spruce (Picea abies) seedlings grown in semi-hydroponic sand culture

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1 New Phytol. (1996), 133, Growth and mineral nutrition of nonmycorrhizal and mycorrhizal Norway spruce (Picea abies) seedlings grown in semi-hydroponic sand culture I. Growth and mineral nutrient uptake in plants supplied with different forms of nitrogen BY LUDGER ELTROP.\ND HORST MARSCHNER Institute of Plant Nutrition, University of Hohenheim, Stuttgart, Germany {Received 26 June 1995; accepted 10 January 1996) S U M M.\ R Y Growth, nitrogen uptake and mineral nutrient concentrations in the plant tissues were determined m nonmycorrhizal and mycorrhizal Norway spruce (Picea abies (L.) Karst.) seedhngs grown under controlled conditions in a semi-hydroponic culture system with quartz sand as substrate and a percolating nutrient solution. The culture system allowed the determination of nutrient uptake rates in mycorrhizal root systems with an intact extramatrical mycelium. The rate of infection of the roots by the mycorrhizal fungi and Laccaria laccata was high but the rate of infection by Paxillus involutus was low. When supplied with ammonium nitrate, the d. wt of the roots and particularly of the shoots was.significantly lower in mycorrhizal than in non-mycorrhizal plants. Despite the lower root d. wt, the number of root tips and the root branching ratio (number of root tips per unit root length) were significantly higher in mycorrhizal plants infected with L. laccata and P. tinctorius than in non-mycorrhizal plants. The depletion of ammonium in the external solution was faster than the depletion of nitrate. Nitrate uptake rates increased at ammonium concentrations below 400/«l. The maximal N uptake rates (I^,,as)i calculated after Lineweaver-Burk. were significantly higher for ammonium than for nitrate. The N uptake rates did not differ significantly between nonmycorrhizal and mycorrhizai plants. The concentrations ot N, P, K, Ca and Alg tended to be higher in the smaller mycorrhiza! than in the larger non-mycorrhizal plants.. \ significant increase in mineral nutrient concentration in mycorrhizal compared with non-mycorrhizal plants was found only for N concentrations in the needles of mycorrhizal plants infected with P. tinctorius. When they were supplied with ammonium ((NHj),SOj) as source of N, but not when they were supplied with nitrate (KNO,,), the d. wt was lower in mycorrhizal plants infected with P. tinctorius than it was in nonmycorrhizal plants, llierefore, N uptake rates were increased in mycorrhizal plants with P. tinctorius only when they were supplied with ammonium but not with nitrate. The insignificant differences in uptake rates of N, P, K, Ca and Mg between non-mycorrhizal and mycorrhizal plants indicate that at unlimited spatial nutrient availability the contribution of the extramatrical mycelium to nutrient uptake by mycorrhizal plants was small. It is suggested that the decreased growth of mycorrhizal plants is due to the demand of the mycorrhizal fungus for photosynthates, i.e. source limitation. Key words: Ectomycorrhiza, growth, mineral nutrient uptake, nitrogen, semi-hydroponic sand culture. 1983; Jones, Durall & Tinker, 1990; Tam & INTRODUCTION.Q,,.,nn,\ r^ r l r 'UI Griffiths, 1994). One of the major factors responsible In solid substrates like soil, peat, or soil-sand for this growth enhancement is the increased uptake mixtures, where nutrient availability can be spatially of mineral nutrients which is brought about by the limited, mycorrhizal infection of tree seedlings as a increase in the absorptive surface area of mycorrhizal rule increases growth of mycorrhizal compared with roots because of the extramatrical mycelium (Harley, non-mycorrhizal plants (Reid, Kidd & Ekwebelam, 1989). In particular, P uptake is enhanced by the

2 470 L. Eltrop and H. Marschner enlargement of tbe depletion zone around tbe roots and by increased hyphal transport of P to tbe roots (Bolan, 1991), In mycorrbizal Pi?ius sylvestris increased N uptake was also largely attributed to absorption and transport by tbe extramatrical mycelium (Finlay et al., 1988). Higber nutrient uptake in mycorrbizal plants may also be caused by greater specific nutrient uptake rates in mycorrbizal tban in non-mycorrbizal roots. Increased specific uptake rates per unit d, wt or surface area m mycorrbizal roots were found for N (Boxman & Roelofs, 1988; Hogberg, 1989) and P (Langlois & Fortin, 1984), However, ammonium and nitrate uptake might be niore affected by changes m the ph of tbe external solution than by mycorrhizal infection (Rygiewicz, Bledsoe & Zasoski, 1984a, b). By contrast, Ingestad, Arveby & Kahr (1986) and Scheromm, Plassard & Salsac (1990) found no significant differences in N uptake rates between non-mycorrhizal and mycorrhizal pine seedlings. These results are in line with observations that N uptake differs considerably between species (Littke, Biedsoe & Edmonds, 1984) and strains (Kieliszewska-Rokicka, 1992) of mycorrhizal fungi, at least when grown in vitro. Nutrient uptake in mycorrhizal roots is also considerably affected b>' the chemical properties of the fungal cell walls, Ashford et al. (1988, 1989) demonstrated the existence of two apoplastic blockages in the sheath and exodermis of eucalypt and Pisonia mycorrhizas, which are likely to restrict the exchange between the roots and the soil. The surface of the fungal mycelium can also be strongly hydrophobic (Unestam, 1991), raising doubt about its general role as the major nutrient-absorbing organ of mycorrhizal root systems. Nutrient and water uptake might thus be confined to certain parts of the funga! mycelium (e.g. growing hypha! tips) and to the symplastic pathway (Ashford et al., 1989), On the other hand, in non-mycorrhizal roots specific nutrient uptake rates might also be high, particularly in white and non-suberised root zones (Haussling et al., 1988), For studying nutrient uptake in mycorrhizal tree seedlings, hydroponic or semi-hydroponic culture systems have been developed. In these systems, materials like quartz sand (Jentschke, Godbold & Huttermann, 1991), brick pellets (Wallander, 1992), perlite (Colpaert, van Assche & Luijtens, 1992) or filter paper (Plassard et al., 1994) were used as support for fungal growth, or a diluted nutrient solution was supplied periodically as a thin film on PVC plates (Ingestad et al., 1986; Kamminga-van Wijk, 1991), The aim of the present study was to compare growth and nutrient uptake in non-mycorrhizal and mycorrhizal Norway spruce seedlings in a culture system which allows measurements in intact mycorrhizal root systems under axenic conditions. In this culture system, the role of the extramatrical mycelium in growth and mineral nutrient uptake in mycorrhizal plants should be eva!uated indirect!y by exc!uding spatia! limitations of nutrient availability. MATERIALS AND METHODS The semi-hydroponic sand culture system The main components of the semi-hydroponic sand culture system are shown in Figure 1. The plants were cultivated in 25 cm-long glass vials (diameter 3 cm), filled with 2-3 mm coarse acid-washed quartz sand. The shoots were exposed to ambient environmental conditions while the roots were grown axenically in the glass via!s, Erlenmeyer flasks containing the nutrient solution were connected to the glass vials with silicone tubing. By means of a peristaltic pump the nutrient solution was percolated through the system at rates which ensured that the roots were covered with a thin layer of nutrient solution and that the glass vials contained mainly air. Prior to the transfer of plants to the glass tubes, the Erlenmeyer flasks and silicon tubes were sterilized by autoclaving. Treatment of plant material Seeds of Picea abies (L.) Karst. from a forest stand on acid soil were surface-sterilized in 35 o H.,O., for Seedling Aeration tube Silicone cap - - Quartz sand Coarse quartz sand and nylon net,,,. Erlenmeyer flask with nutrient solution Coachwork Glass tube Peristaltic pump Figure L Schematic presentation of the semi-hydroponic sand culture system used in the present study. The main parts are (i) glass tubes as culture vessels for the plants, (ii) Erlenmeyer flasks for the supply of nutrient solution and (iii) a peristaltic pump by which the nutrient solution is percolated through the culture system.

3 Growth and mineral nutritiori of Norway spruce, I min then thoroughly rinsed with distilled water. For germination, seeds were plated out on nutrientmalt agar in Petri dishes and were put in the dark for 10 d at 24 C (Eltrop, 1993). Sterile seedlings with well-developed radicles (1-3 cm) were selected and transferred into glass jars filled vi,'ith 500 ml of perlite and 250 ml of fivefold-concentrated nutrient solution. The glass jars were tightly sealed and the seedlings were grown for 3^ wk under full light at 24 C and 16 h day length in a growth chamber. The seedlings were then selected for uniformity and were transferred to the culture vessels by inserting the roots into the glass tubes through a hole in the silicone cap. The plant stems were tightly sealed with autoclavable coachwork putty. After transfer, the seedlings were supplied with fivefold-concentrated nutrient solution for 10 wk to obtain vigorous plant growth. Before inoculation, the concentration of the nutrient solution was reduced to the basic strength, leading to significantly reduced rates of shoot growth. The nutrient solution was permanently percolated through the glass \-ials at a rate of 0-3 ml min"' and was changed once or twice a week, depending on the growth of the seedlings. The composition of the nutrient solution of basic strength was as follows: macronutrients (mm): 0-4, NH4NO3; 0-05, KH.,PO,; 0-25, KCl; 0-1, MgSO^; 0-2, CaSO,,; 0-02, Na.jSO^; micronutrients (/tm): 55, H3BO.,; 5, MnSO^; 6-3, ZnSO,; 0-32, CuSO,; 0-1, Na,MoO.,; 9-5, Fe-EDTA. The ph was adjusted to 5-0"with H.,SO,. Treatment of fungal material and tnycorrhizal inocidation The mycorrhizal fungi were cultured on liquid nutrient-majt media in constantly agitated Erlenmeyer flasks (Eltrop, 1993). Before inoculation, the fungal mycelium was homogenized with an L^ltra Turrax. For inoculation, 5 ml of the mycelial suspension was injected into the glass vials. Three different mycorrhizal fungi, isolated from acid soils, were used in the experiments: L. laccata (Maire) P. D. Orton, strain S 238 H, isolated from Tsuga mertensiana; P. involutus (Batsch.) Fr. strain NAL'; and P. tinctorius (Pers.) Desv. Eight plants were inoculated with each mycorrhizal fungus, and another eight plants received the same amount of the fungal media with autoclaved mycelium (non-mycorrhizal plants). Glass vials containing quartz sand and no plants served as a control. Plants were kept in a growth chamber at 22/18 C (day/night) and 16 h day length at a light intensity of 320/miol m"'" s"'. Nutrient uptake and short-term uptake rates of ammonium and nitrate Long-term nutrient uptake was measured b\' determining the mineral-nutrient content of needles and roots. Short-term uptake rates of nitrogen were measured by determining the depletion of ammonium and nitrate from the media. Experiment 1 : Supply of N as ammonium nitrate {NH^NO.:^). Plants were supplied with a complete nutrient solution containing 800 //m NH4NO3. Mycorrhizal plants were inoculated with L. laccata, P. involutus or P. tinctorius 10 weeks after transfer of the plants into the culture vessels. Short-term uptake rates of ammonium and nitrate were determined in weeks 21 and 22 after the start of the culture. Experiment 2: Supply of N as either ammojiium {{Nli^.,SO^) or nitrate {KNO^). Until successful formation of mycorrhizal roots, plants received a complete nutrient solution containing 800 /J.M NH^NOg. jmycorrhizal plants were inoculated with P. tinctorius in week 14 after transfer of the plants into the culture ^'essels. In week 17, NHjNOg, as source of N in the nutrient solution was replaced by 800//M of N as (NHJ.^SO^ or KNO3. Short-term uptake rates of ammonium and nitrate were determined in weeks 21 and 22 after the start of the culture. Prior to the determination of the short-term uptake rates of ammonium and nitrate, plants were deprived of nutrients for 48 h by supplying them with distilled water only. Thereafter, plants were supplied with a complete nutrient solution (for composition, see above) where nitrogen was varied according to expts 1 and 2. Uptake experiments were carried out at a temperature of 22 C (day and night) over 5 d. Nutrient uptake was determined by measuring the depletion of mineral nutrients in the nutrient solution. Samples of the nutrient solution were taken every 24 h. L'ptake rates were calculated for the period of constant nutrient uptake, w'hich was determined in preliminary experiments. Root f. wt was determined after the end of the experiments. The uptake rates were plotted against the supplied N concentrations. The substrate affinity parameter K^ and the maximal uptake rate I\,,a^ were calculated from the double reciprocal plot (Lineweaver-Burk transformation) of the substrate concentration and corresponding uptake rate. After the uptake experiments, the ph of the nutrient solution was determined with a glass electrode. Owing to the imbalance in cation/anion uptake and the preferred ammonium uptake in Norway spruce, the ph of the NH,^-N-containing solution generally decreased during the course of the experiments. Therefore, in expt 2, the nutrient solution was buffered with 4 nim MES (2-morpholinoethansulphonic acid). Analysis After termination of the short-term uptake experiments, plants were harvested. The roots were rinsed

4 472 L. Eltrop and H. Marsckner with distilled water and the fresh and dry weights of the plants were determined. The number of nonmycorrhizal and of mycorrhizal root tips was counted under a dissecting microscope. Mycorrhizal roots were checked under a microscope for the presence of a Hartig net. For the determination of mineral elements, the plant material was dried at 60 C immediately after harvest. After grinding and dry-ashing at 550 C, P was measured photometrically, K, Ca and Na by flame emission spectrometry, Mg and Mn by atomic absorption spectrometry and N by an automatic nitrogen analyser (ANA). Ammonium and nitrate in the nutrient solution were measured by an autoanalyser (Technicon II). Statistics Diagrams are presented with SE bars. The means of treatment levels were checked for statistically significant differences with one or two factorial analysis of variance (ANOVA) and Scheffe-test. Means followed by the same letter are not significantly different at F < 0-1. RESULTS Supply of N as ammonium nitrate In the first 60 d of culture, plant growth was slow even though the plants were supplied with a fivefoldconcentrated nutrient solution. This was probably caused by the poor contact between the roots and the quartz sand, which resulted in a low nutrient supply to the seedlings. After inoculation in week 10, shoot length increment decreased in mycorrhizal compared with non-mycorrhizal plants (data not shown). The decrease was most pronounced in mycorrhizal plants with L. laccata. After 22 wk of growth, shoot length of mycorrhizal plants was between 62% (with L. laccata) and 70 b (with P. tinctorius) of that of nonmycorrhizal plants. Plant dry weight at harvest was considerably lower in mycorrhizal than in non-mycorrhizal plants (Table 1). Shoot d. wt had decreased more than root d. wt, thus the shoot: root ratio was slightly lower in mycorrhizal than in non-mycorrhizal plants. At 65 "o of the d. wt of non-mycorrhizal plants, the dry weight reduction was most distinct in mycorrhizal plants with L. laccata. Despite the significantly lower root d. wt, the total number of root tips and the root branching ratio (number of root tips per unit root length) was significantly higher in these plants than in the non-mycorrhizal plants (Table 2). In mycorrhizal root tips with L. laccata and P. tinctorius the Hartig net was well developed and a completely closed hyphal sheath at the root tip had formed. In mycorrhizal plants with L. laccata, 83-3 %, and in mycorrhizal plants with P. tinctorius, 85-6 o of lateral roots were mycorrhizal (Table 2). Plants inoculated with P. involutus were less infected (32-3 b), and well-developed mycorrhizas were restricted to the upper and drier parts of the culture vessels. The concentration of N in needles, stems and roots was generally higher in mycorrhizal than in non-mycorrhizal plants, but to a significant degree only in the needles of mycorrhizal plants with P. tinctorius (Table 3). The total N accumulation after 22 wk of growth was not significantly different between the treatments, with the exception of mycorrhizal plants with L. laccata in which, owing to the decrease in d. wt compared with that of the non-mycorrhizal plants, total N accumulation was significantly lower (data not shown). The distribution of N between roots and shoots was affected by mycorrhization only with P. tinctorius, where 41 "o of total accumulated N was found in the needles compared with 35 "o in non-mycorrhizal plants. The P concentrations in roots and shoots did not differ significantly between non-mycorrhizal and mycorrhizai plants with the exception of significantly higher P concentrations in mycorrhizal roots with L. laccata (Table 3). Owing to the lower d. wt of mycorrhizal plants (Table 2), the total accumulation of P (data not shown) was lower than in nonmycorrhizal plants, particularly in mycorrhizal plants with L. laccata. Nor did the concentrations of K, Ca and Mg differ Table 1. Shoot and root dry weight and the shoot j root ratio in 22-wk-old non-mycorrhizal and mycorrhizal Norway spruce seedlings grown in semihvdroponic sand culture and sitpplied with NH^A^O^ (means+ SD, n = 6) Dry weight (g) Shoots Roots l^otal Shoot/Rootratio Laccaria laccata Paxillus involutus 2-11 ±0-19 a* 1-61 ±0-26 a 3-72±0-4a 1-.3 a b l-09±0i6b b 1-2 a l-65±0-s6ab l-36±o-36ab ab 1-2 a 1-61 ±0-33 ab l-3o±o-23ab ab 1-2 a * Means followed by the same letter are not significantly difterent at P < 0-1 in the Scheffe test.

5 Growth and mineral niitriticm of Norway spruce, I 473 Table 2. Number of root tips, mycorrhizal infection and the root branching ratio in 22-wk-old non-mycorrhizal and mycorrhizal Norway spruce seedlings grown in semi-hydroponic sand culture and supplied loith N D, n = 6) Root tips Total Mycorrhizal Mycorrhizal infection rate - ( o root tips of total) Branching ratio (tips cm"') Laccaria laccata Paxillus involutus 3548 ±1046 a* S168±2327b 2445 ±892 a 3869±1879a 4305 ±1087 b 790±223 a 3312±867b 83-3 b 32-3 a 85-6 b 1-4 a 2-6 b 1-5 a 2-3 ab * Means followed by the same letter are not significantly different at P sj 0-1 in the Scheffe test. Table 3. Concentrations {mg g ^ d. wt) of N, P, K, Ca and Mg in Jieedles, stems and roots of 22-wk-old nonmycorrhizal and mycorrhizal Norway spruce seedlings grown in setni-hydroponic sajid culture and supplied with {means ±SD, n = 6) N K Ca Needles Non-inycorrhizal Laccaria laccata Paxillus involuttis Stems Laccarta laccata Paxillus iitrolutus Roots Non-niycorrhizaJ Laccaria laccata Paxillus ittvolutus 7-9 ±0-9 a 9-9±2-2ab ll-6±2-] ab 13-3±24b 5-5±0-8a 7-2 ±0-6 a 7-9±M a 7-7±l-6 a 12-5 ±0-4 a 13-5 ±3-0 a 15-7±5-5 a O-51±O-12a 0-65 ±0-07 a 0-53±0-08 a 0-55 ±0-10 a 0-54 ±0-09 a 0-67±0-16a 0-55 ±0-07 a 0-62±0-l] a 0-81 ±0-08 ab 0-99 ± 0-10 b 0-78 ±0-09 a ab 3-9±0-8a 3-9±0-4a 4-2±0-6ab 4-4±M b 3-6±0-6a 5-1 ±0-9 b 4-3±0-2ab 4-4±0-7ab 7-2±l-0a 8-0±l-2a 6-3 ±1-5 a a l-8±o-3a 2-3 ±0-7 a 2-1 ±0-9 a I-9±0-6a 3-O±O-8 a 2-4±0-l a 2-4±0-2a 2-3 ±0-2 a 1-5 ±0-1 ab 2'l±O-3b 1-6 ±0-3 ab a 1-63 ±0-2 a 2-11 ±0-5 a 1-99 ±0-8 a l-74±0-5a 0-78±0-l a 0-97 ±0-1 a 0-75±0-2 a ()-8]±0-2a 0-60 ±0-1 ab 0-66±0-l b 0-49 ±0-1 a 0-49 ±0-1 a Means followed by the same letter are not significantly different at P s^0-\ in the Scheffe test. Table 4. Shoot and root dry weight, shoot: root ratio, and mycorrhizal itifection in 22-ivk-old non-mvcorrhizal and mycorrhisal Noricay spruce seedlings groivn in senii-hydropoitic sand culture and supplied with {N or KNO.^ {means ±SD, n = 6). Ammonium supply Pisolithtts tinctorius Nitrate supply Non-mycorrhiza! Pisolithtts tinctorius Dry weight (mg) Shoots Roots ± 87-9 a* ± 60-4 a 1-5 a ±93-3 a 560-4±75-6 a 1-4 a ±86-4 a ±94-7 a 1-2 a 852-3±131-6 a 556-0±50-8a 1-5 a Root tips (No.) Shoot: Root ratio Mvc. Total 1195±143a 954±17!a 1346±239 a 70-9 a Myc. inf. ("o root tips of total) I053±193a a a 54-8 b Differential nitrogen supply started in week 17; before then the plants were supplied with NH^NO., * Means followed by the same letter are not significantly different at P ^ 0-1 in the /-test. markedly between non-mycorrhizal and mycorrhizal plants. The concentrations of these nutrients in the needles were similar or slightly higher in the smaller mycorrhizal than in the larger non-mycorrhizal plants. Only in the needles of mycorrhizal plants with P. tinctorius and in the stems of mycorrhizal plants with L. laccata were the concentrations of K significantly increased. The distribution of these

6 474 L. Eltrop and H. Marschner Table 5. Concentrations (mg g'^ d.wt) of N, P and K in needles and roots of 22-wk-old non-mycorrhizal and tnvcorrhizal Norivav spruce seedlings grown in se7ni-hydroponic sand culture and supplied with {NH^),,SO^ or KNO.j (means+ SD, n = 6). Needles N Roots K N K Ammonium supply Nitrate supply Pisolitliiis tinctorius 14-8±2-2a* b a 12-5±2-6 a O-53±O-lOa 0-59 ±0-04 a 0-61 ±0-15 a a 4-5 ±0-7 a 4-8 ±0-6 a 3-9±0-7a 4-6 ±0-8 a 14-1 ±1-1 a 16-1 ±0-9 a 11-5 ±0-9 a a 0-74 ±0-11 a l-08±0-07b 0-90 ±0-27 a a 7-8±0-8a 8-5 ±0-7 a 6-9 ±1-2 a Oa Differential nitrogen supply started in week 17, before then the plants were supplied with NHjNO., * Means followed by the same letter are not significantly different at P ^ 0-1 in the t test. nutrients between roots and shoots was not significantly different in non-mycorrhizal and mycorrhizal plants. Supply of N as either ammonium or nitrate When ammonium and nitrate were supplied separate!y, the differences in shoot length increment between non-mycorrhizal and mycorrhizai plants with P. tinctorius were less distinct. Compared with non-mycorrhizal plants, mycorrhizal plants tended to be smaller with ammonium supply but larger with nitrate supply. The smal!er shoot length increment in the nitrate-supplied non-mycorrhizal plants was in part due to the formation of buds, which occurred in half of the plants of this treatment. The dry weights of non-mycorrhizal and mycorrhizal plants (Table 4) differed according to plant size. Compared with that of non-mycorrhizal plants, shoot d, wt was c. 12 "o lower in mycorrhizal plants (P. tijictorius) supplied with ammonium but c. 30 o higher in mycorrhizal plants supplied with nitrate. Root d, wt did not differ significantly among treatments. Mycorrhizal infection tended to increase the total number of root tips (Table 4). In ammonium- as wel! as in nitrate-supplied plants the total number of root tips was higher in mycorrhizal than in non-mycorrhizal plants. However, the mycorrhizal infection rate ( mycorrhizal of total root tips) was significantly lower in nitrate- than in ammonium-supplied plants, indicating an inhibiting effect of nitrate or an enhancing effect of ammonium on the development of mycorrhizal root tips. The concentrations of N in needles and roots were higher in ammonium- than in nitrate-supplied plants, particularly when mycorrhizal (Table 5). By contrast, the N concentrations in the needles of the nitrate-supplied plants were not increased by mycorrhizal infection. In the roots, the N concentrations of non-mycorrhizal and mycorrhizal plants were not significantly different. NO3- NH/ 1000 r A Laccaria laccata Figure 2. Depletion of ammonium (open symbols) and nitrate (closed symbols) in the nutrient solution by nonmycorrhizal and mycorrhiza! Norway spruce seedlings. The plants were initiahy supp!ied with 800 /IM NH^NO,,, In general, the concentrations of P and K in the needles were not affected by the treatments (Table 5). Only in plants supplied with ammonium were the P concentrations in the roots significantly higher in mycorrhizal than in non-mycorrhizal plants. Short-term uptake rates of ammonium and 7iitrate In the short-term studies, the kinetics of ammonium and nitrate uptake were investigated in more detail. With supply of NH^NOg, ammonium was taken up in preference to nitrate (Fig, 2), In the first 3 d, the ammonium concentration in the nutrient solution declined more rapidly than the nitrate concentration, A significant nitrate depletion did not occur until days 2-3, until the concentration of ammonium had decreased to less than c. 400 //M. Mycorrhizal infection did not significantly influence the depletion of either N form.

7 Growth and mineral fiutrition of Norway spruce, I r 0-32 NO3- -Q- Laccaria laccata 1-5 r NO NH Non-mycorrhizai Pisotithus tinctocius o o 7" 0-24 u> Z "0 ^ D o o Dl z "o E a) 4-' CD k_ a j^(d +^D L N concentration Figure 3, Uptake rates for ammonium (open symbols) and nitrate (closed symbols) in non-mycorrhiza! and mycorrhizal Norway spruce seedlings supplied with different concentrations of NI Table 6. Kinetic uptake parameters {substrate affinity K^ in jjtm, and maximal uptake rate V^^^^ in /imolg^^f.wtlf\ after Lineweaver Burk) for ammonium and nitrate in intact root systems of 22-wk-old non-mycorrhizal and mycorrhizal Norway spruce seedlings Supply of N as NH^NO,, 227 Laccaria laccata 229 Paxillus ittvolutus Ammonium Supply of N as (NHj).,SO, or KNO,, Nitrate K V Nitrogen was supplied either as NH^NOg or SOj or KNO3 at a total N concentration of 800 With increasing supply of the external N concentration, the uptake rates of ammonium and nitrate became markedly diflferent. In the low concentration range, the uptake rates of both ammonium and nitrate increased, that of nitrate to a lesser extent, however (Fig. 3). At higher external concentrations (>200/;M), the uptake rates of an-imonium continued to increase, although less markedly, whereas the uptake rates of nitrate more or less levelled oft. Over the whole concentration range tested, the uptake rates of ammonium in mycorrhizal plants clearly exceeded those in non-mycorrhizal plants (Fig. 3). The maximal uptake rates (I^,,;,x) were generally higher for ammonium than for nitrate (Table 6). 0-0 L N concentration 1200 Figure 4. Uptake rates for ammonium (open symbols) and nitrate (closed symbols) in non-mycorrhizal and mycorrhizal. Norway spruce seedlings supplied with either SO^ or For ammonium, l\.^^^ was in the range /imol Nli/S g~' root f. wt h"' and tended to be higher in mycorrhizal than in non-mycorrhizal plants. This was particularly evident in mycorrhizal plants with L. laccata. The affinity constant K^ for ammonium did not differ between non-mycorrhizal and mycorrhizal plants. For nitrate, V,^^.^^^ was in the range of 0-3 /imol g'' root f. wt h"' and was similar in non-mycorrhizal and mycorrhizal plants. Only in m}-corrhizal plants ^ith P. tinctorius was the Pj,,.,,. for nitrate (0-51 //mol g"' root f. wt h"') distinctly higher than in ali other plants, as was the K. (367 //M), indicating a less efficient uptake rate for nitrate at concentrations of 400 /IM and below. When ammonium and nitrate were supplied separately, the uptake rates of the two N forms were similar in the low concentration range (Fig. 4), in contrast with the lower uptake rates of nitrate when N was supplied as NH^NO, (Fig. 3). At higher concentrations of KNOg, however, there was a levelling oft of uptake rates of nitrate similar to that in the experiments with supply of NH4NO3. In contrast, the uptake rates of ammonium continued to increase. At the highest external ammonium concentration, the uptake rates were significantly higher in mycorrhizal than in non-mycorrhizal plants (Fig. 4). DISCUSSION Mycorrhizal infection of Norway spruce seedlings decreased shoot and root d. wt considerably (Tables 1, 4) and did not significantly enhance mineral nutrient uptake (Tables 3, 5). Of the three fungi tested, only P. tinctorius tended to increase N uptake, more specifically ammonium uptake. Reduced growth in mycorrhizal compared with non-mycor-

8 476 L. Eltrop and H. Marschner rhizal plants when grown in hydroponic or seniihydroponic culture has been reported by other authors before (Ingestad et al., 1986; Nylund & Wallander, 1989; Colpaert et al, 1992). Growthstimulating effects of mycorrhizal infection, as reported by Jentschke et al. (1991) in Norway spruce and Lactarius rufits, appear to be an exception in such culture s\'stems. The lack of enhancement of plant growth and nutrient uptake by mycorrhizal infection reported here contrasts with the stimulation by mycorrhizal infection of growth in plants grown in solid substrates like soil, peat or soil/peat and soil/sand mixtures (Reid et al, 1983; Jones et al, 1990; Tam & Griffiths, 1994). This difference in growth response IS likely to be related to the different spatial availability of mineral nutrients in the two types of culture. In hydroponic and semi-hydroponic culture, nutrient delivery to the root surface is ensured, whereas in solid substrates, nutrient depletion zones develop around the roots, limiting nutrient uptake. In contrast to solid substrates, where the extramatrical mycelium is considered to be responsible for the increased nutrient uptake in mycorrhizal plants owing to the increase in the spatial availability of nutrients (Harley, 1989; Marschner & Dell, 1994), in hydroponic or semi-hydroponic culture the extramatrical mycelium appears to be of small or even no importance for nutrient uptake. The development and maintenance of the extramatrical myceliuni, however, requires energy in the form of carbohydrates. The d. wt of the extramatrical mycelium can even exceed the d. wt of the roots (Colpaert e? a/., 1992). In the present study, however, the amount of extramatrical mycelium in the mycorrhizal plants was less than 3 o of root d. wt (Eltrop & Marschner, 1996). The decreased growth of mycorrhizal plants was therefore most likely due to the fungal demand for carbohydrates for the maintenance of the fungal metabolism rather than for fungal growth. This withdrawal of carbohydrates from the plant was not compensated for by higher nutrient uptake. The slightly increased nutrient concentrations in the roots and needles of mycorrhizal plants (Tables 3, 5) were presumably the result of concentration effects in the dry matter of the mycorrhizal plants which were smaller than the non-mycorrhizal plants. The reduced d. wt in mycorrhizal compared with non-mycorrhizal plants supplied with ammonium compared with those supplied with nitrate (Table 4), indicates that with ammonium nitrate supply also the growth reduction was related to ammonium uptake. In the other studies with hydroponic or semi-hydroponic culture, where similar d. wt reductions in mycorrhizal compared with non-mycorrhizal plants were observed (Nylund & Wallander, 1989; Kamminga-van Wijk, 1991), N was also supplied as ammonium nitrate. With nitrate supply, however, the enhanced bud formation suggests, that hormonal effects may have contributed to the reduced growth in non-mycorrhizal compared with mycorrhizal plants. In nitratesupplied plants compared with ammonium-supplied plants, the cytokinin concentrations in the shoots may decrease (Buban et al, 1978). As cytokinins are mainly produced in the root meristems, mycorrhizal infection might significantly influence cytokinin concentrations in the plants, either directly, by stimulating the production or transfer to the roots, or indirectly by increasing the number of production sites (Nylund, 1988; Rupp, Mudge & Negm, 1989). In the present study, mycorrhizal infection increased the number of root tips in both the ammonium and the nitrate treatments (Table 4). The lower shoot d. wt and the formation of buds in the nonmycorrhizal plants supplied with nitrate might thus be related to decreased cytokinin concentrations in the shoots, whereas the increase in d. wt in mycorrhizal plants might have been due to increased cytokinin concentrations brought about by the higher number of root tips or by the production of growth regulators by the mycorrhizal fungus {P. tinctorius). The infection rate of the roots with P. tinctorius was significantly decreased when the plants were supplied with nitrate (Table 4), confirming similar results by other authors (Theodorou & Bowen, 1969; Alexander, 1983). It is not clear whether the decrease in mycorrhizal infection by nitrate is due to the inability of some mycorrhizal fungi to utilize and grovi' on nitrate per se (Ho & Trappe, 1980) or whether inhibitors of mycorrhizal infection are produced under nitrate supply (Alexander, 1983). In nodulating legumes, nitrate supply in comparison with ammonium supply decreases the exudation of isoflavonoids by roots (Wojtaszek, Stobiecki & Gulewicz, 1993). Phenolics were also found in root exudates of forest trees (Smith, 1976) and were considered to be responsible for the reducing activity of the mycorrhizal mycelium of P. tinctorius and P. involutus (Cairney &.Ashford, 1991). The decrease in mycorrhizal infection by nitrate supply might thus be related to decreased concentrations of phenolics in the root exudates, which act as signal substances for the establishment of the symbiosis. However, the role of root-borne plant hormones mycorrhizal infection is still uncertain. In the short-term uptake experiments, ammonium was taken up at higher rates than nitrate by both non-mycorrhizal and mycorrhizal plants, whether it was supplied simultaneously or separately (Figs 3, 4). Higher uptake rates of ammonium than of nitrate are a general characteristic of forest trees (Alexander, 1983 ; Marschner, Haussling & George, 1991) as well as of mycorrhizal fungi (Littke et al, 1984; Scheromm et al, 1990). Inhibition of nitrate uptake by ammonium is also commonly observed in tree

9 Groivth and mineral nutrition of Norway spruce, I All seedlings (Runge, 1983; Kamminga-van Wijk & Prins, 1993). It might be caused b}' inhibition of nitrate reductase activity by ammonium (Peuke & Tischner, 1991), accumulation of amino acids in the roots (Muller & Tourraine, 1992) or by direct inhibition of nitrate influx (Lee & Drew, 1989). From experiments in split root systems, Chaillou et al. (1994) concluded that the inhibition of nitrate uptake by ammonium is the result of enhanced nitrate efflux rather than of inhibited nitrate influx. With nitrogen supplied as ammonium nitrate, nitrate uptake rates remained low until the ammonium concentration dropped below //,M (Fig. 2). This threshold value for the onset of significant nitrate uptake might vary according to the type of roots and the culture system used. In nonmycorrhizal long roots of mature Norway spruce trees, net uptake of nitrate started at an ammonium concentration below //M (Marschner et al., 1991). However, the long roots were excavated from soil substrate and then incubated in the nutrient solution, whereas in the present study intact and complete root systems were percolated with the nutrient solution in situ. In the latter culture system, the roots were coated with a thin la\-er of nutrient solution only and the depletion of ammonium at the root surface might thus have been higher than measured in the bulk solution in the Erlenmeyer fiask. The threshold concentration of ammonium for the onset of significant nitrate uptake might therefore be lower m nutrient solution culture than determined in the present study. In any case, the threshold concentration did not difier between non-mycorrhizal and mycorrhizal plants. The K,,a^- values for ammonium and nitrate in Norway spruce (Table 6) w-ere of the same order of magnitude as those reported for mycorrhizal Douglas fir (Kamminga-van Wijk, 1991) and maritime pine (Plassard ei al., 1994). According to the similar uptake rates in the short-term experiments (Figs 2, 3), the I'J,,,,^ did not differ consistently between nonmycorrhizal and m3-corrhiza] plants. Similar J-'^^i.^^ values for non-mycorrhizal and mycorrhizal plants were also found by Kamminga-van Wijk (1991) with Douglas fir and L. laccata (S 238). In the present studv, the I-''... values for ammonium and nitrate were higher compared with the non-mycorrhizal plants only in the mycorrhizal plants with P. tinctorius (Table 6). By contrast, in maritime pine, the I/^jj,,,, values for nitrate uptake were markedly lower in mycorrhizal (with Hebeloma cylindrosportim) than in non-mycorrhizal plants (Plassard et al., 1994), which might be caused by the much greater amount of fungal biomass in maritime pine (c. 20"o of root d. wt) than in the Norway spruce seedlings of the present study, and by the better adaptation by pine than by spruce to nitrate supply. The increased nitrogen uptake rates (Fig. 4) and needle N concentrations (Tables 4, 5) w-hich occurred in mycorrhizal plants with P. tinctorius but not m mycorrhiza] plants with P. involutus and L. laccata, indicate that the stimulating mycorrhizal effect on nutrient uptake depends on the species of mycorrhizal fungi. Large differences in N uptake rates also exist between different species of mycorrhizal fungi grown?«vitro (Littke et al., 1984) and between different strains of the same mycorrhizal fungus (Kieliszewska-Rokicka, 1992). The results of the present study show that specific nutrient uptake rates per unit root dry weight are not per se higher in mycorrhizal than in non-mycorrhizal roots. Stimulating effects of mycorrhization on nutrient and, more specifically, N uptake appear to be restricted to certain species or strains of mycorrhizal fungi. In this sense, the results support the view that non-mycorrhizal roots might also play a considerable role in mineral nutrient uptake of forest trees (Marschner et al. 1991)..ACKNOWLEDGEMENTS This work was supported by the European Community in the framework of the ' STEP-program'. The authors thank Dr Z. Rengel for critical reading of the manuscript and valuable suggestions and corrections of the English. REFERENCES Alexander IJ The significance of ectomycorrhizas in the nitrogen cycle. In: Lee JA, McXeiU S, Rorison IH, eds. Nitrogen as an Ecological Factor. The 22nd Symposium of the British Ecological Society. Oxford, London: Blackwell Scientific Publications, Ashford, AE, Peterson CA, Carpenter JL, Cairney JWG, Alla-way WG Structure and permeability of the fungal sheath in the Pisonia mycorrhiza. Protoplasma 147: Ashford AE, Alla-way WG, Peterson CA, Cairney JWG Nutrient transfer and the fungus-root interface. Australian Journal of Plant Physiology 16: Bolan NS A critical re\-ie\v on the role of mycorrhizal fungi in the uptake of phosphorus by plants. Plant and Soil 134: Boxman AW, RoelofsJGM Some effects of nitrate versus animoniuit) nutrition on the nutrient fluxes in Pinus sylvestris seedlings. Effects of mycorrhisal infection. Canadian Journal of Botany 66: Buban T, Varga A, Tromp J, Knegt E, Bruinsma J Effects of ammonium and nitrate nutrition on the level of zea tin and amino nitrogen m xyiem sap of apple rootstocks. Zeitschrift fiir Pflamenphysiologie 89: Cairney, JWG, Ashford AE Release of a reducing substance by the ectomycorrhiza] fungi and Paxillus involutus. Plant and Soil 135: Chaillou S, Rideout JW, Raper CD Jr., Morot-Gaudry J-F Responses of soybean to ainmonium and nitrate supplied in combination to the whole root system or separately in a splitroot system. Physiologia Plantarum 90: Colpaert JV, van Assche JA, Luijtens K The growth of the extramatrical mycelium of ectomycorrhizal fungi and the growth response of Pinus sylt:estris L. Neit: Phytologist 120: Ellrop L Role of ectomycorrhiza in the mineral nutrition of Noricay spruce seedlings (Picea abies L. Karst.). Ph.D. thesis. University of Hohenheim, Germany. Eltrop L, Marschner H. Growth and mineral nutrition of nonmycorrhizal and mycorrhizal Norway spruce {Picea abies (L.) Karst.) seedlings grou-n m semi-hydroponic sand culture. 12. Carbon partitioning in plants supplied with ammonium or with nitrate. A'fEt Phytologist 133:

10 478 L. Eltrop and H. Marschner Finlay RD, Ek H, Odham G, Soderstom B Mycelial uptake, translocation and assimilation of nitrogen from 15 N- labelled ammonium by Pinus sylvestris plants infected with four different ectomycorrhizal fungi. Neii; Phytologist 110: Harley JL The significance of mycorrhiza. Mycological Research 92: Haussling M, Jorns CA, Lehmbecker G, Hecht-Buchhoiz Ch, Marschner H Ion and water uptake in relation to root development in Norway spruce {Picea abies (L.) Karst,). Journal of Plant Physiology \Z2: 486^91. Ho I, Trappe JM Nitrate reductase activity of nonmycorrhizal Douglas fir rootlets and some associated mycorrhizal fungi. Plant and Soil 54: Hogberg P Growth and nitrogen inflow rates in mycorrhizal and non-mycorrhizal seedlings of Pinus sylvestris. Forest Ecology and Management 28: Ingestad T, Arveby.4S, Kahr M The mhuence of ectomycorrhiza on nitrogen nutrition and growth of Pnius svlvestris seedlings. Physiologia Plantarum 68: Jentschke G, Godbold DL, Huttermann A Culture of mycorrhizal tree seedlings under controlled conditions: effect of nitrogen and aluminium. Physiologia Plantarum 81: Jones MD, Durall DM, Tinker PB Phosphorus relationships and production of extramatrical hyphae by two types of willow ectomycorrhizas at different soil phosphorus levels. Neiv Phytologist 115: Kamminga-van Wijk C Mycorrhizal and non-mycorrhizal Douglas fir grozcn in hvdroculture. Ph.D. thesis, Rijksunn-ersiteit Groningen, The Netherlands. Kamminga-van Wijk C, Prins HBA The kinetics of NH^* and NOg" uptake by Douglas fir from single N-solutions and from solutions containing both NHj~ and NOg. Plant and Soil 151: Kieliszewska-Rokicka B Effect of nitrogen level on acid phosphatase acti\-ity of eight isolates of ectomycorrhizal fungus Paxillus inz'olutus cultured in vitro. Plant and Soil. 139: Langlois CG, Fortin JA Seasonal variations in the uptake of ^"P phosphate ions by excised ectomycorrhizae and lateral roots of Abies balsamae. Canadian Journal of Forest Research 14: 412^15. Lee RB, Drew MC Rapid, reversible inhibition of nitrate influx in barley by ammonium. J'ow!'?m/ of Experimental Botany 40: ' Littke WR, Bledsoe CS, Edmonds RL Nitrogen uptake and grow-tb in vitro by Hebeloma crustulinifornie and other Pacific Northwest mycorrbizal fungi. Canadian Journal of Botany 62: Marschner H, Deli B Nutrient uptake in mycorrbizal symbiosis. Plant and Soil 159: Marschner H, Haussling M, George E Ammonium and Nitrate uptake rates and rbizospbere-ph in non-mycorrhizal roots of Norway spruce (Picea abies [L.] Karst.). Trees 5: Muller B, Touraine B Inhibition of NO,," uptake by various phloem-translocated amino acids in soybean seedlings. Journal of Experimental Botany 43: Nylund J-E Tbe regulation of mycorrhiza formation - Carbohydrate and Hormone theories reviewed. Scandinavian Journal of Forest Research 3: Nylund J-E, Wallander H., Effects of ectomycorrhiza on bost growth and carbon balance in a semi-bydroponic cultivation system. New Phytologist 112: Peuke AD, Tischner R Nitrate uptake and reduction of aseptically cultivated spruce seedlings, Picea abies (L.) Karst. Journal of Experimental Botany 239: Plassard C, Barry D, Eltrop L, Mousain D Nitrate uptake in maritime pine (Pinus pinaster Soland in Ait.) and the ectomycorrbizal fungus Hebeloma cylindrosporum: effect of ectornycorrbizal symbiosis. Canadian Journal of Botany 72: Reid CPP, Kidd FA, Ekwebelam SA Nitrogen nutrition, photosynthesis and carbon allocation in ectomycorrbizal pine. Plant and Soilll: 415^32. Runge M Pbysiology and ecology ot nitrogen nutrition. In: Lange OL, Nobel PS, Osmond CB, Ziegier H, eds. Encyclopedia of Plant Physiology. 12 C. Berlin: Springer Verlag, Rupp LA, Mudge KW, Negm FB Involvement of etbyiene in ectomycorrbiza forination and dicbotomous brancbing of roots of mugo pine seedlings. Canadian Journal of Botany 67: Rygiewicz PT, Bledsoe CS, Zasoski RJ. 1984a. Effects of ectomycorrbizae and solution ph on ('"''N) nitrate uptake by coniferous seedlings. Canadian Journal of Forest Research 14: Rygiewicz PT, Bledsoe CS, Zasoski RJ Effects of mycorrbizae and solution ph on (' N) ammonium uptake by coniferous seedlings. Canadian Journal of Forest Research 14: Scheromm P, Plassard C, Saisac L, 1990, Nitrate nutrition of maritime pine {Pinus pinaster Soland in Ait.) ectomycorrbizal witb Hebeloma cvlindrosporum Romagn. New Phytologist 114: Smith WH Cbaracter and significance of forest tree root exudates. Ecology 57: Tam PCF, Griffiths DA Mycorrhizal associations in Hong Kong Fagaceae. VI. Growtb and nutrient uptake by Castanopsis fissa seedlings inoculated witb ectomycorrbizal fungi. Mycorrhiza 4: Theodorou C, Bowen GC. 1969, Tbe influence of ph and nitrate on mycorrbizal associations of Pinus radiata D. Don. Australian Journal of Botany 17: Unestam T, Water repellency, mat lorniation, and leafstin-iulated growtb of some ectomycorrbizal fungi. Mvcorrhiza 1: Wallander H Regulation of ectomycorrhizal symbiosis in Pinus syk-estris L. seedlings. Infiuence of mineral nutrition. Pb.D. tbesis, Swedisb University of Agricultural Sciences, Department of Forest Mycology and Pathology, Uppsala, Sweden. Wojtaszek P, Stobiecki M, Guiewicz K Role of nitrogen and plant growtb regulators in tbe exudation and accumulation of isoflavonoids by roots of intact wbite lupin {Lupinits albus L.) plants. Journal of Plant Physiology 142:

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