Key words: Arbuscular myeorrhiza, cadmium, extraradical hyphae, heavy metals, hyphal transport.

Transcription

1 ^ ew Phytol. (997), 35, Uptake of ^^^Cd by roots and hyphae of a Glomus mosseae/ Trifolium subterraneum myeorrhiza from soil amended with high and low eoneentrations of eadmium BY E.J. JONER* AND C. LEYVAL Centre de Pedologie Biologique, CNRS, Laboratoire Associe a V Universite de Nancy I, 7, rue N.D. des Pauvres, BP 5, 545 Vandoeuvre-les-Nancy Cedex, France {Received 7 ay 996; accepted 7 October 996) SUARY Subterranean clover {Trifolium subterraneum L.) in symbiosis with Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe was grown in compartmented pots. Side compartments werefilledwith soil where recently added ( wk) radio-labelled cadmium (Cd) at three levels of non-labelled Cd could be accessed by either roots or arbuscular mycorrhizal hyphae. All treatments were replicated with non-mycorrhizal plants. After a growth period of 52 d roots and shoots were analysed for ^'^Cd, and lengths of roots and hyphae in respective labelled compartments determined. uptake by roots was not significantly influenced by the mycorrhizal status of the plant. Uptake of Cd from hyphal compartments was higher in mycorrhizal than in non-mycorrhizal plants, corresponding to 96, 27 and 3 % of that in non-mycorrhizal plants when, and mg Cd kg'^ was added, respectively. A large proportion of the increased Cd content of mycorrhizal plants was sequestered in the roots. It is concluded that extraradical hyphae of A fungi can transport Cd from soil to plants, but that transfer from fungus to plant is restricted due to fungal immobilization. No reduction of hyphal growth into soil with up to 2 mg extractable Cd kg"' was observed. Key words: Arbuscular myeorrhiza, cadmium, extraradical hyphae, heavy metals, hyphal transport. of their host plants in this respect (see, e.g., Bradley, INTRODUCTION. ^,... Wilkins, 99). For A the The ability of arbuscular mycorrhizas (A) to results are conflicting. Some reports indicate higher enhance plant uptake of nutrient elements through concentration of trace elements in plants owing to extraradical hyphal transport has been demonstrated A, even resulting in toxic levels in the plants for P (Hattingh, Gray & Gerdemann, 973), NH/ (Killham & Firestone, 983; Weissenhorn et al., (Ames et al., 983), Cu (Li, arschner & George, 995), whereas others have found a reduced plant 99) and Zn(Burkert&Robson, 994). By contrast, concentration of, e.g., Zn and Cd in mycorrhizal the uptake of other elements such as Fe and n is plants (Schuepp, Dehn & Sticher, 987; Elsometimes reduced when plants are colonized by Kherbawy et al., 989; Heggo, Angle & Chaney, A (George, Romheld & arschner, 994). The 99; Weissenhorn ef a/., 995). We have chosen to interest in environmental pollution has increased focus on Cd in the following study, as it is a highly over the past decades, and with it the interest in toxic pollutant in soil that is to some extent possible mycorrhizal influence on excess plant up- accumulated by food plants of domesticated animals take of essential and non-essential trace elements, and man. Other types of myeorrhiza confer a virtual protection Studies of the influence of A on Cd uptake by plants have so far been performed as pot experiments * To whom correspondence should be addressed at (present comparing mycorrhizal and non-mycorrhizal plants, address): Department of Biotechnological Sciences, icrobiology The results of such experiments can be ambiguous?2t;s^ntr^^s"'^"'''"'"^ ^^ '^^^'^ '^ ''^ '^ ^ regarding the mechanism of element uptake as erik.joner@ibf.nlh.no mycorrhizal colonization might cause differences in

2 354 E.J. Joner and C. Leyval length, architecture and exudation of roots, even if the shoot growth is matched by adding extra nutrients to non-mycorrhizal plants (Atkinson, Berta & Hooker, 994). To quantify the hyphal contribution in uptake of any nutrient in question, labelling with stable or radioactive isotopes in compartmented systems has been used repeatedly with success (Jakobsen, Abbott & Robson, 992; Johansen, Jakobsen & Jensen, 992). For nonessential trace elements similar investigations have not yet been reported. It was the objective of the present investigation to quantify the mycorrhizal contribution to Cd uptake in subterranean clover (Trifolium subterraneum L.), by using the radioisotope ^"^Cd and compartmented pots, thereby allowing differentiation between uptake by roots and hyphae and correction for ^"^Cd movement by mass flow and diffusion. ATERIALS AND ETHODS Experimental design The experiment was a 2 x 2 x 3 factorial with mycorrhizal colonization (+ / ) combined with two modes of access to labelled Cd (roots or hyphae), and three concentrations of added Cd (, or mg Cd kg"'). There were three replicates per treatment. Pots Pots consisted of PVC tubes (56 mm diameter) and fittings forming a vertical root compartment (RC; 5 cm'') and either a horizontal root-hyphae compartment (RHC; 35 cm^) or a horizontal hyphal h \ RC \ mg Cd kbq lo^ 37 ftm mesh (HC) or mm mesh (RHC) H HC/RHC Figure. Diagram of compartmented pot showing the arrangement of the root compartment (RC), the hyphal compartment (HC)/the root-hyphae compartment (RHC), the interface mesh and the placement of Cdamended soil labelled with "»Cd. Table. Some physical and chemical characteristics of the experimental soil before dilution ( :, zu/zv) with fine quartz sand Soil type NaHCOg-extractahle P (mg P Org C (%) ph (H^O) CEC* (m.e. g-i) Sand (%) Silt (%) Clay (%) * easured at ph 7 in NH^AC. Hapludalf compartment (HC; 35 cm^) separated by mm and 37/^m nylon mesh, respectively (Fig. ). The former mesh size allowed roots to enter the side compartment, while the latter restricted the entry of roots, but allowed free passage of mycorrhizal hyphae. Soil and nutrients A P-deficient clay soil (the top 25 cm of a forest {Fagus sylvatica, Carpinus betulus) brown soil (Table ) from Vandoeuvre, France (48 38'34" N- 6 T 24" E)), was sieved (< 2 mm) and sterilized by gamma-irradiation (27 kgy) prior to mixing : (w/w) with heat-sterilized fine (< -5 mm) quartz sand. Nutrient salts were added in the following amounts (mg kg~^ dry soil): Ca(NO3)2. 4H2O (253), NH4NO3 (86), K2SO4 (56), gso4.7h2o (23), nso^.h^o (5), ZnSO4.7H2O (), CUSO4.5H2O (8), H3BO3 (2), COSO4.7H2O (-5), NagoO^. 2H2O (-5). Soil that was placed in RC (65 g) of mycorrhizal pots, and all soil in HC (8 g) and RHC (8 g), received 25 mg P kg"^ (as KH2PO4); soil in RC of non-mycorrhizal pots received 35 mg P kg"^. Soil in the side compartments consisted of three layers; first a 4 mm buffer layer closest to the mesh, then a 8 mm layer containing, or loomgcdkg-i (as CdCl^.H^O) uniformly labelled with "'Cd ( kbq '"^CdCl^ per pot), and finally a 23 mm layer to top the compartment. Layers were separated by disks of nylon mesh ( mm) to ease separation during harvest, and packed to a soil density of -33 g cm~*. Soil in RC and each layer in RHC/HC received water individually to 6% of water holding capacity (WHC). Small pots (6 g soil) without plants were prepared with soil with the same water content, concentrations of nutrients and CdCla.H^O (but without ' *Cd) to monitor Cd availability. Thirty-six days after sowing all pots received an additional 7 mg of NH4NO3. Plants and soil inoculation All pots received 2x ml of a soil filtrate ( g of the original non-irradiated soil in 25 ml water, shaken h and filtered through a paper filter) in RC and

3 ycorrhizal influence on Cd uptake 355 RHC/HC, respectively, as an inoculum of soil micro-organisms except A. ycorrhizal pots were inoculated with 4 spores of a Cd-tolerant isolate of Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe (isolate P2, BEG 69, propagated on leek (Weissenhorn, Leyval & Berthelin, 993)) placed in RC adjacent to the RHC/HC. Two imbibed seeds of subterranean clover {Trifolium subterraneum L., cv. ount Barker) were sown in each pot and thinned to one plant per pot 5 d later. Plants were grown under light tubes (Sylvania Grolux; /imol m"^ s'^ PAR) with a light/dark cycle of 6/8 h and a temperature of C, and watered daily by weight. Harvest and analyses Plants were harvested 52 d after sowing. Subsamples of roots were taken to determine total and colonized root length. Shoots and remaining roots were dried for 24 h at 8 C and shoot and root d. wt recorded. To measure movement of Cd and hyphal growth, soil in HC was cut into layers of 7-9 mm width, representing soil with a distance from the rootconfining mesh of -7 mm, 8-4 mm (two sections of the buffer soil) and 5-23 mm (the half of the labelled soil closest to the roots). Water content and activity of ^""Cd were determined in duplicate samples from these three layers of both mycorrhizal and non-mycorrhizal treatments. Hyphal lengths were measured in duplicate samples from all three layers of mycorrhizal treatments and from the 8-4 mm layer of non-mycorrhizal treatments. The activity of ^"^Cd in soil samples from HC, roots and shoots was measured on a Packard Cobra Auto- Gamma counter, correcting for isotopic decay. Correction for counting efficiency was made using standard samples with known activity of ^"'Cd. Total and mycorrhizal root length was determined for RC and RHC by a line-intersect method (Tennant, 975) after staining root subsamples in trypan blue (Kormanik & cgraw, 982). Length of mycorrhizal hyphae was measured using a membrane filter-grid intersect method modified after Abbott, Robson & De Boer (984). odifications included the use of 2 ml aliquots and 2 mm diameter filter area. Duplicate filters were made from each soil sample from HC, and hyphae were counted in 25 fields of vision at x magnification. A common hyphal background was calculated similarly (two subsamples, duplicate filters) from HC of the nine non-mycorrhizal pots, and subtracted from the values measured in mycorrhizal samples. Plant available Cd was estimated by extraction in NH4NO3 (Symeonides & crae, 977) and NH^-acetate/O-l Na-EDTA, ph 7 (AFNOR, 99). Treatment effects were tested by two- or three-factor analysis of variance, and means compared by the F test, by estimated values for the least significant difference (P = -5), or by standard error of mean. RESULTS A colonization and plant growth Inoculation with G. mosseae resulted in A colonizing on an average 57% of the root length in samples taken in RC adjacent to the labelled compartment (the site of inoculum placement. Table 2), but only 2-5% of the roots sampled in RC 5- cm from the site of inoculum placement (top and bottom of pot; data not shown). Roots in RHC were poorly colonized in the buffer layer (< -6%), and largely uncolonized in the labelled soil layer (Table 3). The presence of hyphae and juvenile A spores among the stained roots were however noted in all layers of mycorrhizal RHC irrespective of the level of Cd addition. Plants receiving no mycorrhizal inoculum remained uncolonized, and neither spores nor hyphae were observed among stained roots. ycorrhizal inoculation resulted in higher d. wt of roots and shoots (Table 2). Concentration of Cd or mode of access to Cd-amended soil did not influence shoot d. wt, but in non-mycorrhizal plants root d. wt was lower in pots with HC than in pots with RHC. ean root length in RHC was lower in mycorrhizal than in non-mycorrhizal pots in all soil layers, except in the treatments with loomgcdkg"^ where root lengths were similar (Table 3). The addition of and lomgcdkg"^ to RHC generally resulted in a moderate reduction of the root length in the two distal soil layers compared with that in soil layer closest to RC. At mg Cd kg~^ root lengths were reduced in all soil layers compared with treatments with lower Cd additions, and the reduction was most pronounced in the two distal layers. Root:shoot (R/S) ratios were not affected by the mycorrhizal status of plants nor by Cd addition, whereas plants with roots accessing the labelled soil (RHC) had higher R/S ratios than plants grown in pots with only hyphal access to the labelled soil (Table 2). Only small and pale nodules were found on the roots, with no differences between treatments (data not shown). Plant uptake of Plant uptake of ^"*Cd and its distribution between roots and shoots varied greatly depending on mode of access to the labelled soil and in some cases also depending on Cd concentrations in the labelled soil (Tables 4, 5). Thus when roots accessed the labelled soil shoot concentration of ^*"Cd reached the highest values and exceeded by far the concentrations in the roots. When roots were excluded from the labelled

4 356 E. J. Joner and C. Leyval Table 2. Shoot and root d. wt, root: shoot (R/S) ratio and mycorrhizal colonization in root compartments (RC) of mycorrhizal () and non-mycorrhizal () Trifolium subterraneum with roots (RHC) or A hyphae (HC) partly exposed to three concentrations of Cd (n = 3) Compartmentation Cd added (mg kg-i) ycorrhizal status Shoot d. wt (g) Root d. wt (g) R/S ratio Root colonization in RCt (%) HC RHC LSD«.o, Analyses of variancej ycorrhiza Compartmention yc. X Cd Cd X compartment yc. X compartment yc. X Cd X compartment #** ** **# * *««#** *# N.D. * t easured at the site of inoculum placement. X, not significant; *P< -5; **P < -; p < -; N.D., not determined. Table 3. Total and colonized root length in Cd-amended soil and adjoining soil layers of root-hyphae compartments (RHC) as influenced by added Cd and mycorrhizal inoculation in the root compartment (RC) (n = 3) Root length in RHC (cm g-') ycorrhizal inoculation Cd added to soil 5-33 mm (mg kg-') Soil -4 mm Total Colonized Soil 5-33 Total mm Colonized Soil mm Total Colonized yc LSD Analyses of variancef ycorrhiza Distance Interactions Total # ** root length < Colonized N.D. *#* - - root length t, not significant; *P < 5, **P < -; P < -; N.D., not determined. soil (HC treatments) the concentrations of reached the highest values in roots. Further, shoot concentrations of ""Cd decreased with increasing concentrations of Cd in labelled soil in the RHC treatments, whereas the ""Cd concentrations in the roots outside RHC were unaffected by soil Cd concentration. By contrast, both root and shoot concentrations of "*Cd increased significantly with increasing Cd concentrations in labelled soil of the HC treatments. The mycorrhizal status of the plants did not influence shoot concentrations of ^"^Cd, but concentration in roots were increased in mycorrhizal plants (HC treatments). When the labelled soil was accessed by roots no differences between mycorrhizal and non-mycorrhizal plants were significant. No significant correlation was found between plant ^""Cd

5 ycorrhizal influence on Cd uptake 357 Table 4. ean concentration of ^"^Cd in shoots and roots, and Cd-root -.shoot {R/S) ratio in non-mycorrhizal ) and mycorrhizal {) Trifolium subterraneum, having roots and/or A hyphae partially exposed to equal amounts of ^'^^Cd at three concentrations of Cd (n = 3) Cd placed inhc Cd placed inrhc Cd added (mg kg"i) ycorrhizal status Shoot (Bq g-^) Root (Bq g-') R/S ratio Shoot (Bq g-^) Roott (Bq g-^) R/S ratiof LSD Analyses of variance^ yeorrhiza yc. X Cd ** * * #*# t Roots in RHC not included. X, not significant; *P< -5; **P < -; #**P < -. Table 5. ean content of ^"^Cd in shoots and roots and Cd-root:shoot (R/S) ratio of ^ ^Cd in non-mycorrhizal {) and mycorrhizal () Trifolium subterraneum with roots and/or A hyphae partly exposed to equal amounts of ^'^^Cd at three concentrations of Cd (n = 3) Cd placed inhc Cd placed in RHC Cd added (mg kg"^) ycorrhizal status Shoot (Bq) Root (Bq) Cd-R/S ratio Shoot (Bq) Roott (Bq) Cd-R/S ratiof LSD Analyses of variance^ yeorrhiza yc. X Cd #*# #** *# #*# #*# t Roots in RHC not included. I, not significant, **P < -; P < -. uptake and hyphal length in the corresponding HC (see below) using linear regression. The recovery in plants of initially added ^"''Cd to HC/RHC ranged from -2 to 9-5%. Hyphal growth Length of hyphae in HC was measured in soil sections at three distances from roots. A common background measured in non-mycorrhizal HC constituted -48 m g"^, and was subtracted from all values in mycorrhizal HC. The resulting hyphal lengths are presented in Table 6. No significant differences in hyphal length as a function of distance from roots or as a function of soil Cd concentration were found. Even in soil with mg of added Cd kg"^ juvenile spores were observed during hyphal measurements. No such spores were encountered in non-mycorrhizal samples. Soil Cd concentrations Safety precautions did not permit determination of Cd in soil or plants containing ^"'Cd, so for calculations of Cd concentrations specific activities of 685, 685 and 68-5 Bq/tg"^ Cd are assumed.

6 358 E. J. Joner and C. Leyval Table 6. Hyphal lengths in hyphal compartments (HC) of mycorrhizal pots as infiuenced by Cd addition to soil 5-33 mm from roots and distance from roots Cd added to labelled soil (mg kg-^) LSD Analysis of variancef Distance from roots Interactions Hyphal length in HC (m -7 mm 8-4 mm from root! 5 from roots mm from roots f, not significant (P > -5). Hyphal background as measured in non-mycorrhizal HC has been subtracted. calculated from the amounts of Cd and ^"'Cd added in the treatments with, and mg of added Cd kg~s respectively. Availability of added Cd (as estimated by NH^NOg extraction) increased with increasing amounts of initially added Cd from 4 to 65 % (data not shown). ore firmly bound Cd (85 % of initially added Cd; estimated by NH4AC/EDTA extraction) was not influenced by initial Cd concentration. Values in Table 7 were determined in soil without extraction (total ^ Cd content) and show that Cd mobility in the experimental soil was high and increased with increasing Cd additions. The presence of A hyphae had no effect on concentrations or mobility of ^""Cd in HC. DISCUSSION Compartmented pots were used in an attempt to separate and compare Cd uptake by extraradical A hyphae and plant roots from soil with high or low Cd concentrations. This compartmentation also allowed us to study plant uptake from soil with high Cd concentrations without concomitant adverse effects on plant size. The mobility of freshly added Cd in the soil of the present experiment was high, and increased with increasing amounts of added Cd, as shown in Table 7. The use of a 4-mm layer of soil without added Cd between RC and labelled soil did not prevent movement of Cd to RC, since plants of the nonmycorrhizal/hc treatments contained measurable quantities of "*Cd. The recovery of ^"^Cd in these plants as a percentage of Cd added to HC ranged from -9 % at mg of added Cd kg-\ increasing up to -56% at loomgkg-i of added Cd. The high mobility of added Cd might have resulted from the low cation exchange capacity of the experimental soil (Levi-inzi, Soldatini & Riffaldi, 976) and the short time between mixing Cd into the soil and the start of the experiment (Schmitt & Sticher, 986). The increasing Cd mobility at high levels of added Cd might similarly result from saturation of the fixing capacity of the soil. Both diffusion and mass flow seem to be involved as transport mechanisms for Cd in soil. ions are more mobile in soil than ions of Zn and Cu (Hornburg, Welp & Brummer, 995), and since diffusion rates of these metals decrease with increasing ionic diameter (Schmitt & Sticher, 99) Cd being the greater, mass flow should be important in Cd transport. The uptake of ^ *Cd by non-mycorrhizal plants after supply of ^"'Cd to the HC was attributed to root Table 7. Soil ^'^^Cd content in hyphal compartments as infiuenced by distance from the root compartment (RC), addition of Cd to soil and absence/presence of mycorrhizal hyphae {figures in parentheses are SE of mean, n = 3) Activity of '»Cd in soil (Bq g"') mg Cd kg""' mg Cd kg-' mg Cd kg-' No hyphae Hyphae No hyphae Hyphae No hyphae Hyphae -7 mm 394 (23) 8-4 mm 23(53) 5-23 mm 7636(3) Analysis of variancef added Distance Hyphal presence Cd X distance Other interactions 398 (2) 268 (238) 84(27) #** 588 (28) 225 (232) 6393 (6) 582(8) 2287 (26) 66 (237) 399(5) 2986 (24) 4993 (45) 46 (96) 39 (42) 54 (48) f, not significant, P < -. Soil sections -4 mm from roots are parts of initially unlabelled soil, whereas the 5-23 mm section is part of the soil initially labelled with 685 Bq g"'.

7 ycorrhizal influence on Cd uptake 359 uptake at the mesh interface following diffusion/ mass fiow from the site of Cd addition. In mycorrhizal plants significantly higher "*Cd concentration and ^"^Cd content were found in the roots compared with roots of non-mycorrhizal plants. This difference between mycorrhizal and non-mycorrhizal plants might be attributed to hyphal uptake, but confounding effects might be found in plant size, which was also significantly higher for mycorrhizal plants. At harvest, the water content of the soil layer closest to the mesh (-7 mm) was not significantly different from that of the labelled soil layer (5-23 mm), nor were there any differences between mycorrhizal and non-mycorrhizal pots (data not shown). This indicates little movement of water (and thus mass fiow of Cd) over the mesh interface, a phenomenon commonly observed in our experiments of this kind (e.g. Joner & Jakobsen, 995). Data for soil ^^^Cd concentration (Table 7) do not indicate differences between mycorrhizal and non-mycorrhizal plants in movement of Cd, nor any depletion due to hyphal uptake. However, a high proportion of ^"^Cd was not accounted for when recovery of ^ *Cd in plants and in HC soil was calculated (range: %). If higher uptake in mycorrhizal plants was due to more Cd reaching these plants by diffusion, a steeper gradient in Cd between soil layers in mycorrhizal HC than in non-mycorrhizal HC would be expected. In the case of mass fiow contributing more to uptake in the larger mycorrhizal plants, lower total amounts of "*Cd should have been found in mycorrhizal HC. Neither was the case, and though the measurements of soil Cd movements were not very accurate, we conclude that some hyphal transport took place. The ^""Cd concentrations in mycorrhizal plants corresponded to 96, 27 and 3% of those in nonmycorrhizal plants when, and mg Cd kg"^ was added to HC, respectively. Similarly, hyphal Cd uptake might have constituted a maximum of -6, 4 and 55% of corresponding uptake in plants when roots accessed the labelled soil (calculated from Table 5). The latter increase is probably due to root uptake being gradually impaired as the Cd concentration in soil rose. Conversely, the hyphae seemed unaffected in this respect. Hyphal growth was not influenced by soil Cd concentrations up to what might have been c. 2 mg of NH4NO3- extractable Cd kg"^ soil (when mg Cd kg"^ was added, 3% of the tracer remained in the 5-23 mm soil layer, and 65% of this was extractable). The extraradical mycelium thus seems to constitute a robust part of the symbiosis, being far less sensitive to Cd than e.g. roots (see below). Roots: shoot (R/S) ratios of ^"^Cd were highest in mycorrhizal plants (Tables 4, 5), though R/S ratios of plant dry matter were not. This suggests an efficient immobilization of Cd in the roots of mycorrhizal plants, as concentration and content of ^"^Cd in corresponding shoots were not influenced by rnycorrhiza. Our results thus confirm the findings of Schviepp et al. (987) and their proposal of a plant protection mechanism involving Cd retention in fungal structures, similar in principle, but probably not in extent, to that found in ectomycorrhiza (e.g. Turnau, Kottke & Dexheimer, 996). Uptake of ^ *Cd from RHC by mycorrhizal plants was not different from uptake by non-mycorrhizal plants whether comparisons were made on the basis of concentration or total content. Differences in root densities in RHC between mycorrhizal and nonmycorrhizal treatments were significant, but correlations between root length and ^"^Cd in plants were generally not (results not shown). The lower root length in mycorrhizal plants resulted in higher specific uptake of Cd (root length basis) at the two lowest levels of soil Cd. Considering the high mobility of Cd in soil in the present experiment and the high amounts of Cd commonly immobilized by roots (Cd-R/S ratios in Trifolium spp. of 7-, e.g. Jarvis, Jones & Hopper, 976), it is possible that an increased root length in the labelled soil above that found in mycorrhizal treatments would contribute as much to immobilization of Cd as to increased plant uptake. The Cd-R/S ratios of < in RHC treatments probably resulted from the split root-type of Cd addition and subsequent measurements of ^"^Cd in roots in RC. The observed internal distribution of Cd indicates that aerial parts are the preferred organs for deposition of Cd that is mobile within the plant. As growth reductions were only observed for roots inside Cd-amended compartments the translocated Cd did not seem to confer any toxic effect upon the plants though mean values reached 26 /ig Cd g"^ in shoots (calculated from Table 4). This is (for example) three times higher than threshold values given for Cd toxicity in barley by Beckett & Davis (977). Commonly the whole root system encounters Cd in soil, and Cd is then largely immobilized on cell walls or in vacuoles of roots (Lindsay & Lineberger, 98; Vazquez, Poschenrieder & Barceld, 992) at the site of Cd uptake,, with only a small fraction being so mobile within the plant as to reach the shoots. This, along with fungal immobilization, can explain why Cd-R/S ratios were always > in HC treatments where Cd was taken up by roots that constituted a part of the roots analysed for ^""Cd. The growth of roots in RHC was reduced in the labelled soil layer by addition of Cd. Roots of mycorrhizal plants were more severely affected than those of non-mycorrhizal plants, though roots at the site of Cd placement were largely uncolonized. The poor spread of root colonization from the site of inoculum placement is a phenomenon that seems unrelated to the presence of Cd, for it has been found in similar experiments without added Cd (Joner & Jakobsen, 994). It did, however, prevent assessment of effects of Cd toxicity on the colonization process.

8 36 E. J. Joner and C. Leyval but more detailed studies on this are published elsewhere (Weissenhorn & Leyval, 995). To what extent the choice of endophyte (a heavymetal-tolerant strain of G. mosseae) might have influenced hyphal uptake of Cd is uncertain. The fungal tolerance to heavy metals probably influenced the ability of the hyphae to grow into soil with high Cd concentrations, and it might have retained more of its metabolic activities than would a non-tolerant fungus. This hypothesis is currently under investigation. ACKNOWLEDGEENTS We thank Gilbert Belgy for performing analyses of soil extracts, and arie-jose Belgy for help with ""Cd analyses. EJ Joner received a grant from the European Community programme Human Capital and obility (ERBCHBI CT9473) for the duration of this investigation. REFERENCES Abbott LK, Robson AD, De Boer G The effect of phosphorus on the formation of hyphae in soil by the vesicular arbuscular mycorrhizal fungus, Glomus fasciculatum. New Phytologist 97: AFNOR. 99. Determination du cuivre, manganese et zinc. Extraction par I'acetate d'ammonium en presence d'edta. In: Recueil de normes frajifaises. Qualite du sol, Paris: AFNOR, X3-2. Ames RN, Reid CPP, Porter LK, Cambardella C Hyphal uptake and transport of nitrogen from two '^N-labelled sources by Glomus mosseae, a vesicular-arbuscular mycorrhizal fungus. New Phytologist 95: Atkinson D, Berta G, Hooker JE Impact of mycorrhizal colonization on root architecture root longevity and the formation of growth regulators. In: Gianinazzi S, Schuepp H, eds. Impact of Arbuscular ycorrhizas on Sustainable Agriculture and Natural Ecosvstems. Basel: Birkhauser Verlag, Beckett PHT, David RD Upper critical levels of toxic elements in plants. New Phytologist 79: Bradley R, Burt AJ, Read DJ The biology of mycorrhiza in the Ericaceae. VIII. The role of mycorrhizal infection in heavy metal resistance. New Phytologist 9: Biirkert B, Robson A "''Zn uptake in subterranean clover {Trifotium subterraneum L.) by three vesicular arbuscular mycorrhizal fungi in a root-free sandy soil. Soil Biology and Biochemistry 26: El-Kherbawy, Angle J, Heggo A, Chaney R Soil ph, rhizobia, and vesicular-arbuscular mycorrhizae inoculation effects on growth and heavy metal uptake in alfalfa {edicago sativa L.). Biology and Fertility of Soils 8: George E, Romheld V, arschner H Contribution of mycorrhizal fungi to micronutrient uptake by plants. In: anthey JA, Crowley DE, Luster DG, eds. Biochemistry of etal icronutrients in the Rhizosphere. Boca Raton, FL, USA: CRC Press, Inc., Hattingh J, Gray LE, Gerdemann JW Uptake and translocation of "^P-labelled phosphate to onion roots by endomycorrhizal fungi. Soil Science 6: Heggo A, Angle JS, Chaney RL. 99. Effects of vesiculararbuscular mycorrhizal fungi on heax'j' metal uptake by soybeans. Soil Biology and Biochemistry 22: Hornburg V, Welp G, Brummer GW Verhalten von Schwermetallen in Boden. 2. Extraktion mobiler Schwermetalle mittels CaClj und NH4NO3. Zeitschrift fur Pflanzenerndhrung und Bodenkunde 58: Jakobsen I, Abbott LK, Robson AD External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterraneum L. 2. Hyphal transport of '^P over defined distances. New Phytologist 2: Jarvis SC, Jones LHP, Hopper J uptake from solution by plants and its transport from roots to shoots. Plant and Soil AA: Johansen A, Jakobsen I, Jensen ES Hyphal transport of '^N-labelled nitrogen by a vesicular-arbuscular mycorrhizal fungus and its effect on depletion of inorganic soil-n. New Phytologist 22: Joner EJ, Jakobsen I Contribution by two arbuscular mycorrhizal fungi to P uptake by cucumber {Cucumis sativus L.) from ^^P-labelled organic matter during mineralization in soil. Plant and Soil 63: Joner EJ, Jakobsen Uptake of '^P from labelled organic matter by mycorrhizal and non-mycorrhizal subterranean clover {Trifolium subterraneum L.). Plant and Soil 72: Killham K, Firestone K Vesicular-arbuscular mycorrhizal mediation of grass response to acidic and heavy metal deposition. Plant and Soil 72: Kormanik PP, cgraw AC Quantification of vesiculararbuscular mycorrhiza in plant roots. In: Schenck NC, ed. ethods and Principles in ycorrhizal Research. St Paul: American Phytopathological Society, Levi-inzi R, Soldatini G, Riifaldi R adsorption by soils. Journal of Soil Science 27: -5. Li X-L, arschner H, George E. 99. Acquisition of phosphorus and copper by VA-mycorrhizal hyphae and rootto-shoot transport in white clover. Plant and Soil 36: Lindsay P, Lineberger R. 98. Toxicity, cadmium accumulation and ultrastructural alterations induced by exposure of Phaseotus seedlings to cadmium. Horticultural Science 6: 434. Schmitt H, Sticher H Prediction of heavy metal contents and displacement in soils. Zeitschrift fur Pflanzenerndhrung und Bodenkunde 49: Schmitt HW, Sticher H. 99. Heavy metal compounds in the soil. In: erian E, ed. etals and Their Compounds in the Environment. Occurrence, Analysis and Biological Relevance. Weinheim: VCH Verlaggesellschaft mbh, Schuepp H, Dehn B, Sticher H Interaktionen zwischen VA-ykorrhizen und Schwermetallbelastungen. Angewandte Botanik 6: Symeonides C, crae S The assessment of plantavailable cadmium in soi\s. Journal of Environmental Quality 6: Tennant D A test of a modified line intersect method of estimating root length. Journal of Ecology 63: Turnau K, Kottke I, Dexheimer J Toxic element filtering in Rhizopogon roseolus/pinus sylvestris mycorrhizas collected from calamine dumps. ycological Research : Vazquez, Poschenrieder C, Barcelo J Ultrastructural effects and localization of low cadmium concentrations in bean roots. New Phytologist 2: Weissenhorn I, Leyval C Root colonization of maize by a Cd-sensitive and a Cd-tolerant Glomus mosseae and cadmium uptake in sand culture. Plant and Soil 75: Weissenhorn I, Leyval C, Belgy G, Berthelin J Arbuscular mycorrhizal (A) contribution to heavy metal uptake by maize {Zea mays L.) in pot culture with contaminated soil. ycorrhiza 5: Weissenhorn I, Leyval C, Berthelin J Cd-tolerant arbuscular mycorrhizal (A) fungi from heavy-metal polluted soils. Plant and Soil 57: Wilkins D. 99. The influence of sheathing (ecto-) mycorrhizas of trees on the uptake and toxicity of metals. Agriculture, Ecosystems and Environment 35:

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