A role of vesicular-arbuscular (VA) mycorrhizal fungi in facilitating interplant nitrogen transfer

Soil Biol. Biochem. Vol. 25, No. 6, pp. 651-658, 1993

0038-07I7/93 56.00+ 0.00 Copyright 0 1993Pergamon Press Ltd

Printed in Great Britain. All rights reserved

A ROLE OF VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI IN FACILITATING NITROGEN TRANSFER

(VA) INTERPLANT

B. FREY and H. SCHUEPP Department of Phytopathology and Soil Microbiology, Swiss Federal Research Station, CH-8820 Wldenswil, Switzerland (Accepted

I5 January 1993)

Summary-Two

greenhouse experiments were carried out to study the transfer of nitrogen from berseem to associated non-legumes via the hyphae of VA mycorrhizal fungi using lSN as tracer. A special cuvettemembrane system was used to study the effects of restricted and unrestricted hyphal growth between the legume and the non-legume on nitrogen transfer. The roots of berseem plants, either inoculated with the VA mycorrhizal fungus Giomus intrurudices or non-inoculated, were separated by a 3cm root-free zone from the roots of the non-legume. In the first experiment, a split-root technique was employed to label the legume with “N. Maize was chosen as the non-legume. 50-day old berseem plants were supplied with a rSN-enriched N source, which was added to the outer side of the divided root system of the legume. In the second experiment apple was the non-legume chosen. The legume was labelled with ‘sN by injection into the leaf petioles after a 51-day growth of berseem. Both methods of r5N-labelling of the legume were effective in enriching all plant parts with lsN. Transfer of 15Nfrom berseem to the non-legume infected by the VA mycorrhizal fungus was significantly higher than in the non-infected non-legume over a 28-day period. However, the patterns of “N transfer differed between the two experiments. By correcting the value of ‘rN in mycorrhizal receiver plants for the background values in the corresponding non-mycorrhizal treatments, 4.7% of the 15Ncontent of berseem was transferred to apple. In contrast, the amounts of “N transferred to mycorrhizal maize were smaller with 0.1% of the “N derived from berseem. (Trifolium alexandrinum)

INTRODUCTION It is well documented growth resulting from

that the increase vesicular-arbuscular

in plant

(VA) mycorrhizal symbiosis is usually associated with increased nutrient uptake by the hyphae from the soil (Rhodes and Gerdeman1980; Harley and Smith, 1983). The external mycelium can spread away from the roots over considerable distances. In fact, Li et al. (1991a) found that during 49 days’ growth, hyphae of Glomus mosseae in association with Trifolium repens L. extended at least 11 cm. It is widely accepted that a hyphal network associated with the roots of a living plant is capable of infecting the roots of other plants growing in its vicinity (Chiariello et al., 1982; Francis and Read, 1984; Newman, 1988). Francis et al. (1986) indicated that mycorrhizal connections are likely to be involved in nutrient transfer between plants. Interconnections may also have an effect on nutrient recycling from dying to younger plants (Eason et al., 1991). Adverse effects arising from the disturbance of VA mycorrhizal mycelial systems on plant nutrition emphasize the role of fungal hyphae in nutrient transfer (Evans and Miller, 1990). Legumes are important in mixed cropping (Blevins et al., 1990; Latif et al., 1992). A key factor is the input of N from N,-fixation by means of the legume-Rhizobium symbiosis. Several reports have documented that a non-legume can benefit from N

supplied by an intercropped legume (Ledgard et nl., 1985; Boller and Niisberger, 1987; Ta and Faris, 1987). Experiments using “N as a tracer have shown that VA mycorrhizal hyphae may be involved in nitrogen transfer from a legume to a non-legume (Van Kessel et al., 1985; Haystead et al., 1988; Barea et al., 1989; Hamel et al., 1991a). However, all of these studies have been carried out under conditions in which the plant root systems were not separated by an interposed compartment. Studying the hyphal transport of nutrients between plants is facilitated by a novel method of separating root compartments by a root-free zone and restricting root penetration by a fine mesh (Schiiepp et al., 1987; Camel et al., 1991; Bethlenfalvay et at., 1991). Our objective was to assess the effect of mycorrhizal hyphae on N-transfer from the legume berseem to the non-legumes maize or apple through a 3-cmwide root-free soil zone. In the first experiment a split-root system was employed in which half the root of the legume was labelled with “N. In the second experiment 15N was supplied by injection into the petioles of the legume. MATERIALS AND

METHODS

Soil mixture

The soil used was a loamy sand of low fertility collected from a field trial in Hessen (Wldenswil) 651

652

B. FREYand H.

selected for its low P content. It was sieved (c 2.0 mm), y-sterilized (10 kGray) and combined 3 : 1 by volume with sieved ( < 1 mm) acid-washed and autoclaved quartz-sand. Nutrient analysis of the final mixture gave the following characteristics: pH-H,O 7.2, NaHCO,-extractable (Olsen) P 9.Opg total N 0.28% with KCl-extractable g-‘soil, concentrations of N03-N 21 pg g-l and NH4-N 23 pg g -‘. The bulk density of the soil mixture was 1.35 g cm.-3 VA mycorrhizal inoculum

The fungal inoculum was G. intraradices Walker and consisted of sand containing colonized roots of Tagetes sp. The inoculum was prepared from 16week-old pot cultures, maintained on 7’agete.s by cutting the roots into 2cm long segments, and then mixing these with sand containing spores and hyphae. Experiment

1 (split-root application)

Berseem (Trifolium alexandrinum L. cv. Landsorte) and maize (Zea mays L. cv. Honeycomb-Fl) were used. Seeds of both plant species were surface steril-

Berseem

Maize

P 43

B Solid N-15

plate divider

B

~ Rootonly (I or RootFUllgUS (II and III)

40 pm net

/\r

Root-free mne (I, II) or Fungus(III)

Root only (I, II) or RootFungus

system in experiment I used for studying 15N-transfer between berseem (Trifolium alexandrinum) and maize (split-root experiment). Plant compartments were separated from the 3-cm-thick root-free zone by a 40 pm nylon net which restricted root growth. 15N was applied to the outer (A) compartment of the divided root-system of the legume. The experimental design was as follows: I-non-mycorrhizal T. alexandrinum, non-mycorrhizal 2. MCZYS : II-mycorrhizal T. alexandrinum: VAM confined by a 6.45 pm-membrane, no colonization of Z. mays; III-mycorrhizal T. alexandrinum:VAM growing through root-free zone and colonizing Z. mays.

Fig. 1. The cuvette-membrane

SCHOEPP

ized in 5% sodium hypochlorite for 5 min, rinsed three times with sterile water and sown in moist, oven sterilized sand which was fertilized once after 8 days with 30 ml of $ strength Hewitt’s nutrient solution (1966) lacking P. The seeds were inoculated at sowing with a dense suspension of R. leguminosarum biovar trifolii (RCR 5) in 1% sucrose. The Rhizobium isolate was kindly supplied by Dr U. Hartwig, ETH Ziirich. It had been originally isolated at Rothamsted Experimental Station, Harpenden, U.K. The split-root experiment was conducted in a system which allowed compartmentation of roots and hyphae similar to that outlined by Schiiepp et al. (1987) with some modifications (Fig. 1). Cuvette sections for root growth were 2 cm thick, 15 cm wide and 15 cm deep. The section for root-free soil was 3 cm thick. For compartmentation a 40 pm meshsize nylon net (Zi,irich Bolting Cloth Mfg Co. Ltd, Riischlikon, Switzerland) was used which restricted root growth but allowed fungal hyphae to pass through. The root compartment of the legume consisted of two 2-cm-thick compartments with a solid plate as a barrier between the two parts of the split-root system. Three treatments according to Fig. 1, two mycorrhizal ones with (treatment II) and without (treatment III) a membrane restriction and a non-mycorrhizal one (treatment I), were established. In order to set up a control treatment with a mycorrhizal legume (treatment II), the 40 pm net of the berseem compartment was replaced by a membrane of 0.45 pm pore size (Millipore filter type HV). This membrane cannot be penetrated by VA mycorrhizal hyphae (Li et al., 199lb). The root compartments were filled with 570 g of dry, sterilized soil: sand mixture, and the root-free soil compartments with 721 g of the mixture. After 20 days, the berseem plants were large enough to be transferred to the split-root units. At transplanting, the root system of each plant was divided in half along its length and placed in a plastic Y-tube (Fig. 1) to protect the roots from evaporation and damage. The main root was placed down one side and the oldest lateral roots were placed down the other. The tube was then filled with the growing medium and two seedlings were replanted with their divided root systems in the appropriate sections of the split-root compartment. Leakage and contact between soil and water from each section were avoided. At transplanting the inner (B) root compartments of berseem (Fig. 1) were inoculated with the mycorrhizal fungus (treatments II and III) by mixing uniformly 25 g of fresh weight inoculum with the entire growth medium, or left uninoculated (treatment I). 20 ml of a 25 pm mesh filtrate containing microorganisms other than the VA mycorrhizal fungus were applied to the non VAM-inoculated control compartments to ensure the presence of the same microflora in all treatments. There were six replicates of each treatment.

Mycorrhiza

on interplant N-transfer

Five weeks later two maize seedlings were planted into the other outer compartment (Fig. 1). The bet-seem plants were all fertilized at 3539 and 43 days after transplanting with 20ml of a l/4 strength Hewitt’s solution containing P applied to the outer (A) compartment. The non-legume received no fertilizer throughout the experiment. The experiment was carried out in a greenhouse between January and April. A 16 h photoperiod was provided by supplemental high pressure sodium vapour lamps at 350 pE m-2 s-I in the range of 40s700 nm throughout the 11 weeks of the experiment. The mean glasshouse temperature regime was 23°C during the day and 18°C at night. The pots were watered daily to field capacity with deionized water. The cuvette systems were frequently rotated to minimize positional effects. “N labelling of berseem was initiated 50 days after transplanting. 2.44mg of “N were applied as 50% enriched KNO, (Isotec Inc., Miamisburg, U.S.A., purchased from Numelec SA, Geneva, Switzerland) in 20ml distilled water. The label was injected by syringe into the outer (A) root compartment. A second application was made 1 week after the first application. Additional cuvette systems were held without “N labelling of berseem for the determination of the natural abundance of i5N in berseem and maize tissue. Plants were harvested 28 days after initiation of 15N labelling. Experiment

2 (foliar application)

Berseem (Trifolium alexandrinum L. cv. Landsorte) and apple (Ma/us x domestica Borkh.) collected from “Golden Delicious” fruits were used. Seeds of both were surface sterilized as described above and germinated on moist, oven-sterilized sand. At sowing, berseem was inoculated with Rhizobium as described in experiment 1. The experimental design was similar to that used in experiment 1 except in the outer (A) compartment (Fig. 1). The cuvette system consisted of three compartments, two outer ones for root growth and a central one for hyphal growth only. A control treatment with a mycorrhizal legume (treatment II) was established as described in experiment 1. The size of the cuvette sections and the quantities of soil mixture used were the same as in experiment 1. Seeds of apple and berseem were germinated for 20 days and then transplanted into the appropriate root compartments. Each plant compartment contained two plants. Soil and mycorrhizal inoculum were prepared as above. The legume was inoculated with the same mycorrhizal fungus (treatment II and III) as in experiment 1 or left uninoculated (treatment I). An inoculum filtrate as described above was given to the non VAM-inoculated control compartments. Treatments and replicates were established as in experiment 1. The berseem plants were all fertilized at 36,40 and 44 days after transplanting with 20 ml of a l/4 strength Hewitt’s solution containing P. The non-

653

legume received no fertilizer throughout the experiment. Greenhouse conditions and watering were the same as in experiment 1. 51 days after initiation of the experiment, berseem was labelled with 0.1 mg of 15N as 99% enriched KNO, (Isotec Inc., Miamisburg, U.S.A., purchased from Numelec SA, Geneva, Switzerland) in 10~1 distilled water. The label was injected into leaf petioles of berseem using a Hamilton syringe (F. Trefny, personal communication). This injection was repeated daily for 10 days with the addition of 100 pg of “N on each occasion. The application of i5N was carried out carefully to avoid contamination of soil and non-legume plants with ‘5N-enriched solution. Additional cuvette systems were held without “N labelling of berseem for the determination of the natural abundance of 15N in the plant material. The plants were harvested 17 days after the last r5N application. Plant harvest and analysis

The cuvette holders were removed to separate root, hyphal and bulk soil compartments. The roots were separated from the shoots, and thoroughly washed. A subsample of 0.5 g fresh roots of each root compartment was used for estimating mycorrhizal infection using the gridline intersect method described by Giovannetti and Mosse (1980) after clearing and staining of the roots (Kormanik et al., 1980). Infection was expressed as the percentage of root length infected. Plant components were dried at 60°C for 48 h, weighed and finely ground using a cyclone mill. Total N in plant tissues was determined after a semi-micro Kjehldahl digestion using an automated indo-phenol blue method (Varley, 1966) and the 15Nenrichments were determined using a Micromass 622 mass spectrometer (VG Isogas, Cheshire, U.K.). Samples from unlabelled plants were also collected and analysed to provide background 15Nconcentrations. Atom% i5N excess was calculated by subtracting the natural abundance of 15N in unlabelled plant material from atom% “N. Soil cores (1 cm dia) from each compartment were taken for the determination of mycelium length using the membrane filter technique (Hanssen et al.,1974). Following this technique, the dried sample was stained for 15 min with 0.1% Trypan blue in lactic acid-glycerol-water (4: 2 : 1) at 90°C with stirring. After l/500 dilution of the soil with water, a 20ml sample was taken using a syringe with a 4 mm calibre tip and filtered by vacuum through a 0.45 pm cellulose Millipore filter with a gridline scored on its surface. The length of VA mycorrhizal mycelium was recorded as m g-i dry soil, calculated using Newman’s (1966) formula from the number of hyphae gridline intersections observed under the microscope ( x 200 magnification). Four replicates were analysed per treatment. The normality of all of the experimental data was tested

654

B. FREYand H.

SCH~~EPP

Table 1. Mycorrhizal colonization (percentage of colonized root length), dry weight, N-concentration and atom % “N excess in plants parts of (a) berseem (Trifolium alexundrinum) and (b) maize (experiment 1). The proportion of “N added to the donor which was subsequently recovered in the receiver plant is expressed as the percent recovery of applied i5N. For treatment code see Fig. 1 Treatment Parameter

I

II

III

0

54 a*

56a

(a) In berseem

Infection (%) of total root length colonized Shoot dry weight (g) Root dry weight (g)t Shoot N concentration (%) Root N concentration (%) Shoot atom % “N excess Root atom % r5N excess @) In maize Infection (%) of total root length colonized Shoot dry weight (g) Root dry weight (g) Shoot N concentration (%) Root N concentration (%) Shoot atom % i5N excess Root atom % i5N excess Recovery (%) of applied i5N

6.4 a 0.7 a 1.9a 2.3 a 2.3352 a 0.1132a 0 0.1 a 0.3 a I.7 a 1.3a 0.0071 a 0.0143 a 0.03 a

6.1 a 0.9 ab 2.0 a 2.3 a 2.6432 a 0.7857a

1.3 a 1.3b 2.2 a 2.2 a 2.6041 a 0.7810 a

0

62

0.8 a 0.4 a 1.6a 1.2a 0.0065 a 0.0147 a 0.03 a

0.8 a 0.3 a 2.4 b 1.7b 0.0197 b 0.0358 b 0.12 b

*Values are means of four replicate treatments. Different letters within the same row indicate a significant difference at the P < 0.05 level. tData from the inner (B) root compartment, see Fig. 1. using the Kolmogorov-Smimov test. Data were then further analysecl using a standard analysis of variance. Unless otherwise stated, the effects discussed are significant at the P ~0.05 probability level. Least significant differences (LSD) were calculated for all

significant F-ratios to allow comparison of treatment means (Steel and Torrie, 1980). RESULTS Experiment

1

Colonization by G. intraradices reached over 50% of the root length of inoculated (treatments II and III) berseem roots and over 60% of the root length of maize in treatment III (Table l), while no mycorrhizal colonization was found in the controls (treatments I and II). At harvest, the Millipore membrane had not been broken down and no perforations were visible. The membrane acted as an effective barrier to the hyphae in both the root-free and the maize compartments (treatment II). The background hyphal density in these compartments amounted to 0.3 m g-’ of dry soil. The root-free zone of the non-membrane restriction (III) treatment contained 3.1 f 0.2 m of fungal hyphae g-r of dry soil. Mycelium length in the inner (B) root compartment of infected berseem did not differ between the membrane restriction (II) (5.1 m + 0.3) and the non-membrane restriction (III) treatment (4.5 + 0.2 m gg’ of dry soil). Shoot dry weights of berseem were similar in control plants (treatment I) and in plants colonized

by G. intraradices (treatments II and III), whereas dry weights of uninoculated roots (treatment I) in the inner (B) compartment were significantly lower than in non-membrane restriction (III) treatment (Table 1). Mycorrhizal infection (treatment III) in maize did not affect the dry matter yield compared to non-infected maize (see treatments I and II, Table 1). However, infected maize (treatment III) showed higher N concentrations in shoots and roots than non-infected maize (Table 1). Transfer of lsN from berseem to maize was detectable from the “N enrichment of all shoots and

shoot

root

Fig. 2. Amounts of 15N in excess bg) transferred from berseem (T. alexandrinum) to maize through a 3-cm-thick root-free compartment as affected by the different treatments (experiment 1). For treatment code see Fig. 1. Bar heights represent the mean and standard error of four replicates.

Mycorrhiza on interplant N-transfer

655

Table 2. Mycorrhizal colonization (percentage of colonized root length), dry weight, N-concentration and atom % “N excess in plant parts of (a) berseem (Trifoliwn ulexandrinum) and (b) apple seedlings (experiment 2). The proportion of “N added to the donor which was subsequently recovered in the receiver plant is expressed as the percent recovery of applied rsN. For treatment code see Fig. 1 Treatment Parameter (a) In berseem Infection (%) of total root length colonized Shoot dry weight (g) Root dry weight (g) Shoot N concentration (%) Root N concentration (%) Shoot atom % i5N excess Root atom % 15Nexcess (b) In apple seedhgs Infection (%) of total root length colonized Shoot dry weight (g) Root dry weight (g) Shoot N concentration (%) Root N concentration (%) Shoot atom % i5N excess Root atom % “N excess Recovery (%) of applied i5N

I

II

III

0

48 a*

52a

1.6a 0.2 a 2.?a 3.6 a 2.2107 a 0.2428 a 0 2.1 a l.Oa 1.5a 1.1 a 0.0164 a 0.0174 a 0.64 a

1.8a 0.2 a 2.7 a 2.9 b 2.1066 a 0.5326 b

2.0 a 0.2 a 2.5 a 2.8 b 1.8315 a 0.4070 ab 72

0

3.3b 1.2a 1.5a 1.1 a 0.0241 c 0.2639 b 4.45 b

2.0 a l.Oa 1.5a 1.2a 0.0085 b 0.0095 a 0.45 a

*Values are means of four replicate treatments. Different letters within the same row indicate a significant difference at the P < 0.05level. roots of maize. However, maize infected by the mycorrhizal fungus (treatment III) showed significantly higher atom% excess lSN (Table 1) and “N contents (Fig. 2) than non-mycorrhizal maize (treatment I and II), indicating that 15N had been transferred from &seem to maize via VA mycorrhizal

hyphae. Non-mycorrhizal control plants (treatments I and II) showed low but significant enrichment of lSN suggesting that small amounts of “N were transferred by mechanisms other than direct transfer through mycorrhizal hyphae. Recovery of “N in non-mycorrhizal maize (treatments I and II) was 0.04% of the 15N in the legume. In contrast, 0.12% of the 15N in the legume was found in mycorrhizal maize (treatment III). Experiment

be 0.35 m g-’ dry soil. Average hyphal length in the root compartment of the mycorrhizal legume in the membrane restriction (II) treatment was 6.4 m g-’ dry soil and 5.7 m in the non-membrane restriction one (III). Shoot and root biomass of berseem were similar in control plants and in plants colonized by G. intraradices (compare treatments I-III, Table 2). Howmycorrhizal infection (treatment III) ever, significantly increased the shoot dry weight in apple compared to non-mycorrhizal apple (treatments I and II, Table 2). N concentrations in the receiver plants were not affected by mycorrhizal infection (see

2

The mycorrhizal fungus colonized 48 and 52% of the total root length of berseem in the membranerestriction treatment (II) and the non-membrane restriction treatment (III), respectively (Table 2). There was no VA mycorrhiza formation in the control cuvettes without mycorrhizal inoculum (treatment I). Root colonization by the VA mycorrhizal fungus in the receiver plants averaged 72% (Table 2). No colonization occurred in apple plants, where mycorrhizal hyphae restricted by a 0.45 pm membrane (treatment II) had no access to the central compartment. Hyphal lengths in the root-free compartment in the non-membrane restriction (III) treatment were 4.5 m g-’ dry soil. The hyphal background in the root-free compartment of treatments I and II was estimated to

shoot

root

Fig. 3. Amounts of 15N in excess &g) transferred from berseem (T. alexandrinum) to apple through a 3-cm-thick root-free compartment as affected by the different treatments (experiment 2). For treatment code see Fig. 1. Bar heights represent the mean and standard error of four replicates.

656

B. FREY and

H.

SCHUEPP

mycorrhizal compared to non-mycorrhizal plants. Complications of releasing more N compounds out of the roots influenced by mycorrhizal infection could be resolved by a mycorrhizal control treatment in which hyphae are restricted by a 0.45 pm pore size membrane (membrane-restriction treatment). In fact, experiment 2 indicated that less 15N was transferred to apple in the mycorrhizal control (II) treatment compared to the non-mycorrhizal (I) treatment (Fig. 3). Hamel et al. (1991a) suggested that the fungal extension of mycorrhizal roots improved the recovery of the N lost by the plants. Preliminary experiments indicated that the membrane did not impose a significant diffusion barrier for nitrogen. Nitrogen compounds could diffuse across the membrane in response to a concentration gradient. A similar membrane had been used in studies on phosphorus transport via VA mycorrhizal hyphae (Li et al., 1991a). Our study clearly demonstrates that, in spite of the 3 cm root-free separating zone, the VA mycorrhizal mycelium is a significant pathway of “N transfer DISCUSSION from a legume to a non-legume. However, the patBoth of our experiments provided evidence of 15N terns of “N transfer differed between the two expertransfer from a legume to a non-legume involving iments. Calculation of the amounts of 15Ntransferred mycorrhiza fungi. This is in agreement with earlier involves assumptions that the 15N incorporated into reports showing that VA mycorrhizal mycelium can berseem has the same opportunity to be transferred increase the transfer of 15N from soybean to interto the non-legume as does all other N in the plants. cropped maize (Van Kessel et a1.,1985; Bethlenfalvay In particular, if the 15N is utilized in structural et al., 1991; Hamel et al., 1991a), from white clover components of the root or in the shoot, this will be to ryegrass (Haystead et al., 1988) and from alfalfa to less likely to be lost from the roots (Giller et al., 1991). ryegrass (Barea et al., 1989). Similar responses have been hypothesized between actinorhizal alder and The occurrence of nutrient flow between linked adjacent plants via connecting ectomycorrhizas plants is likely to depend on whether or not a (Arnebrandt et al., 1990). However, no such a role of physiological imbalance exists (Eason and Newman, VA mycorrhizal fungi in N transfer between alfalfa 1990). Ritz and Newman (1985) suggested a net and bromegrass was found by G’Keefe and Williams transfer of nutrients following the death of one plant (1987) or by Hamel et al. (1991b) in studies of as was demonstrated for phosphorus. Thus, the direcfield-grown soybean and maize. All these earlier tion of nutrient movement between plants is likely to studies except for that of Bethlenfalvay et al. (1991) depend on a source-to-sink relationship. Findings were carried out in systems where roots were not from Bethlenfalvay et al. (1991) indicated that Nseparated by a root-free soil compartment. In some transfer from neighbouring soybean plants to maize cases a complete separation of the two root systems may be driven by such a physiological imbalance. In was not even possible (Van Kessel et al., 1985). our study, differences in the N demand of receiver Analysis of nitrogen transfer between plants via VA plants might influence the pattern of 15N transfer. A mycorrhizal hyphae is very difficult under these cirlarge difference in tissue N concentration between cumstances. Interposing a root-free zone between the berseem and apple was sufficient to set up a sourceroot systems of the donor and receiver plants to-sink relationship suggesting a mycorrhiza-medi(Schiiepp et al., 1987; Camel et al., 1991) reduces ated net transfer of N from berseem to apple. Our nutrient transfer by the soil pool pathway and enables data showed that shoot growth responded strongly to distinction to be made between N transfer by mass fungal colonization. This is in agreement with a flow and diffusion, and by means of mycorrhizal report by Miller et al. (1989). This growth stimulation hyphae (Bethlenfalvay et al., 1991). In our study very probably caused a strong demand for N in mycorsmall amounts of 15N were transferred by means rhizal apple. Likewise, the different developmental other than by VA mycorrhizal hyphae. stages of apple and maize might have affected their Differences in the morphology and physiology of demand for N. The apple plants were 7 weeks old mycorrhizal and non-mycorrhizal plants can further when the “N-1abelling of the berseem was begun compared to 2-week-old maize. complicate the study of nutrient transport by mycorrhizas (Faber et al., 1991). Findings from Hamel et al. Our study did not only show an obvious benefit to (1991a) showed reduced “N in the root exudates of the receiver plants in terms of transfer of increased treatment III, Table 2). However, total N content was significantly higher in infected apple (treatment III) due to the higher dry weights compared to noninfected apple plants (treatments I and II). Data from the 15N analyses in plant tissue of berseem indicated effective translocation of foliarlyabsorbed lSN into the root system (Table 2). Total “N contents in roots of berseem did not differ between treatments (data not shown). However, lSN contents in apple were significantly increased by the mycorrhizal infection (treatment III) compared to noninfected apple (treatments I and II, Fig. 3). There was movement of less than 7 pg of “N (0.7% of the “N in the legume) from berseem to non-mycorrhizal apple (treatments I and II), probably by a mechanism other than translocation through mycorrhizal hyphae. In contrast, there was movement of 49 pg “N (5.4% of the “N in the legume) from berseem to mycorrhizal apple, indicating a mycorrhiza-mediated transfer of 15N.

Mycorrhiza on interplant N-transfer 15N, but also in the total plant N content of mycorrhizal receiver plants. This increase in N content of the non-legume may not be attributable entirely to mycorrhiza-mediated N transfer from the legume, because VA mycorrhizal hyphae are known to play a role in the uptake of NH: from soil and its translocation to the host plant (Ames et al., 1983; Barea et al., 1987; Johansen et al., 1991). The role of direct mycorrhizal links in this interplant N transfer remains to be evaluated. Evidence has been presented that direct mycorrhizal links between roots are involved in phosphorus transfer (Newman and Ritz, 1986). This requires that nutrients pass directly from the host root tissue to its internal fungus. A role of direct links in N transfer could not, however, be confirmed by our study. Findings from Hamel et uZ.(1991a) indicated that N efflux from host to fungus at the symbiotic interface was of little significance. Similarly, Johansen er al. (1991) suggested that hyphal N transfer is one-way, i.e. from soil to host plant only, and not in the opposite direction. It is therefore possible that the pathway of N transfer between plants involves leakage of N from roots of the legumes and its subsequent uptake and transport by the mycorrhizal hyphae. Acknowledgements-Financial support from the Swiss Federal Office for Education and Science is gratefully acknowledged. We would like to thank Dr C. Walker for verifing the identity and purity of the VA mycorrhizal inoculum. We also gratefully acknowledge Dr G. Cadisch of Wye College, (U&ersity of London),Wye, U.K. for his help in the analvsis of the”N samples and Dr P. Christie for critically reading the manuscript.-

REFERENCES

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