souche de L. bicolor fut choisie suite à la sélection en conditions in vitro réalisée par Kernaghan et al. (2002) en contradiction avec les résultats de l’expérience 2, cette dernière ayant été réalisée postérieurement aux expériences 3 et 6. Malgré le fait que cette souche se soit finalement avérée sensible au stress sodique, L. bicolor est une espèce modèle qui a servi dans l’expérience qui suit à démontrer l’influence du mycobiote sur la physiologie de son hôte en conditions stressantes. De plus, cette expérience a permis de sélectionner des indicateurs physiologiques pertinents pour réaliser les expériences des chapitres 5 et 6 plus complexes par une plus vaste gamme de traitements (plusieurs espèces fongiques et végétales).

Une étude détaillée sur la réponse physiologique au stress sodique de semis de Pinus banksiana Lamb. (pin gris) inoculés avec Laccaria bicolor (Maire) Orton UAMH 8232 en conditions de pépinière a été effectuée. Cette souche de champignon ectomycorhizien (ECM) fut sélectionnée préalablement en conditions in vitro pour sa résistance à différents sels. Ce champignon peut potentiellement apporter une plus grande résistance à son hôte par rapport à Thelephora americana Lloyd, le champignon ECM colonisateur dominant dans les pépinières. Pour vérifier cette hypothèse, des semis de pépinière de un an, inoculés ou non avec L. bicolor, ont été exposés à 0, 0,5, 1,0, 2,5 ou 5,0 g Na l-1 de substrat. Après 30 jours, la colonisation par des champignons ECM, la biomasse, la photosynthèse, la fluorescence de la chlorophylle a, le potentiel hydrique, l’hydratation et la composition minérale des tissus ont été mesurés. L’inoculation de semis de pin gris a fait augmenter l’accumulation de biomasse de l’hôte dans les traitements 0,5 et 1,0 g Na l-1 de substrat ; l’efficacité photosynthétique, la croissance racinaire et la colonisation fongique des semis inoculés ont été stimulées, et l’accumulation de Na+ et de Cl- a été réduite par comparaison avec des semis colonisés par T. americana. Au-delà de 1,0 g Na l-1 de substrat, les plantes inoculées ou non ont desséché et montré une perturbation photochimique élevée. Sur le long terme, l’influence de L. bicolor peut améliorer la croissance et la survie de semis de pin gris exposés à des niveaux de NaCl inférieurs à ceux obtenus avec 2,5 g Na l-1 de substrat.

A comprehensive study was performed on the physiological response to sodic stress of Pinus banksiana Lamb. (jack pine) inoculated with Laccaria bicolor (Maire) Orton UAMH 8232 under nursery conditions. This ectomycorrhizal (ECM) strain was selected in vitro for its tolerance to different salts and, therefore may confer a higher resistance to jack pine seedlings than does Thelephora americana Lloyd, the dominant nursery colonizer. To test this hypothesis, one-year-old tree nursery grown seedlings, inoculated or not with L. bicolor, were exposed to 0, 0.5, 1.0, 2.5 or 5.0 g Na l-1 of substrate. After 30 days, colonization by ECM fungi, biomass, photosynthesis, chlorophyll a fluorescence, water potential and content, and mineral composition were characterized. Inoculation of jack pine seedlings improved biomass accumulation in the 0.5 and 1.0 g Na l-1 of substrate; photosynthetic efficiency, root growth and fungal colonization of inoculated seedlings were stimulated, and Na+ and Cl- accumulation was lower compared to T. americana-colonized seedlings. Above 1.0 g Na l-1 of substrate, both inoculated and non-inoculated seedlings dehydrated and showed high photochemical disturbance. In the long-term, the influence of L. bicolor is likely to promote better growth and survival of jack pine seedlings at NaCl exposure levels lower than that of the 2.5 g Na l-1 of substrate treatment.

Glycophytic organisms living in or on sodic soils are adversely affected by the presence of excess Na+ in the soil solution. High sodicity affects plant physiology by causing both hyperosmotic and hyperionic stresses (Levitt 1980, Yeo 1983, Munns & Termaat 1986, Cheeseman 1988, Munns 1993, Neumann 1997, Hasegawa et al. 2000). As an integrative process of all metabolic process acting in response to the stress, growth is generally reduced by salt stress (Levitt 1980). The osmotic stress induces stomatal closure and drought stress signaling (Hasegawa et al. 2000, Zhu 2001, 2002). For osmotic adjustment of the water potential, osmolyte contents must increase in the tissues of the plant exposed. As a result, Na+ and Cl-, being in excess in the environment, are used as low cost osmotica to counteract osmotic deficiencies (Yeo 1983, Niu et al. 1995, Niu et al. 1997, Hasegawa et al. 2000). These ions may accumulate in the cytoplasm and become toxic for cell functioning. In such a case, specific ion effects of Na+ and Cl- may lead to phytohormonal imbalance, alter enzymatic activity and cell membrane integrity, dysfunction of metabolism pathways, and reduce net photosynthesis (Munns 1993, Kozlowski 1997, Neumann 1997, Hasegawa et al. 2000, Mansour & Salama 2004). The resistance of a given plant depends mainly on its ability to vacuolize excess ions and/or to allocate them to mature tissues (Levitt 1980, Niu et al. 1995, Hasegawa et al. 2000), and on the plant’s potential to synthesize compatible osmolytes for osmoregulation and osmoprotection (Yeo 1983, Niu et al. 1997, Yeo 1998, Bohnert & Shen 1999).

The mycorrhizal symbiosis is a plant-fungal association in which the fungus favors mineral nutrition of the host plant in return for energy in the form of sugars (Smith & Read 1997). Mycorrhiza may be considered as a special adaptation of land plants to stresses such as nutrient deficiency (Marschner & Dell 1994, Smith et al. 1994, Read et al. 2004), heavy metal toxicity (Kottke 1992, Hartley et al. 1997, Jentschke & Goldbold 2000, Schützendübel & Polle 2002), and physical constraints such as water deficit (Dosskey et al. 1991, Lamhamedi et al. 1992, Augé 2001), on various plant species. Al-Karaki (2000) and Al-Karaki et al. (2001) showed that arbuscular mycorrhizal (AM) fungi can improve NaCl stress tolerance of tomato plants grown under high saline conditions (e.g. ECe = 7.4 dS m-1). Ectomycorrhizal (ECM) fungi may also be able to reduce the effect of salt stress on their host plant’s physiology. Dixon et al. (1993) showed improved growth and mineral nutrition (i.e., P) of Pinus taeda L. (loblolly pine) colonized by Laccaria laccata (Scop.: Fr.) Cooke and Pisolithus tinctorius (Mich.: Pers.) Coker & Couch grown at five NaCl concentrations (up to 80 mM NaCl) compared to seedlings inoculated with Thelephora americana Lloyd (formerly terrestris). In a ten week experiment, Mushin & Zwiazek (2002) observed improved NaCl tolerance (following application of a 25 mM solution) of Picea glauca (Moench) Voss (white spruce) seedlings colonized by Hebeloma crustuliniforme (Bull) Quel. The later fungi reduced Na+ accumulation in shoot, increased N and P acquisition and increased root hydraulic conductance of its host.

Pinus banksiana Lamb. (jack pine) is a common species used for reclamation of salt (essentially Na+ and Cl-) affected lands produced by the oil sand industry’s activity in northeastern Alberta, Canada (Fung & Macyk 2000); however, compared to other boreal forest species, jack pine has been shown to be sensitive to salt stress (Renault et al. 1999). When exposed to NaCl dissolved in the soil solution, jack pine seedlings readily accumulate Na+ and Cl- and show a weak root control on shoot accumulation, especially for Cl- (Apostol et al. 2002, 2004, Franklin et al. 2002a,b, Franklin & Zwiazek 2004). To help overcome this, seed sources of jack pine have been selected for their salt tolerance (Khasa et al. 2002). Considering the inherent properties of ECM fungi, we hypothesized that certain fungal species should be able to further reduce the salt stress experienced by jack pine. Laccaria bicolor (Maire) Orton is a model ECM fungal species and the salt tolerance of the strain UAMH 8232 has already been reported (Kernaghan et al. 2002). The jack pine - L. bicolor association may be interesting for the revegetation of challenging salt affected soils where ECM inoculum potential is low (Bois et al. 2005c). In order to draw a picture of the "whole plant" physiological response to sodicity - including growth, water relations, net photosynthesis and mineral composition - jack pine seedlings inoculated or not with L. bicolor were subjected to a range of Na+ concentrations. Sodium was provided as NaCl as it has a higher stress potential than when applied as Na2SO4 (Franklin et al. 2002b). This is the first insight into the physiological response of the jack pine - L. bicolor symbiosis to a sodic environment.

The mycelium of L. bicolor UAMH 8232 was grown for 30 d in a bioreactor in a modified Melin Nokrans (MMN) (Marx 1969) liquid medium. After harvesting, the mycelium was washed with sterilized distilled water to remove any remaining nutrients, and was chopped in a blender. The mycelium was resuspended in 1 l of distilled water (approx. 880 000 active propagules per liter) and stored for 10 d at 4ºC prior to use. The mycelium of L. bicolor was incorporated to the growing medium prior to sowing. The concentrated liquid inoculum was diluted in water (1:39, v:v) and incorporated into a peat based (19:1, peat:perlite, v:v) potting compost (2:3, water borne inoculum: potting compost, v:v). The substrate used for non-inoculated control plants was mixed with water. All seedlings were grown under normal tree nursery conditions. However, the watering and fertilization rates were reduced by one third of the operational fertigation regime to favor root colonization by L. bicolor rather than by other ECM fungi commonly found on nursery stock (e.g. T. americana) which are adapted to wet and nutrient rich conditions (Hacskaylo 1965, Marx 1991). Jack pine seedlings (Syncrude seed source, provided by Alberta Tree Improvement and Seed Center, Smoky Lake, AB, Canada) were grown in 112-77 styrofoam containers, under greenhouse conditions at the Smoky Lake Forest Nursery (AB, Canada). Six months after sowing, height and total dry mass (DM) were recorded on 20 seedlings sampled among inoculated and non-inoculated plants. After eight months of growth, seedlings were transferred to a growth chamber at Université Laval (QC, Canada). In order to induce dormancy and simulate winter conditions, the temperature was progressively reduced from 18/10ºC (day/night) to 8/4ºC over a two week period. The photoperiod was also reduced from 12 to 8 h (RH 60%). The seedlings were kept dormant for 14 weeks. During this period, they were lightly watered and fertilized with 4 mg of N (20-8-20r (Plant Prod Québec, Laval, QC, Canada)). Seedlings were then transplanted into 1 l pots filled with a sand:Turface® MVP (ProfileTM, Buffalo Grove, IL, USA) mix (1:1, v:v) saturated with tap water (380 ml) which contained: 0.03 mM of K+, 0.90 mM of Ca2+, 0.20 mM of Mg2+ and 0.80 mM of Na+. A mineral medium was chosen to avoid the bias generated by the high buffering capacity of peat-based organic substrates. Any residual peat was washed off of the seedling roots before transplanting dormant plants in the new substrate. Seedlings were transferred to a greenhouse (30% RH, 16 h photoperiod, 24-18ºC day/night, photon flux density (PFD) of 100-150 μmol m-2 s-1), and for the first 14 d, plants were watered every 2 d with 100 ml of tap water to maintain the growing medium at field capacity. During the next 14 d, seedlings were watered every 2 d with 100 ml of a NaCl solution such that at day 28, the selected amount of Na (0, 0.5, 1.0, 2.5 or 5.0 g Na l-1 of substrate) was reached for each treatment. The seedlings were grown for an additional 14 d, during which they were watered with tap water; care was taken to avoid loss of elements by percolation.

At the end of the experimental period, shoot height, in vivo chlorophyll a fluorescence (ChFl) and net photosynthetic rates were measured. A portable fluorometer (model PAM-2000, Heinz Walz GmbH, Effeltrich, Germany) was used to evaluate ChFl of growing and mature needles. Growing needles were sampled from the part of the plant above the scar of the last dormant apical bud, and mature needles were sampled below this point. Plants were kept in the dark for 1 h prior to fluorescence induction. The dark-adapted minimal fluorescence (F 0) was obtained under a modulated light. A saturating pulse (0.8 s) of light was applied to obtain the dark-adapted maximal fluorescence (F m). The dark-adapted variable fluorescence was calculated using these two parameters (F v = F m - F 0) in order to determine the F v/F m ratio. For the purpose of the quenching analysis, the photosystems were light-adapted by illumination with an actinic red light (photosynthetic active radiation (PAR) of 340-350 μmol photons m-2 s-1) for 20 min. The actual fluorescence (F t) at this steady state and the light-adapted maximal fluorescence (F m'), induced by a saturating pulse of light at this point, were used to calculate the effective quantum yield of PSII (ΦPSII = (F m’-F t) / F m'). Needles were then illuminated with far red light to obtain the light-adapted minimal fluorescence (F 0'). From this value, the photochemical quenching coefficient (qP = (F m’-F t) / (F m’-F o’)) was calculated.

Net photosynthesis, stomatal conductance and transpiration were measured using a gas exchange system (model Licor 6400, Li-Cor Inc., Lincoln, NE, USA) on mature needles; needles of growing parts were too small for such measurement. Mature needles were enclosed in a 250 ml cuvette (Model LI-6400-05, Li-Cor Inc., Lincoln, NE, USA) and illuminated with a PFD of 1000 μmol m-2 s-1. The projected area of needles used for this measure was determined (Winseedle v. 2002A, Regent Instruments Inc., Montréal, Québec, Canada) after removing all necrotic tissues. Afterward, a branch was removed from each seedling to evaluate water potential with a Scholander pressure chamber (PMS Instrument Co., Corvallis, OR, USA).

For Chla and Chlb contents of needles, 0.1 g of growing and of mature green needles were sampled from each seedling, placed in 15 ml screw-topped plastic tubes containing 10 ml of an 80% acetone solution, and stored at 4ºC. Chlorophyll extraction was improved by grinding the needle tissues using a polytron (Kinematica GmbH, Brinkman Instruments, Switzerland). Tubes were kept at 4ºC for 24 h prior to spectrophometric measurements at 647 nm and 664 nm (zero at 750 nm) as indicated by Arnon (1949). Chla and Chlb contents were determined using the Photosynthesis Assistant software v. 1.1.2 (Dundee Scientific, Dundee, UK).

Survival was evaluated at the time of harvest and totally dehydrated seedlings were considered dead. Roots and shoots of seedling were divided into growing and mature parts, and the fresh mass (FM) recorded. Growing roots were white and grew into the experimental rooting medium beyond the ball of mature roots. The latter were brown due to build up of phenolics and corresponded to root development during the period of growth in the 112-77 containers. Sub-samples of each tissue were oven dried at 65ºC for 48 h in order to calculate the dry mass (DM). The growth rate was calculated as the percentage of DM of the growing parts of NaCl treated seedlings relative to the average DM of growing parts of the controls. The percentage of water in fresh tissues was calculated from FM and DM measurements. Level of root colonization by ECM fungi was evaluated on fresh root sub-samples. A minimum of 300 root tips were counted per plant and 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).

Dried tissues were ground and the amount of N, P, K, Ca, Mg, Na and Cl present in mature (produced prior to dormancy) and growing (after dormancy) shoot tissues was determined. Mineral analyses were performed following the standard methods outlined by Kalra (1998). After harvest of the seedlings, the electrical conductivity (EC), the sodium absorption ratio ( ) and the pHH2O of the growth substrate were measured to ensure the effectiveness of the NaCl treatments. The EC and SAR are relevant soil parameters and have predictive abilities for plant performance on saline soil (Gardner 2004); the EC describes the osmotic stress and the SAR is a measure of the potential sodicity hazard. These indicators were measured on 1:5 (w:v) soil:water extracts (EC1:5 and SAR1:5) a more convenient method than on saturated paste extracts (ECe and SARe). Soil analyses were performed following the standard methods outlined for Canadian forest soils by Kalra and Maynard (1992).

The five NaCl treatments (0, 0.5, 1.0, 2.5 or 5.0 g Na l-1 of substrate) were chosen accordingly to Lichtenthaler’s (1996) concept of plant stress: once the threshold of stress resistance is passed, the damage from long-term low-stress exposure is similar to that from short-term high-stress exposure. Although, NaCl exposure, in the present study, lasted four weeks, the highest treatment corresponded to a concentration of Na+ ranging between 500 mM and 600 mM when the substrate was at field capacity, a concentration equivalent to that of sea water. Glycophytes exhibit intolerance at soil concentrations higher than 50 mM (Orcutt & Nilsen 2000). This latter concentration was reached in the 0.5 g Na l-1 of substrate treatment when the substrate was at field capacity. The 1.0 and 2.5 g Na l-1 substrate corresponded approximately to 110 mM Na+ and 280 mM Na+ when the substrate was at field capacity. Pots were layed out according to a factorial design comprising four completely randomized blocks. Each experimental unit was a pot containing either an inoculated or non-inoculated plant exposed to one of the five levels of NaCl treatments. In total, the experiment comprised 40 plants: 20 inoculated and 20 non-inoculated. Fluorescence and carbon exchange measurements of seedlings within a block were assessed on the same day. Main and interaction effects of inoculation and the gradient of NaCl treatments were investigated using two-way ANOVA analyses (PROC GLM, SAS system, The SAS Institute, Cary, NC, USA) with the following effect model: yijk = µ + τi + βj + (τβ) ij + δk + εijk where yijk is the response, µ is the mean of the response variable, τi is the effect of the ith level of the inoculation treatment, βj is the effect of the jth level of the NaCl treatment, (τβ) ij is the effect of the interaction between τi and βj , δk is the effect of the kth block, and εijk is the error term ((τδ) ik + (βδ) jk + (τβδ) ijk ) (Steel et al. 1997). Where measurements were performed on different parts of each experimental unit (e.g. growing vs mature parts), data were considered as time repeated and that effect was included in the model. Results of polynomial contrasts and linear regression were used to detect differences between inoculated and non-inoculated seedlings in all NaCl treatments. In order to reduce distortion of regression coefficients due to the unequal difference between NaCl doses, the NaCl treatment independent variable was log transformed. The SAS type IV sum of squares was used to evaluate data sets with missing values. All data from variables with non-significant treatment effects were pooled to simplify analysis and graphical presentation.

The following results are given in the light of the growing part response which reflected past growth conditions and response to the NaCl treatment of the seedlings. Mature part responses were used only to complement and to contrast growing part responses.

The pH of the growing medium varied between 5 and 5.7 across all NaCl treatments (Table 4.1). A sandy saline soil has approximately an EC1:5 > 0.2 dS m-1 (equivalent to an ECe > 4 dS m-1 using a conversion factor of 17 (Slavich & Petterson 1993)) and a sodic soil has an SAR1:5 > 10 (equivalent to an exchange sodium percentage higher than 15% (Sumner et al. 1998)). In control NaCl treatment (0 g Na l-1 of substrate), the substrate showed neither sodic nor saline conditions. With the addition of NaCl, the EC1:5 and the SAR1:5 increased (Table 4.1) and the growth substrate exhibited saline and sodic conditions in all NaCl treatments.

A significant (P < 0.05) increase in the Na and Cl content of the shoot of both inoculated and non-inoculated seedlings was recorded with increasing amounts of NaCl applied. However, inoculated plants accumulated, on average, significantly (P < 0.05) less Na+ and Cl- in their shoot tissues than non-inoculated plants (Table 4.2, Figure 4.1a,b). In treatments below 2.5 g Na l-1 of substrate, growing parts of inoculated plants tended to accumulate Na+ and Cl- at an equal or lower rate than that of mature parts. In the 5.0 g Na l-1 of substrate treatment, growing parts of inoculated seedlings had the highest Na (2.5% DM) and Cl (5.5% DM) contents.

Table 4.2 Main contrast results of physiological parameters.

SP/RP: shoot parts/root parts (growing vs mature parts) IN: inoculation (L. bicolor vs T. americana)

Axis labels are non-transformed.

At harvest, of the short roots produced by inoculated plants after transplantation, 65% were colonized by L. bicolor (as confirmed by ITS-RFLP analyses) in the control NaCl treatment and no other fungus was detected. By contrast, in non-inoculated seedlings in the control NaCl treatment, 50% of the fine roots from growing parts were colonized by T. americana (Figure 4.2). Less than 1% of short roots from non-inoculated seedlings were associated with a species of Wilcoxina. Colonization of fine roots by ECM fungi on the growing parts of the root system significantly decreased with increasing NaCl treatments (P < 0.01) (Table 4.2), reaching a minimum (approx. 10%) in the 5.0 g Na l-1 of substrate treatment. Nevertheless, L. bicolor maintained a significantly higher level of colonization than the concert of common nursery fungi along the NaCl gradient tested (P < 0.05). In treatments of 1.0 g Na l-1 of substrate or less, colonization by L. bicolor was stimulated and was approximately 80%.

Axis labels are non-transformed.

Chlorophyll a fluorescence and net photosynthesis – In the 0.5 and 1.0 g Na l-1 of substrate treatments, inoculated and non-inoculated plants exhibited a nearly constant F v/F m ratio close to 0.84 (Figure 4.3a). In the 2.5 and 5.0 g Na l-1 of substrate treatments, the F v/F m ratio declined significantly with increasing sodicity (< 0.01) and inoculated seedlings showed higher stress than non-inoculated seedlings in the 5.0 g Na l-1 of substrate treatment (Table 4.2). The values of ΦPSII significantly decreased with increased sodicity (P < 0.01). Needles from growing parts had a significantly higher quantum yield than those of mature parts (P < 0.01) (Table 4.2, Figure 4.3b). In treatments below the 2.5 g Na l-1 of substrate, inoculated seedlings showed significantly higher values of ΦPSII (P < 0.05). This benefit was reduced with increasing NaCl concentrations. Beyond a threshold level between the 1.0 and 2.5 g Na l-1 of substrate treatments, differences in quantum capture efficiency between inoculated and non-inoculated seedlings became minimal. The photochemical quenching coefficient, qP, gave the same response pattern as for ΦPSII (data not shown).

Axis labels are non-transformed.

The net photosynthesis of mature needles was significantly higher (by up to 31% in the control NaCl treatment) in inoculated seedlings (P < 0.05) compared to non-inoculated seedlings (Table 4.2, Figure 4.4a). However, that difference decreased while net photosynthesis decreased with increasing NaCl treatments (P < 0.001): net photosynthesis of inoculated and non-inoculated plants was close to zero in the 2.5 and 5.0 g Na l-1 of substrate treatment. Stomatal conductance (Figure 4.4b) and transpiration (Figure 4.4c) significantly decreased with increasing NaCl application (P < 0.001) and were similar in inoculated and non-inoculated seedlings (Table 4.2). The Chla and Chlb shoot content significantly decreased (P < 0.001) in mature parts and remain unchanged in growing parts with increasing NaCl treatments (data not shown).

Axis labels are non-transformed.

Water status - There was no effect of inoculation on shoot xylem water potential and this parameter showed a linear decrease (P < 0.001) with increasing NaCl treatments (data not shown). That parameter exhibited values of -0.4 MPa in the control NaCl treatment and decreased until -0.8 in the 5.0 g Na l-1 of substrate treatment. Water content decreased with increasing NaCl treatments, and it was lower in growing and mature shoots of inoculated plants than in those of non-inoculated seedlings (P < 0.05) (Table 4.2, Figure 4.5). Of the 40 plants assessed in the present experiment, two inoculated seedlings dehydrated and died in the 5.0 g Na l-1 of substrate treatment. Non-inoculated seedlings at this treatment level showed similar water content but were still alive at the end of the experimental period.

Six months after sowing (during the tree nursery growth phase), inoculated seedlings were significantly smaller (11.9 cm and 1.6 g DM) than non-inoculated seedlings (12.7 cm and 2 g DM) (P < 0.05). At the time of harvest following the experiment, inoculated seedlings were still smaller (data not shown) and their growing shoots and roots (Table 4.3) exhibited a lower DM than non-inoculated seedlings (P < 0.05). In general, increasing NaCl treatments significantly reduced both height (< 0.05) and DM of shoots (< 0.05) and roots (< 0.05) (Table 4.3). However, the height of inoculated seedlings showed a slight increase with increasing NaCl treatments and the values observed converged with those of non-inoculated seedlings. Although increased NaCl did not significantly affect the shoot:root ratio, L. bicolor-inoculated seedlings invested more in biomass accumulation of growing roots (in percentage of the control NaCl treatment), which resulted in a significantly (P < 0.001) lower shoot:root ratio of growing parts (Table 4.3). Shoot biomass accumulation significantly decreased with increasing NaCl treatments (< 0.05) and was similar in inoculated and non-inoculated seedlings (Table 4.2, Figure 4.6). Root biomass accumulation declined linearly by increasing the amount of NaCl applied and inoculated seedlings maintained a higher accumulation than non-inoculated seedlings (P < 0.05) (Table 4.2, Figure 4.6). The regression curves indicated that biomass accumulation in roots was stimulated (> 100%) in inoculated seedlings in the 0.5 g Na l-1 of substrate treatment. Above this treatment, root biomass accumulation of both inoculated and non-inoculated seedlings was repressed and converged in the highest NaCl treatment (around 50-60%) (Figure 4.6).

SP/RP: shoot parts/root parts (growing vs mature parts); PP: plant parts (mature vs growing); IN: inoculation (L. bicolor vs T. americana)DM: dry mass

Axis labels are non-transformed.

The N, P, K, Ca, Mg content significantly (with at least P < 0.05) increased in shoot tissues with increasing NaCl treatment (data not shown). The main effect of L. bicolor on mineral accumulation was a significantly (P < 0.001) higher P content in shoot tissues of inoculated seedlings in all NaCl treatments (Table 4.4). In a lesser extent, N content was significantly higher in growing shoots of inoculated seedlings in the 0.5 and 1.0 g Na l-1 of substrate treatments (Table 4.4). The K content was also higher in inoculated seedlings than in non-inoculated seedlings although it was not significant. Growing and mature shoot parts of non-inoculated seedlings exhibited a significantly higher Na/Cl ratio than that of inoculated plants (P < 0.001) (data not shown). The ratios of Na/P, Cl/P, Na/K, Cl/K, Na/Ca, Cl/Ca, Na/Mg and Cl/Mg indicated that, relatively to the other elements, significantly more Na+ and Cl- were absorbed with increasing NaCl stress (data not shown). These ratios were also significantly higher (with at least P < 0.05) in non-inoculated than in inoculated seedlings.

SP: shoot parts (growing vs mature parts); IN: inoculation (L. bicolor vs T. americana)DM: dry mass

Inoculation with L. bicolor UAMH 8232 increased the resistance of nursery-grown jack pine seedlings to sodic stress until a threshold amount of NaCl applied. The photosynthetic, ChFl (i.e., F v/F m and ΦPSII) and water relation parameters indicated the level of stress (Kramer & Boyer 1995, Lichtenthaler 1996, Rohacek & Bartak 1999, Maxwell & Johnson 2000) and the rest of the measured variables served to describe the response of seedlings to each level of NaCl application. From this, three patterns of responses were detected in L. bicolor-inoculated seedlings compared to T. americana-colonized seedlings along the gradient of NaCl. First, in the control NaCl conditions, inoculated seedlings showed a lower biomass accumulation than non-inoculated seedlings while they exhibited a higher net photosynthesis and mineral acquisition (e.g., P). Second, in saline (hyperosmotic) and sodic (excess Na+) conditions below a threshold level situated somewhere between 1.0 and 2.5 g Na l-1 of substrate, inoculated seedlings showed increased root biomass accumulation, increased L. bicolor colonization, higher C fixation, light energy and mineral acquisition, and they accumulated less Na+ and Cl- in shoot tissues. Third, beyond that threshold level, the stress was intense and L. bicolor-inoculated seedlings tended to dehydrate faster than T. americana colonized seedlings.

In non-limiting conditions, roots colonized by L. bicolor, when compared to those of seedlings colonized by common nursery ECM fungi (e.g., T. americana), seemed to be a greater sink for fixed C. The host-fungus source-sink relationship is already widely accepted (Dosskey et al. 1990, 1991, Wu et al. 2002). It is possible that the sink effect was driven by fungal growth as suggested by seedling responses in the control NaCl treatment, in which, higher net photosynthetic rates and photochemical quenching values of mature needles of inoculated seedlings were not correlated with increased plant growth. The induced growth depression of jack pine seedlings following colonization with L. bicolor was also described by Kropp & Mueller (1999). A similar response has been recorded in other host - ECM fungus associations: L. laccata and H. crustuliniforme reduced Pinus sylvestris L. (scots pine) growth (Nylund & Wallander, 1989), and H. crustuliniforme reduced Pseudotsuga menziesii (Mirb.) Franco (Douglas fir) growth (Dosskey et al. 1990, 1991). Laccaria bicolor is a fast growing fungus that can develop an extensive energy demanding extraradical phase and may be a greater sink than T. americana. Furthermore, the higher derivation of resources from plant growth may have been generated by the larger portion of seedling short roots colonized by L. bicolor in inoculated seedlings. The present results suggest that inoculated seedlings allocated more resources to maintaining the efficiency of their resource collecting organs (i.e., chloroplasts and mycorrhizas) in mature and growing parts. By contrast, non-inoculated seedlings growing in non-limiting conditions showed increased shoot and root DM, with a higher investment of resources in shoot growth.

Without taking into consideration the inoculation effect, the general response of jack pine seedlings for the range of treatments from 0 to 1.0 g Na l-1 of substrate in the present experiment confirmed the results obtained by Apostol et al. (2002, 2004), Franklin et al. (2002a,b), and Franklin & Zwiazek (2004). Within the above range, the root biomass accumulation and physiological state of inoculated seedlings were greater than those of non-inoculated seedlings. There are three main reasons which may explain the potential beneficial effects of inoculation on the host physiology. Firstly, Na+ and Cl- contents were lowered in shoots of seedlings inoculated with L. bicolor. The L. bicolor membranes may have either exhibited a higher ion selectivity (Kottke 1992) or the fungal mycelium may have accumulated excess ions in its tissues (e.g., by vacuolization). It is also possible that the influence of the inoculated fungus modified the organic osmolyte (e.g., sugars) accumulation in its host tissues (Wedding & Harley 1976, Pfeffer et al. 2001) which favored the limited accumulation of excess ions in shoot (Niu et al. 1995, 1997). Secondly, in conjunction with the lower Na+ and Cl- shoot content, the growth improvement of inoculated seedlings was likely to be promoted by the higher production of carbohydrates. As stomatal conductance was similar, the main factor for the improvement of photosynthetic efficiency was possibly the higher quantum capture efficiency (ΦPSII) of the photochemical system of inoculated seedlings. Thirdly, L. bicolor increased content of N and P in growing shoots. The higher N (a major constituent of chlorophyll and rubisco) and P content of growing tissue of inoculated seedlings possibly indicated a higher potential for growth and metabolic activity under excess NaCl conditions. A consequence of this may be an improvement in the photochemical efficiency (i.e., ΦPSII, qP) of inoculated seedlings compared to non-inoculated controls.

Photosynthetic and ChFl responses, and Na+ and Cl- accumulation patterns changed at a threshold situated somewhere between 1.0 and 2.5 g Na l-1 of substrate. Above this threshold, the strategy of resistance (adaptive consequences) was obscured by the outset of degeneration of overly stressed seedlings (pathological consequences). As net photosynthesis nearly ceased above this threshold, it can be considered that the seedlings were in a different physiological state. Validity of statistical inference about plant resistance to lethal levels of NaCl was compromised as not all plants may have started to die off at the same rate and/or moment. Nevertheless, some conclusions can be drawn as the highest disturbance of the chlorophyll antenna coincided with a greater increase of Na and Cl contents in shoots of inoculated seedlings compared to non-inoculated ones. Possibly, to avoid a high physiological dehydration, inoculated seedlings accumulated toxic levels of Na+ and Cl- (Apostol et al. 2002, Franklin et al. 2002b, Franklin & Zwiazek 2004), which in turn affected photochemistry and eventually other essential mechanisms (Hasegawa et al. 2000, Papageorgiou & Murata 1995). Therefore, we suggest that this threshold corresponds to the physiological point where osmotic stress becomes intolerable and where Na+ and Cl- reach toxic concentration levels in shoot tissues (i.e., between 1.0 and 1.5% DM of Na, and/or between 2 and 3% DM of Cl). Beyond this point, toxicity increases or the plant dies from dehydration.

Beyond that hypothetical threshold, the production of organic osmolytes was possibly reduced to a minimum as a result of limited C fixation and disturbance of the light harvesting system. Inorganic osmolyte (e.g., Na+ and Cl-) contents of inoculated and non-inoculated seedling shoots differed with increasing NaCl treatments but xylem water potentials between the two types of seedlings kept similar. As L. bicolor-colonized roots may have maintained a stronger sink for sugars (which are potential osmolytes) from the shoot than T. americana-colonized roots, the importance of osmoregulation in the shoot via Na+ and Cl- may have increased faster in inoculated plants to maintain a similar xylem water potential. Laccaria bicolor is known to be sensitive to water stress (Coleman et al. 1989). This trait may have entered into conflict with the host’s requirements and thus limited benefits of the symbiosis once the fungus reached its limit of resistance to osmotic stress. Our study suggests that L. bicolor may be, in certain conditions, an exploitative ECM fungus (Bronstein 2001, Egger & Hibbet 2004). In addition, T. americana may have favored better water relations at these high osmotic stress levels. Guelh et al. (1992) observed that, although L. laccata improved turgor potential of inoculated Douglas fir, seedlings colonized with T. americana exhibited a higher hydraulic conductance and showed more resistance to drought stress. Furthermore, inoculated seedlings were also disadvantaged in the 5.0 g Na l-1 of substrate treatment by their smaller size at the outset of the experiment.

Finally, the present results highlight the need to consider using mycorrhizal inoculum in nursery stock production for revegetation of salt-affected sites. Thelephora americana and Wilcoxina sp. are common ECM fungi in tree nursery that readily colonize non-inoculated plants (Kernaghan et al. 2003). These fungi are adapted to the wet and nutrient rich conditions prevalent in nurseries (Hacskaylo 1965, Marx 1991) and should be considered as opportunistic species capable of colonizing roots of plants grown in inoculum poor substrate. They could be rapidly out competed after out-planting (Marx 1991). We suggest that, in the short-term, L. bicolor-inoculated nursery seedlings outplanted on sites in the range of tolerable sodicity and salinity (SAR1:5 of 12 to 18 and EC1:5 of 0.6 to 1 dS m-1, in the present study) will show increased root growth, mineral acquisition and improved light energy capture and net photosynthesis. Site adapted fungi have the capability to reduce those stresses perceived by plants at transplantation (Trappe 1977, Kropp & Langlois 1990, Malajczuk et al. 1994). Therefore, a potentially salt resistant fungus such as L. bicolor, should improve plant survival under saline and sodic conditions (dispersion of soil colloïds sets apart) and thus compensate for the initially smaller size of inoculated seedlings. If the fungus used is more resistant/tolerant to the main site stressor, it has the potential to rapidly colonize the new adjacent substrate and both protect and enhance the nutrient content of its host. Considering that soil resources and edaphic stressors are the main constraints for revegetation of degraded lands, the depressed biomass accumulation from the nursery phase may, with time, be compensated for by the greater overall efficiency of the mycorrhizal plant (Trappe 1977, Kropp & Langlois 1990). That strategy is likely to improve survival and overall growth in the long-term if drought periods (major determinant of salt concentration in the top soil) are not too long and/or intense. Otherwise, if the suggested threshold of Na+ and Cl- concentrations in the substrate and in the tissues is passed, L. bicolor inoculation would accelerate wilting of seedlings. T. americana-colonized seedlings were also likely to die off at these lethal levels of stress, being bigger they had resources to survive longer to extreme NaCl stress but not to grow. Therefore, revegetation of sites showing excess NaCl above the detected threshold or lightly salt-affected sites exposed to intense drought periods will require inoculation with a more resistant fungus.

This research was funded by Syncrude Canada Ltd. and NSERC (CRDPJ 250448-01 to D.P. Khasa). We wish to thank Julie Talbot and Yves Dubuc for all their help with plant physiological measurements, Alain Brousseau for elemental analysis, Michèle Bernier-Cardou for advices in statistical analyses, and Andrew Coughlan and Jean-Luc Jany for help in solving scientific problems. We also wish to thank Erin Bergrand and Claude Fortin for technical help and advice.

© Gregory Bois, 2005