Plant and Soil 238: 217–225, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
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Colonization with Hebeloma crustuliniforme increases water conductance and limits shoot sodium uptake in white spruce (Picea glauca) seedlings Tawfik M. Muhsin & Janusz J. Zwiazek∗ Department of Renewable Resources, 4–42 Earth Sciences Bldg., University of Alberta, Edmonton, Alberta, Canada T6G 2E3 Key words: Root hydraulic conductance, root respiration, sodium chloride, transpiration, N, P, K
Abstract White spruce [Picea glauca (Moench) Voss] seedlings were inoculated with Hebeloma crustuliniforme and treated with 25 mM NaCl to examine the effects of salinized soil and mycorrhizae on root hydraulic conductance and growth. Mycorrhizal seedlings had significantly greater shoot and root dry weights, number of lateral branches and chlorophyll content than non-mycorrhizal seedlings. Salt treatment reduced seedling growth in both nonmycorrhizal and mycorrhizal seedlings. However, needles of salt-treated mycorrhizal seedlings had several-fold higher needle chlorophyll content than that in non-mycorrhizal seedlings treated with salt. Mycorrhizae increased N and P concentrations in seedlings. Na levels in shoots and roots of salt-treated mycorrhizal seedlings were significantly lower and root hydraulic conductance was several-fold higher than in non-mycorrhizal seedlings. A reduction of about 50% in root hydraulic conductance of mycorrhizal seedlings was observed after removal of the fungal hyphal sheath. Transpiration and root respiration rates were reduced by salt treatments in both groups of seedlings compared with the controls, however, both transpiration and respiration rates of salt-treated mycorrhizal seedlings were as high as those in the non-mycorrhizal seedlings that had not been subjected to salt treatment. The reduction of shoot Na uptake while increasing N and P absorption and maintaining high transpiration rates and root hydraulic conductance may be important resistance mechanisms in ectomycorrhizal plants growing in salinized soil.
Introduction Mycorrhiza is a mutualistic association between fungi and plants. The main contribution of mycorrhizae has been thought to be the uptake of nutrients to the host plants (Allen, 1992; Harley and Smith, 1983; Smith and Read, 1997). Most forest trees form ectomycorrhizal associations which greatly improve tree survival and growth in boreal forests by improving nutrient uptake and water relations (Perry et al., 1987; Read, 1991). Little emphasis has been placed on the role of mycorrhizae in preventing ionic toxicity and salt stress. In some studies, arbuscular mycorrhizal fungi have been shown to reduce sodium uptake by plants and reduce plant yield losses in saline soils (Al-Karki, ∗ Corresponding author: Fax No: +1780 492-1767; E-mail:
[email protected]
2000; Azcon and ElAtrash, 1997). However, Hartmond et al. (1987) and Graham and Syversten (1989) found no differences in salt tolerance between citrus seedlings inoculated with vesicular–arbuscular mycorrhizae and non-mycorrhizal seedlings. The effects of ectomycorrhizal associations on salt stress resistance in forest plants are largely unknown. Elevated salinity is a major land reclamation problem following oil sands mining in the northern boreal forest (Renault et al., 1998, 1999). Therefore, if ectomycorrhizae were shown to be effective in alleviating salt effects, they could be used to help with the revegetation of salt-impacted areas. Salinity can have a dramatic effect on plants due to its osmotic action and direct ionic toxicity (Greenway and Munns, 1980). Sodium chloride has been shown to inhibit both stomatal conductance (Renault et al., 1999) and root hydraulic conductance (Carvajal
218 et al., 2000). The results of recent studies (Carvajal et al., 2000; Martinez-Ballesta et al., 2000) suggest that NaCl decreases the passage of water through roots by reducing the activity of water channel proteins. Plant water flow is controlled by two principal factors, the driving force and the hydraulic resistance of the flow pathway. The greatest hydraulic resistance in plants is present in the root system (Steudle and Peterson, 1998). In our earlier studies (Kamaluddin and Zwiazek, 2001; Wan and Zwiazek, 1999, 2001), we demonstrated that root water flow in woody plants is regulated by water channel proteins and that the function of root water channel proteins is strongly linked to root metabolism. Root water flow is affected by numerous internal and environmental factors including root surface area and volume, root anatomy (Steudle and Peterson, 1998), nutrients (Clarkson et al., 2000), and temperature (Wan et al., 1999). It has also been reported that water flux in both endomycorrhizal (Koide, 1985) and ectomycorrhizal (Coleman et al., 1990) plants is greater than that in non-mycorrhizal plants, which may be attributed to the indirect effect of nutrient status (Coleman et al., 1990). Therefore, it is conceivable that ectomycorrhizal associations may be effective in both reducing salt uptake and also maintaining high root water flow rates in plants exposed to salt stress conditions. The present study examined the effects of ectomycorrhizal associations of Hebeloma crustuliniforme (Bull. Ex St. Amans) Quel. with white spruce [Picea glauca (Moench) Voss] seedlings on root hydraulic conductance and growth of seedlings subjected to salt stress. White spruce is moderately sensitive to salt (Renault et al., 1998, 1999) and is among the principal species that will be used to revegetate salt-affected areas following oil-sands mining in northeastern Alberta, Canada (Renault et al., 1998). We hypothesized that ectomycorrhizae would help maintain balanced water relations under salt stress conditions by increasing root water flow rates. However, to prevent salt injury, the increase in the water delivery rates to shoots should be accompanied by a reduction in plant salt uptake.
Materials and methods
5247) was obtained from Dr. D. Khasa, Department of Renewable Resources, University of Alberta, Edmonton, Alberta. The fungus inoculum used in this study was prepared in a modified Melin Norkrans (MNN) liquid medium according to the method described by Mason (1980). Plant growth conditions Seeds of white spruce [Picea glauca (Moench) Voss] were germinated and seedlings grown for 10 months in 55 cm3 Spencer-Lemaire containers (Spencer-Lemaire Industries Ltd., Edmonton, Alberta) filled with mixture of peat moss and sand (3:1 by volume). The containers were placed on the bench of a growth chamber under a day/night thermal regime of 20/15◦C and 16-h photoperiod, photosynthetically active radiation (PAR) of approximately 350 µmol m−2 s−1 . After germination, seedlings were fertilized weekly with half-strength Hoagland’s mineral solution (Epstein, 1972) and watered every second day. One week prior to the mycorrhizal inoculation, fertilization was stopped. Seedling roots were periodically checked for fungal contaminants in plants randomly removed from the containers. Fungal inoculation and salt treatment The design of this experiment was 2×2 factorial with two levels of salt, 0 and 25 mM NaCl, for mycorrhizal and non-mycorrhizal seedlings. Fungal inoculation and salt treatment commenced simultaneously. Forty uniform seedlings were inoculated with a prepared culture of H. crustuliniforme by injecting with a syringe 20 ml of the mycelial suspension into each seedling container cell. The remaining 40 seedlings were left as non-mycorrhizal control. Both mycorrhizal and non-mycorrhizal seedlings were divided into two groups, each containing 20 seedlings. One group was treated with 25 mM NaCl, and the second, salt control, group received water. There was 50 ml of NaCl solution (water) added to each seedling cell at biweekly intervals, and between salt treatments the seedlings were regularly watered. The seedlings were subjected to this intermittent NaCl treatment for 10 weeks.
Fungal inoculum
Seedling morphology, elemental and chlorophyll analyses
A pure culture of the ectomycorrhiza Hebeloma crustuliniforme (Bull. Ex St. Amans) Quel. (isolate no.
Mycorrhizal and non-mycorrhizal roots were prepared for cryo-scanning electron microscopy (cryo-SEM).
219 Roots, 1-cm long segments, were frozen in liquid nitrogen using a cryophilizer (Emitek 1250, UK) and fractured. The samples were transferred to a cryostage for coating and viewed with a cryo-SEM (JSM 6301, JEOL Ltd., Tokyo, Japan) kept at −150◦C. At the end of 10 weeks of growth, the seedlings were harvested for dry weight assessments. Roots and shoots were separated and dried in an oven at 60◦C for 24 h. Dry root and shoot samples were subjected to Kjeldahl digestion (Richard, 1993) and tissue N and P contents were determined using a color photometric analyzer (Technicon Autoanalyzer II, Tarrytown, NY). Na and K concentrations were determined with a Model 503 Atomic Absorption Spectrophotometer (Perkin-Elmer, Norwalk, CT). Chlorophyll contents in fresh needles were determined in 80% acetone extracts as described by (Sestak et al., 1971). For determination of mycorrhizal infection, soil was gently removed from roots and the mycorrhizal infection percentages per root segment length were estimated by direct microscopic examination of the new roots colonized by hyphal sheath (Schenck, 1982). Root hydraulic conductance Root hydraulic conductance (Kr ) was measured in intact roots using a High Pressure Flow Meter (HPFM) (Dynamax Inc, Houston, TX) as described by Tyree et al. (1995). This method allows for measurements of undisturbed roots since the water is applied under increasing pressures through an excised stem into the root system (Tyree et al., 1995). In the present experiment, the stems were excised at 2 cm above the root collar and flow rates were measured over a range of pressures from 0 to 2.75 MPa to obtain a linear pressure – flow relationship (Tyree et al., 1995). Flow rate measurements were made for six seedlings per treatment. After the measurements, roots were gently washed in water to remove the mycelial sheath, and Kr measured again. This was done in order to examine the contribution of external fungal sheath to root hydraulic conductance. A similar washing treatment was also applied to non-mycorrhizal roots and Kr was measured before and after washing the roots. Transpiration Transpiration rates were measured with a steady state porometer (LI – COR 1600 Lincoln, NE, USA) as described earlier (Wan and Zwiazek, 1999). Measure-
ments were performed on the uppermost branches, 5–8 h following the onset of the photoperiod. Root respiration Root respiration was measured using model 5000 dissolved oxygen meter (YSI, Yellow Springs, OH) as previously described (Wan and Zwiazek, 1999). Root samples were removed from the containers, washed and placed in a tightly closed cylinder with distilled water that was gently stirred with a magnetic stirrer. Oxygen uptake was measured in 5-min intervals for 30 min. Statistical analysis The data were analyzed by analysis of variance to determine the main and interactive effects of two factors: mycorrhizal inoculation and salt treatment on growth and physiological variables. Means were compared for significant differences at P < 0.05 using the Tukey’s test.
Results Seedling morphology and growth Although all inoculated salt treated and control roots were mycorrhizal, the amount of root tissue infected by fungus was reduced by almost 50% in salt-treated seedlings compared with untreated seedlings. Cross sectional views of roots of non-mycorrhizal and mycorrhizal seedlings are shown in Fig. 1. Mycorrhizal roots showed profuse growth of the fungal hyphae around the root epidermis and had hyphal elements of the Hartig net located in the apoplast, mostly in the intercellular spaces between the cortical cells. Nonmycorrhizal roots did not show presence of fungal hyphae in and around roots (Fig. 1). The shoot and root dry weights, number of lateral branches and total needle surface area were significantly (P < 0.05) greater in mycorrhizal seedlings than in non-mycorrhizal seedlings (Table 1). With the exception of root dry weight, salt treatment significantly decreased growth parameters in mycorrhizal and nonmycorrhizal seedlings. This resulted in an increase in root to shoot dry weight ratios of salt-treated seedlings (Table 1). With few exceptions, the effects on mycorrhizal inoculation and salt treatment on growth and morphological variables were the results of main effects rather than interactive effects (Table 2).
220 Table 1. Growth variables, chlorophyll contents and mycorrhizal infection estimates of white spruce seedlings in response to mycorrhizal inoculation and 25 mM NaCl. Means followed by different letters in the same row are significantly different at P < 0.05 (Tukey’s test of ANOVA) Variable
N
Number of branches Needle surface area (cm2 ) Shoot DW (g) Root DW (g) Total DW (g) Root:shoot DW ratio Chlorophyll (mg g−1 FW) Mycorrhizal infection (%)
20
Nonmycorrhizal
22 b
Nonmycorrhizal + NaCl 16 c
Mycorrhizal
Mycorrhizal + NaCl
25 a
19 b
10
9.7 c
8.2 d
14.6 a
10.9 b
10 10 10 10
6.4 b 4.1 b 10.5 b 0.65 b
5.0 c 4.3 b 9.3 c 0.85 a
7.3 a 4.5 a 11.9 a 0.62 b
5.9 b 4.9 a 10.8 b 0.82 a
6
6.4 c
2.3 d
13.7 a
9.2 b
10
0.0 c
0.0 c
52 a
29 b
Table 2. Results of factorial analysis for different variables in response to mycorrhizal inoculation and salt treatment showing the levels of significance (P) Variables
Mycorrhizal inoculation
NaCl treatment
Interaction
Number of branches
<0.001
<0.001
0.510
Needle surface area
<0.001
<0.001
0.083
Shoot DW Root DW Root-shoot DW ratio Total plant DW Chlorophyll Mycorrhizal infection estimate Shoot N Root N Shoot P Root P Shoot K Root K Shoot Na Root Na Transpiration Root respiration Root hydraulic conductance (Kr )
0.001 0.033 0.502 0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.043 0.034 <0.001 <0.001 <0.001 <0.001
<0.001 0.265 <0.001 0.008 <0.001 <0.001 0.086 0.388 0.239 0.365 0.013 0.054 0.001 <0.001 <0.001 <0.001
0.873 0.686 0.935 0.897 0.325 <0.001 0.029 0.114 0.218 0.611 0.969 0.270 0.951 0.026 <0.001 0.060
Intact roots Washed roots
<0.001 <0.001
0.017 <0.001
0.275 0.664
221
Figure 1. Cross-sections of roots viewed with a cryo-scanning electron microscope showing (A) cortical cells of the non-mycorrhizal root, (B) mycorrhizal root with fungal hyphae surrounding the epidermis, (C, D) fungal hyphae in the intercellular spaces of the root cortex (arrows).
Tissue chlorophyll content and elemental analysis
Mycorrhizal inoculation and salt stress had strong effects on needle chlorophyll content. Needles of mycorrhizal seedlings had more than 2-fold higher chlorophyll content than non-mycorrhizal seedlings (Table 1). Salt drastically lowered needle chlorophyll content of mycorrhizal seedlings, but in mycorrhizal
some decline of chlorophyll needle chlorophyll content was also measured (Table 1). Both roots and shoots of mycorrhizal seedlings had higher N and P contents than those of non-mycorrhizal seedlings (Fig. 2A,B). In mycorrhizal seedlings, shoot P exceeded root P concentration (Fig. 2B). Salt treatment had little effect on N and P levels in mycorrhizal and non-mycorrhizal seedlings (Fig. 2A,B). In con-
222
Figure 2. Effects of NaCl treatment on nitrogen (A), phosphorus (B), potassium (C) and sodium (D) contents in shoots and roots of mycorrhizal and non-mycorrhizal white spruce seedlings. Means + SE (n=6) are shown. Different letters above the bars indicate significant differences at P < 0.05 (Tukey’s test). N, non-mycorrhizal seedlings; NS, non-mycorrhizal seedlings treated with 25 mM NaCl; M, mycorrhizal seedlings; MS, mycorrhizal seedlings treated with 25 mM NaCl.
trast, K levels in seedlings increased as a result of both fungal inoculation and salt treatment (Fig. 2C). Only traces of Na were detected in seedlings that were not treated with salt (Fig. 2D). Na concentration in salt-stressed non-mycorrhizal seedlings measured approximately 15 mg g−1 DW in the roots and 7.5 mg g−1 DW in the shoots (Fig. 2D). In the salt-treated mycorrhizal seedlings, there was approximately 6 mg g−1 DW of Na present in the roots and 0.4 mg g−1 DW present in the shoots (Fig. 2D).
Figure 3. Transpiration rate (A), root respiration (B), root hydraulic conductance (C) in mycorrhizal and non-mycorrhizal seedlings treated with NaCl. Means + SE (n=6) are shown. Different letters above the bars indicate significant differences at P < 0.05 (Tukey’s test). N, non-mycorrhizal seedlings; NS, non-mycorrhizal seedlings treated with 25 mM NaCl; M, mycorrhizal seedlings; MS, mycorrhizal seedlings treated with 25 mm NaCl.
Transpiration, root respiration and root hydraulic conductance Transpiration rates in mycorrhizal seedlings were almost 2-fold higher than non-mycorrhizal seedlings (Fig. 3A). Salt treatment reduced transpiration rates in both mycorrhizal and non-mycorrhizal seedlings. The
223 reduction in transpiration rates of mycorrhizal seedlings by salt brought them to the level measured in non-mycorrhizal plants that had not been treated with salt. Similarly to transpiration, root respiration rates were higher in mycorrhizal than the non-mycorrhizal plants (Fig. 3B). Respiration rates declined in salttreated roots, but were still higher in mycorrhizal seedlings than in the non-mycorrhizal seedlings. Root hydraulic conductance (Kr ) was approximately 4-fold higher in mycorrhizal seedlings than in non-mycorrhizal seedlings (Fig. 3C). In mycorrhizal seedlings, Kr declined to about half of the initial value after the roots were washed to remove the fungal sheath (Fig. 3C). Salt treatment reduced Kr in nonmycorrhizal and mycorrhizal seedlings; however, Kr in both unwashed and washed mycorrhizal seedlings that had been treated with salt was still higher than that measured in non-mycorrhizal untreated seedlings (Fig. 3C).
Discussion In the present study, white spruce seedlings received weekly supply of mineral nutrients prior to the ectomycorrhizal inoculation and no additional nutrients were supplied following inoculation. This was done to promote ectomycorrhizal establishment (Wallander and Nylund, 1992). Similarly to other studies (Jentschke et al., 2000; Molina, 1982; Quoreshi and Timmer, 1998; Shaw et al., 1982), our data showed a pronounced effect of ectomycorrhizal inoculation on growth of white spruce seedlings. Higher absorption of N and P by mycorrhizal plants increases their growth rates is well documented (for review see Harley, 1989). It is also well established that salinity can inhibit growth and cause injury and death of plants (Hagemeyer, 1997; Munns, 1993). Salt can affect plants directly due to ionic toxicity, or indirectly, due to osmotic imbalance (Munns, 1993). In our study, Na levels increased dramatically in the shoots and roots of NaCl-treated plants. The presence of ectomycorrhizal associations with Hebeloma crustuliniforme almost fully prevented Na accumulation in the shoots and reduced Na root content to less than 50% of that present in the non-mycorrhizal roots (Fig. 2D). It appears that the role of ectomycorrhizae in alleviating salt stress of white spruce was partly by preventing Na transfer from roots to shoots. Na can alter a number of cell processes including enzyme activity, protein synthesis
and membrane permeability and trigger tissue necrosis (Alam, 1994; Kuiper, 1984). In our study, chlorophyll content dramatically declined and leaf chlorosis was observed in salt-treated plants. This chlorosis was not present in mycorrhizal seedlings treated with salt. Although there may be numerous reasons for low chlorophyll contents in salt-treated plants, it has been suggested that Na has an antagonistic effect on Mg uptake (Bernstein, 1975). Mg is required in relatively large quantities for chlorophyll synthesis and mycorrhizal seedlings are known to be effective in the absorption of Mg under nutrient limiting conditions (Jentschke et al., 2000). In our study, root hydraulic conductance (Kr ) was about 4-fold higher in ectomycorrhizal seedlings than in non-mycorrhizal seedlings. Park et al. (1983) hypothesized that variable responses of plant water relations to mycorrhizae may be related to the different fungal species associated with plant roots. Mineral nutrients can also profoundly alter root hydraulic conductance through their effect on water channel proteins (Clarkson et al., 2000). Relatively high P and N levels that were measured in mycorrhizal seedlings could be partly responsible for high root conductance values. However, root hydraulic conductance declined by about 50% after removing the fungal hyphae. This suggests that the mycorrhizal extraradical mycelium directly contributed to root hydraulic conductance, contrary to the study by Sands et al. (1982) which reported an increase in the resistance to root water flow by the mycelium. It is also possible, that the ectomycrorrhizal hyphae extending to the root cortex reduced resistance to root water flow associated with the apoplastic transport (Steudle and Peterson, 1998). Future studies will be conducted with different mineral nutrition levels to address this issue. In our study, Kr was also reduced as a result of salt treatment. Salinity can have a remarkable effect on plant water relations (Bolanose and Longstreth, 1984; Greenway and Munns, 1980; Renault et al., 1999). It has been demonstrated that NaCl reduces hydraulic conductance due to its direct effect on water channel proteins (Carvajal et al., 2000; Martinez-Ballesta et al., 2000). Although we did not examine the function of root water channels in salt-stressed plants, it is possible that the effect of NaCl on the activity of water channels was also responsible for the reduction in Kr values observed in our study. It is noteworthy that root hydraulic conductance in salt-treated mycorrhizal seedlings was at the level of non-mycorrhizal seedlings that had not been treated with salt. This indicates
224 that ectomycorrhizae can be effective in maintaining relatively high root water flow rates in intermittently or moderately saline soils. In conclusion, our results demonstrated that Hebeloma crustuliniforme was effective in reducing sodium uptake by white spruce seedlings and helped maintain high root respiration rates, root hydraulic conductivity and transpiration. Both the extraradical mycelium and the hyphae of the Hartig net contributed to increasing root hydraulic conductance. In the present study, the increase in N and P uptake by mycorrhizal roots was the most likely mechanism of salt stress reduction and responsible for high rates of water conductance. However, more studies involving different fertilization regimes will be necessary to clearly separate direct and indirect effects of ectomycorrhizae on plants exposed to elevated salt levels. The reduction of shoot Na uptake while increasing N and P absorption and maintaining high transpiration rates and root hydraulic conductance may be important in helping ectomycorrhizal plants survive in salinized soil.
Acknowledgements We gratefully acknowledge research funding for this project from the Natural Sciences and Engineering Research Council of Canada and the Environmental Sciences and Technology Alliance Canada. We also thank Dr. D. Khasa for providing fungal culture and Dr. M. Kamaluddin for help with the manuscript and statistical analysis of the data.
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