Radiocesium transfer between Medicago truncatula plants via a common mycorrhizal network

Environmental Microbiology (2010) 12(8), 2180–2189

doi:10.1111/j.1462-2920.2009.02118.x

Radiocesium transfer between Medicago truncatula plants via a common mycorrhizal network emi_2118

Veronika Gyuricza,1 Yves Thiry,2† Jean Wannijn,2 Stéphane Declerck1 and Hervé Dupré de Boulois1* 1 Université catholique de Louvain, Unité de Microbiologie, Croix du Sud 3, 1348 Louvain-la-Neuve, Belgium. 2 SCK CEN, Foundation of Public Utility, Biopshere Impact Studies, Boeretang 2200, 2400 MOL, Belgium. Summary Common mycorrhizal networks of arbuscular mycorrhizal fungi have been reported to transfer cesium between plants. However, a direct hyphae-mediated transfer (via cytoplasm/protoplasm) cannot be distinguished from an indirect transfer. Indeed, cesium released by the roots of the donor plant can be taken up by the receiver plant or fungal hyphae. In the present study, Medicago truncatula plants were connected by a common mycorrhizal network and Prussian Blue (ammonium-ferric-hexacyano ferrate) was added in the growth medium to adsorb the released radiocesium. A direct transfer of radiocesium to roots and shoots of the receiver plant was clearly demonstrated for the first time. Even though this transfer was quantitatively low, it suggested that shared mycorrhizal networks could contribute to the redistribution of this radionuclide in the environment, which otherwise would be restricted both in time and space. This finding may also help to understand the behaviour of its chemical analogue, potassium.

Introduction Arbuscular mycorrhizas are symbiotic associations between the roots of the vast majority of terrestrial plants and the mycelium of fungi belonging to the phylum Glomeromycota (Schüßler et al., 2001). These microorganisms, termed arbuscular mycorrhizal (AM) fungi, influence plant production, biodiversity and ecosystem functioning (van der Heijden et al., 1998) via an intricate network of hyphae providing nutrients to their hosts. The extraradical hyphae can also connect the roots of plants that belong to Received 8 July, 2009; accepted 21 October, 2009. *For correspondence. E-mail [email protected]; Tel. (+32) 10 47 22 26; Fax (+32) 10 45 15 01. †Present address: ANDRA, 1-7 rue Jean Monnet, 92298 Châtenay-Malabry Cedex, France.

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the same or different species and families, in a common mycorrhizal network (CMN) (for a review see Simard and Durall, 2004). Hyphal connections represent possible pathways for the transfer of soil nutrients as well as for plant-derived C between plants. These CMNs could play a role in plant competition and nutrient cycling in the ecosystems (see Smith and Read, 2008). A number of studies have been conducted on N, P and C transfer between donor and receiver plants. So far, evidence for the transfer of limited amounts of P was obtained from dying donor roots to receiver plants (Johansen and Jensen, 1996), between individual mycelia of two plants (Mikkelsen et al., 2008) and even between donor and receiver plants (Wilson et al., 2006). However, the importance of this transfer is probably ecologically negligible and mechanisms supporting the bi-directional transfer of nutrients between plants have not yet been revealed (Smith and Read, 2008). Carbon transfer between plants via a CMN of AM fungi remains controversial. While it was observed by Grime and colleagues (1987), Lerat and colleagues (2002) and Carey and colleagues (2004), such transfer was not found in other studies (Fitter et al., 1998; Zabinski et al., 2002; Voets et al., 2008). The absence of clear-cut data on the involvement of CMNs in nutrient and C transfer supports the fact that cautiously designed experiments are needed in this area (Smith and Read, 2008). Transfer of radionuclides between plants has been seldom considered, even if several studies have demonstrated the role of AM fungi in the transport of radiocesium to plants (Dupré de Boulois et al., 2006). Radiocesium, a major contaminant of the environment, remains in the upper soil layers due to its low mobility (Delvaux et al., 2001) and accumulates in the food chain mainly through uptake by plants. Air-borne pollution can also provide a source of contamination by the release of radiocesium through re-suspension from contaminated soil particles (Fujiwara et al., 2007). The interception of radiocesium by plant leaves and its transfer into the fruits and roots has been studied extensively (e.g. Zehnder et al., 1995; Carini and Lombi, 1997; Sabbarese et al., 2002; Fortunati et al., 2004), but a subsequent transfer to another plant via a CMN has only been investigated recently, and only for stable Cs (Meding and Zasoski, 2008). These authors observed

Radiocesium transfer between plants via a CMN 2181

Fig. 1. Photographic representation of the experimental system used at harvest (i.e. 4 weeks after radiocesium leaf labelling of the donor plant). The system consisted of a donor and receiver plants interconnected through a CMN (Voets et al., 2008). Both plants grew in a 90 mm Petri plate containing MSR1 medium (see details in Experimental procedures) supplied with 100 p.p.m. of AFCF. AFCF was used as a blocking agent of radiocesium to ensure that radiocesium was transferred exclusively via the CMN.

the transfer of stable cesium between two plants interconnected by a CMN. They used two-chambered pots containing plants separated by stainless steel screens excluding root growth but allowing hyphal connection. Hyphae had direct access to the growing media, thus leakage from the roots and AM fungi and subsequent fungal and plant uptake directly from this releasedsource could not be excluded. Indeed, for radiocesium (behaving similarly as stable cesium) the release from the roots of contaminated plants was reported as a source of re-contamination (Zehnder et al., 1995; Carini et al., 2003). A possible way of eliminating this pool of radiocesium available to the AM hyphae and plants is by blocking the released amount by ammonium-ferrichexacyano ferrate [NH4Fe(III)Fe(II) (CN)6, AFCF, Prussian Blue]. AFCF was shown to be suitable for reducing radiocesium transfer from soil to plants (see Vandenhove et al., 2000 and reference therein), thus acting as a blocking agent of this radionuclide. It was reported to have no effect on plant growth in soil (Vandenhove et al., 1996; 1998; 2000). However, the potentially formed free cyanide (Kang et al., 2007) could possibly have toxic effects on plants under our experimental conditions. Moreover, the influence of AFCF on AM fungi has never been investigated so far. Recently, Voets and colleagues (2008) developed an in vitro culture system associating two plants via a CMN to investigate the transfer of C from a donor to a receiver plant. This system was adapted in the present study to investigate whether radiocesium could be transferred from a labelled donor plant to a receiver plant via a CMN under strict in vitro culture conditions (Fig. 1). AFCF was applied to ensure that radiocesium was transferred exclusively via the CMN.

Results Dosage of AFCF The adsorption rate at the surface of the medium supplied with 100 p.p.m. AFCF was estimated from the amount of 137 Cs removed by water. After 5 min of exposure the adsorption rate was 30 ⫾ 1.17% and increased to 77 ⫾ 2.54% and 86 ⫾ 3.60% after 1 and 3 h respectively. The radiocesium adsorption process was characterized by a very low reversibility. After 5 min of radiocesium exposure, the retention rate of this element in the medium was 99.7 ⫾ 0.05%. Foliar interception and translocation of radiocesium The 134Cs activity in roots and medium of early-labelled (2-week-old) and late-labelled (4-week-old) plants was measured 3, 4 and 5 weeks after labelling, and calculated as a percentage of the total activity applied to the plants (Fig. 2). In all treatments and plants 134Cs was translocated from shoot to roots. However, translocation to roots was significantly lower in the late-labelled plants as compared with the early-labelled plants (Fig. 2A), regardless of the length of the growing period (i.e. 3, 4 and 5 weeks) following labelling with 134Cs (termed as ‘labelled period’). Identically, the 134Cs activity measured in the medium of the late-labelled plants was significantly lower as compared with the activity measured in the medium of the early-labelled plants (Fig. 2B). In both treatments, independently of the length of the labelled period (i.e. 3, 4 and 5 weeks) a significant (P < 0.05) positive correlation was observed between the activity measured in the medium and the activity measured in the roots (r2 = 0.817 and 0.821 in the late- and early-labelled plants respectively).

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2182 V. Gyuricza et al. Fig. 2. Percentage (mean ⫾ SE) of 134Cs activity measured in plant roots (A) and in the medium (B). ‘Early-labelled plants’ refers to plants that were 2 weeks old at the time of labelling, whereas ‘late-labelled plants’ were 4 weeks old at the time of labelling (n = 3 for each length of labelling and for both treatments). Bars with the same letter did not differ significantly (P < 0.05).

The activity measured in the roots of early-labelled plants did not differ significantly after 3, 4 and 5 weeks of labelling and varied between 5.8 ⫾ 1.8% (week 4) and 8.2 ⫾ 1.6% (week 5) of the initially applied activity (Fig. 2A). However, 134Cs released in the medium was significantly higher after 5 weeks (7.2 ⫾ 2.3%) as compared with 3 or 4 weeks (Fig. 2B). For the late-labelled plants the activity measured in the roots and medium never exceeded, respectively, 2.5% and 2.0% of the initially applied activity, regardless of the labelled period. Effects of AFCF on plant and AM fungi Plant and fungal growth parameters in the control and AFCF treatment were estimated after 4 weeks (6-weekold plants) and 6 weeks of growth (8-week-old plants) in AFCF-containing medium (Table 1). For the measurement after 4 weeks, no significant differences were

observed between the control and AFCF treatments for any of the parameters evaluated. However, after 6 weeks the shoot length and the shoot fresh weight were significantly higher in the control treatment, while no differences were noted in fungal growth parameters and the number of leaves. The extraradical mycelium (ERM) developed in the HC for a period of 2 weeks. Within this period, P was taken up by the AM fungi in both the control and AFCF treatments and no significant differences were noted between the two treatments. In the control treatment, the MSR1 medium contained 1.31 ⫾ 0.45 mg g-1 of P, while in the AFCF treatment the medium contained 1.55 ⫾ 0.28 mg g-1 of P. This represented less than half of the original P-content of the medium (3.8 mg g-1 P in the MSR1). In both the control and the AFCF treatments, most hyphae contained cytoplasm, were non-septate and showed metabolic activity. The percentage of vital hyphae as well as the proportion

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Radiocesium transfer between plants via a CMN 2183 Table 1. Plant and AM fungal growth parameters in the control medium and medium containing 100 p.p.m. of AFCF of 6-week-old plants (non-destructive measurements after 4 weeks of growth in AFCF/control conditions) and 8-week-old plants and AM fungi (destructive and non-destructive measurements after 6 weeks of growth in AFCF/control conditions). 6-week-old plants

Shoot length (cm) Number of leaves Root length (cm) Shoot fresh weight (mg) Root fresh weight (mg) Number of spores RC Number of spores HC Hyphal length RC (cm) Hyphal length HC (cm)

8-week-old plants

AFCF

Control

AFCF

Control

12 ⫾ 1aa 5.5 ⫾ 0.6a 83 ⫾ 5a – – 1003 ⫾ 79a – 310 ⫾ 23a –

13 ⫾ 1a 6.6 ⫾ 0.3a 91 ⫾ 6a – – 1262 ⫾ 146a – 334 ⫾ 21 –

17 ⫾ 2a 5.7 ⫾ 1.0a 95 ⫾ 10a 231 ⫾ 24a 480 ⫾ 124 1224 ⫾ 102a 98 ⫾ 31a 369 ⫾ 35a 168 ⫾ 65a

28 ⫾ 1b 7.0 ⫾ 0.8a 126 ⫾ 16a 530 ⫾ 120b 532 ⫾ 120a 1556 ⫾ 179a 115 ⫾ 43a 406 ⫾ 49a 184 ⫾ 64a

a. For plants of the same age (i.e. 6 or 8-week-old plants), values within the same row followed by an identical letter are not significantly different (P < 0.05). Values are means ⫾ SE of five replicates. RC, root compartment; HC, hyphal compartment.

of succinate-dehydrogenase (SDH)-active hyphae did not differ significantly between the AFCF-and the control treatments (Fig. 3). Radiocesium transfer between plants Four weeks after labelling the plants were harvested. Fresh and dry weight of roots and shoots of the donor and receiver plants as well as the number of spores and hyphal length, root colonization in the donor and receiver plants were estimated (Table 2). No differences were found between donor and receiver plants within the same treatment. Identically, no differences were observed between the mycorrhizal and non-mycorrhizal treatments. In the control treatment (i.e. plants that were not connected by a CMN) no activity was detected in either the roots or the shoots of the receiver plants whereas activity was detected in the shoots (88.41 ⫾ 2.57% of the total applied activity) and roots (6.89 ⫾ 1.41% of the total applied activity) of all the donor plants and in the medium (4.70 ⫾ 1.18% of the total applied activity). It was also

Fig. 3. Vitality and metabolic activity of hyphae assessed by SDH activity in the AFCF and control treatment. Columns correspond to the means ⫾ SE of five replicates. NS indicates that no significant difference (P < 0.05) was found between the treatments for the different parameters.

Table 2. Plant and AM fungal growth parameters for plants connected by a CMN and controls without mycorrhiza (n = 6 where one replicate consist of a donor and receiver plants connected or not by a CMN). Non-mycorrhizal

Shoot fresh weight (mg) Root fresh weight (mg) Number of spores Hyphal length (cm) Frequency (%F) Intensity (%I)

Mycorrhizal

Donor

Receiver

Donor

Receiver

130 ⫾ 24aa 508 ⫾ 91a – – – –

108 ⫾ 41a 488 ⫾ 80a – – – –

159 ⫾ 62a 424 ⫾ 85a 978 ⫾ 216 90 ⫾ 39 66 ⫾ 21a 18 ⫾ 12a

195 ⫾ 56a 475 ⫾ 102a 70 ⫾ 31a 20 ⫾ 9

a. Values of the same treatment within the same row followed by an identical letter are not significantly different (P < 0.05) (mean ⫾ SE of six replicates). The plants from the two treatments (mycorrhizal and non-mycorrhizal) were also compared and were not significantly different (P < 0.05).

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2184 V. Gyuricza et al. Table 3. Distribution of

134

Cs in donor and receiver plants linked or not by a common mycelial network. Mycorrhizal plants

Shoot Roots Medium

Non-mycorrhizal plants

Donor

Receiver

Donor

Receiver

84.01 ⫾ 4.95 5.08 ⫾ 1.48

0.41 ⫾ 0.22 1.27 ⫾ 0.54

88.41 ⫾ 2.57 6.89 ⫾ 1.41

0 0

3.63 ⫾ 1.26

4.70 ⫾ 1.18

Average percentage of the total applied activity in different plant parts and medium at the end of the experiment (i.e. after 4 weeks of labelling). Values are means ⫾ SE of six replicates.

shown in an independent assay in which 134Cs was applied on MSR1 medium containing 100 p.p.m. AFCF that no 134Cs was taken up by mycorrhizal plants after 3 weeks of exposition (data not shown). In the systems where donor and receiver plants were connected via a CMN, a transfer of 134Cs was observed. The repartition of 134 Cs between mycorrhizal donor and receiver plants and its release in the medium is shown in Table 3. Activity was detected in the roots of all the receiver plants (1.27 ⫾ 0.54% of the applied activity) and in the shoots of four receiver plants out of the six replicates (0.41 ⫾ 0.22% of the applied activity). In the hyphae, 134 Cs was detected in trace amounts (< 0.2% of the total applied activity) of five replicates and in the medium of all six replicates (3.63 ⫾ 1.26% of the total applied activity). In the donor plants the shoots contained 84.01 ⫾ 4.95% and the roots 5.08 ⫾ 1.48% of the total applied activity. Discussion The role of AM fungi in the transfer of cesium between plants via a CMN has been investigated recently (Meding and Zasoski, 2008). However, no definite discrimination could be made between a direct (via the cytoplasm/ protoplasm of the AM fungi) and an indirect (via re-uptake of released cesium by roots or hyphae) transfer. Here, it was shown that AFCF was an efficient blocking agent of released radiocesium. Indeed, no radiocesium was measured in the receiver plants of the control treatment (i.e. plants not connected by a CMN) while it was found in the mycorrhizae-connected receiver plants. Therefore, a direct transfer of radiocesium between plants connected by a CMN was demonstrated for the first time. In order to demonstrate the role of AM fungi in the direct transfer of 134Cs between plants, we first tested whether AFCF was harmless to plants and AM fungi under our in vitro culture conditions. Although negative side-effects of AFCF application to soil were found to be unlikely (Vandenhove et al., 2000) potential detrimental effects on plants and fungi cannot be excluded. Indeed, information about the degradation of Prussian blue is limited (Jahn et al., 2006) and formation of free cyanide, a phytotoxic compound and a potent metabolic inhibitor (Ebbs et al.,

2003) might occur (Kang et al., 2007). Although iron cyanide complexes (such as in AFCF) are more resistant to biodegradation than simple cyanides and have lower toxicity even at high levels of exposure (Barclay et al., 1998), daylight may increase their decomposition rate (Meeussen et al., 1994). In our experiment, a decrease in shoot length was noted after 6 weeks of contact with AFCF, while such impact was not observed after 4 weeks. Interestingly, neither the percentage of vital hyphae nor the proportion of SDH-active hyphae differed significantly whether AFCF was present in the growth medium or not, and regardless of the duration of contact with this molecule. This indicated that the viability and metabolic activity of the AM fungal mycelium was not affected by the addition of AFCF. However, in order to avoid any detrimental effect of AFCF on plants, the duration of the experiment on radiocesium transfer was limited to a period of 4 weeks. Even though the adsorption of radiocesium by AFCF was initially limited (see preliminary experiment), no radiocesium was detected in the roots and shoots of the receiver plants of the controls in the experiment of radiocesium transfer between plants. This suggested that the amount of radiocesium released from the donor roots was instantly adsorbed by the AFCF and therefore could not be taken up by the receiver plant. The concentration of 100 p.p.m. of AFCF appeared thus suitable to investigate transfer of radiocesium between donor and receiver plants via a CMN. The optimal age of the plants at the time of labelling with 134Cs and the length of the labelling period was investigated in order to maximise leaf interception of 134Cs and subsequent translocation to the roots. The finding that the early-labelled (2-week-old) M. truncatula plants allowed a maximal translocation of 134Cs into the roots, was similar to earlier results on strawberry, where internal redistribution of 134Cs reached its peak in younger plants (Carini et al., 2003). For our early-labelled plants, even though the translocation of radiocesium after 3 and 4 weeks did not differ significantly, this latter was selected in order to increase the density of mycelium linking the donor to the receiver plants. The duration of 5 weeks was discarded due to a significantly higher amount of 134Cs released into

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Radiocesium transfer between plants via a CMN 2185 the medium and a potentially harmful effect of AFCF on plants observed with increased duration of contact, even though a maximal hyphal density was presumably obtained after 5 weeks of growth. Earlier results (Voets et al., 2009) indeed demonstrated that density of mycelium in interconnected plants increased over time. Based on the findings above, the radiocesium transfer between plants via a CMN was investigated. Several studies have already reported the capacity of CMNs to participate in the long-distance transfer of nutrients (e.g. Wilson et al., 2006; Meding and Zasoski, 2008; Mikkelsen et al., 2008). However, proving that the transfer is genuinely via hyphae and not soil is difficult (Robinson and Fitter, 1999). Interconnection of plants via a CMN is a natural process in soils, occurring either via extraradical hyphae spreading from mycorrhizal plants and colonizing the roots of plants with which they come into contact (see Voets et al., 2008) or by anastomosis, ‘a mechanism by which different branches of the same or different hyphae fuse to constitute a mycelial network’ (Kirk et al., 2001). In our experiment, it was not possible to assess the number of interconnections between the donor and receiver plants, neither via direct connection nor via anastomosis, but it can be assumed that radiocesium transfer between plants was related, at least partly, to the number of hyphae connecting them. This was supported by the absence of radiocesium in the shoots of the two replicates with the lowest hyphal lengths that also presented the lowest activity in their roots. Quantitatively, the radiocesium translocated from the shoots to the roots of the donor plants represents only 12–16% of the initially applied activity. The results of Zehnder and colleagues (1993) for strawberry showed a similar redistribution pattern for 134Cs as more than 75% of the initially applied activity remained in the above-ground plant parts and 10% was not incorporated into the plants (i.e. possible to wash off). In our experiment, the percentage of radiocesium interception could not be determined, as leaf architecture and turnover (i.e. fallen leaves) did not allow the wash-off of the extra activity. In the study of Zehnder and colleagues (1993), 6% of the initial applied activity was found in the surrounding soil following its release by roots, which is the same order of magnitude as in our experiment (i.e. between 2.1% and 8.3%). The activities found in the donor and receiver plants show that the AM fungal mycelium offers an additional pathway for the redistribution of radiocesium in the environment. This also means that even in the absence of a transfer of radiocesium between plants, AM fungi represent a sink of radiocesium and modify its redistribution. However, radiocesium was not detected in the shoots of two mycorrhizal receiver plants and only in trace amounts in the other four replicates. This could be related either to the number of hyphae interconnecting the plants (see above) or to

mechanisms preventing root-to-shoot translocation of radiocesium in mycorrhizal plants. As suggested by Dupré de Boulois and colleagues (2005) abscisic acid, which has been reported in higher concentrations in mycorrhizal roots (Danneberg et al., 1993; Bothe et al., 1994; LudwigMüller, 2000), might negatively affect radiocesium loading into the xylem by influencing the expression or activity of K channels. This might be the cause for the occurring ‘protective’ effect in mycorrhizal plants as it was observed earlier (Dupré de Boulois et al., 2005). The in vitro system used in our experiment with AFCF as blocking agent appeared suitable to study direct transfer of radiocesium between plants (i.e. via the cytoplasm/ protoplasm of the ERM). The amount of radiocesium transferred was not quantitatively important, as it was observed also for other elements (Simard et al., 2002; Wilson et al., 2006). However, the shared network of fastgrowing hyphae might allow the long-distance transport of this radionuclide, which otherwise would be restricted both in time and space. The confirmation of radiocesium transfer between plants via CMNs therefore allows a better comprehension of the distribution of this pollutant in the environment but also that of K (Simard and Durall, 2004), even though using radiocesium could give rise to errors in estimating K fluxes due to the differences in their transport pathways in both plants and AM fungi (White and Broadley, 2000; Gyuricza et al., 2008).

Experimental procedures Biological material A strain of Glomus intraradices Schenck and Smith (MUCL 43194) grown in association with Ri T-DNA transformed carrot (Daucus carota L.) roots was purchased from GINCO (http://www.mbla.ucl.ac.be/ginco-bel). The strain was delivered in a Petri plate (90 mm diameter) on the modified Strullu–Romand (MSR) medium (Declerck et al., 1998), solidified with 3 g l-1 GelGro (ICN, Biomedicals, Irvine, CA, USA) and subsequently subcultured following the methods described by Cranenbrouck and colleagues (2005). Several thousand spores were obtained in a period of 5 months. Seeds of Medicago truncatula L., cv. Jemalong A17 (SARDI, Adelaide, Australia) were surface-sterilized by immersion in sodium hypochlorite (containing 5% active chloride) for 10 min, rinsed in deionized sterile (121°C for 15 min) water and germinated in groups of 10 in Petri plates (90 mm diameter) containing 40 ml of MSR medium lacking sucrose and vitamins, and solidified with 3 g l-1 GelGro (referred as MSR1 throughout the text). Germination was conducted in a growth chamber set at 27°C in the dark for 4 days.

Adsorption and retention of radiocesium Prussian Blue is known to have a high selectivity for cesium adsorption and its related radioisotopes (Faustino et al.,

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2186 V. Gyuricza et al. 2008). Here, the MSR1 medium was supplemented with 100 p.p.m. of AFCF to evaluate its adsorption rate (n = 3). Polyethylene vials were filled with 5 ml of this medium and 1 ml of diluted 137Cs stock solution (900 Bq ml-1) was then added on its surface. The system was left to equilibrate for 5 min, 1 h or 3 h. Vials were rinsed twice with 5 ml and once with 10 ml of deionized water. Following washing the medium from each vial was transferred in a 10 ml syringe (polyethylene) and the solution extracted by centrifugation (1500 g, two times 5 min) before filtration (Acrodisc Tuffryn Membrane, 0.45 mm). The adsorption rate at the surface of the medium was estimated from the amount of 137Cs removed by water. The amount of soluble radiocesium extracted by centrifugation of the contaminated gel was used to further calculate the 137 Cs retention rate of the gel in order to determine the reversibility of the adsorption.

Experimental designs and harvests Foliar interception and translocation of radiocesium. Radiocesium translocation from leaves to other plant parts and in particular roots has been demonstrated for several plant species such as tomato (Sabbarese et al., 2002) and strawberry (Zehnder et al., 1995; Carini and Lombi, 1997; Fortunati et al., 2004). Here, we conducted an experiment with M. truncatula plants to determine: (i) the optimal duration of the labelling period (i.e. the time between labelling and 134Cs counting) and (ii) the optimal age of the plant for labelling, in order to maximize the translocation of 134Cs to the roots following its interception by the leaves. Four-days-old M. truncatula plantlets were placed in monocompartmental Petri plates (90 mm diameter) with their roots placed on the MSR1 medium and shoots extending outside through an opening made at the side of the lid (see Voets et al., 2005 for details). The Petri plates were then sealed using Parafilm (Pechiney, Chicago, IL, USA) and wrapped in black plastic bags to maintain roots in the dark. Ten millilitres of MSR1 medium (cooled to a temperature of approximately 35°C to avoid damage to the roots) was added weekly (starting week 2) to the Petri plates to provide plants with nutrients and to avoid medium depletion caused by plant transpiration. The culture systems were incubated in a controlled growth chamber set at 22°C/18°C (day/night) with a 16/8 h photoperiod and 80% relative humidity. Daylight lamps provided an average photosynthetic photon flux at the level of the shoots of 225 mmol m2s-1. Labelling was performed by placing 10 20 ml droplets of 134 Cs source on plant leaves, containing each approximately 400 Bq of activity. The procedure was repeated a second time, shortly after the first droplets had dried. The total activity of 134Cs applied to the leaves of each plant was 8050 ⫾ 150 Bq. The source of 134Cs was in form of CsCl in aqueous solution, supplied by Polatom (Otwock-Swierk, Poland) with a purity of 98%. CsCl was preferred above CsNO3 as Hasegawa and colleagues (2009) showed that the uptake of Cs was strongly affected by counter anions of Cs in the applied solution. They observed that approximately 80% of Cs was absorbed when applied as CsCl, while only 20% was absorbed when NO3- was the counter anion of Cs. Labelling was conducted on 2-week-old plants (referred as early-labelled plants) and 4-week-old plants (referred as late-

labelled plants). Three, four and five weeks after labelling, three randomly selected plants were harvested in both groups. Shoots, roots and medium were collected separately and were subjected to gamma-counting on a Wallac 1480 Gamma Counter (Wallac, Turku, Finland). Roots were rinsed in citrate buffer to remove all residues of medium and the activity of the buffer was then added to the activity of the medium.

Effects of AFCF on plant and AM fungi The toxicity of AFCF was evaluated on plants and AM fungi grown in vitro. Two-week-old in vitro premycorrhized M. truncatula plants (see Voets et al., 2009) were placed in the root compartments (RCs) of bi-compartmental Petri plates (90 mm diameter). The RCs were filled with 25 ml of MSR1 medium supplemented with 100 p.p.m. of AFCF or without addition of AFCF. Ten millilitres of MSR1 medium was added every week (starting week 2) either with or without AFCF as described above. The culture systems were incubated in a controlled growth chamber under the same growth conditions as above. Five replicates were considered for the AFCF and control treatments for all measurements performed. Four weeks after incubation, the hyphae started to cross the barrier separating the RCs from the hyphal compartments (HCs). At that time, the HCs were filled with MSR1 supplemented or not with 100 p.p.m. of AFCF to favour proliferation in the HCs. The RCs and HCs of the systems contained therefore both MSR1 medium supplemented with AFCF (‘AFCF treatment’) or not (‘control treatment’). Roots that crossed the barrier were regularly trimmed. The plants and AM fungi were grown for two additional weeks under the same conditions as described above. The length of hyphae (Declerck et al., 2003) and number of spores in the HCs were estimated (Declerck et al., 2001) as well as the shoot length and the number of leaves both at filling of the HCs (i.e. when plants were grown for 4 weeks in the systems) and at the end of the experiment (i.e. 2 weeks later). Plant shoots and roots were then collected separately and their fresh weight estimated. Mycorrhizal root colonization of the plants was estimated. The roots were cleared in 10% KOH and stained with 0.2% Trypan blue following the method of Phillips and Hayman (1970). They were cut into 10 mm fragments and 150 were randomly selected and examined under a compound microscope [Olympus BH2, Olympus Optical (Europa) GmbH, Hamburg, Germany] at 50–250¥ magnifications to evaluate the frequency (%F) and intensity (%I) of AM fungal root colonization (Declerck et al., 1996). The uptake of phosphorus (P) by the ERM was also determined in the medium of the HCs. From each Petri plate a plug of 1 g medium was collected with a cork-borer and the ERM removed. Total P-content of the plugs was measured using ICP-AES (ICAP 6000, Thermo Fisher Scientific, Waltham, MA, USA) after consecutive boiling with nitric acid and perchloric acid. To determine the metabolic activity of hyphae in the control and AFCF treatments, the ERM was collected by dissolving the remaining medium in the HCs with citrate buffer following the method of Doner and Bécard (1991). The ERM was then stained for SDH activity following the method of Smith and

© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 2180–2189

Radiocesium transfer between plants via a CMN 2187 Gianinazzi-Pearson (1990). The presence of black precipitates in the hyphae was evaluated using a compound microscope (Olympus BH2, Olympus Optical GmbH, Hamburg, Germany) at 50–250¥ magnifications. For each sample (one sample per replicate), approximately 150 intersections of hyphae were counted and divided into two groups based on the presence (non-viable hyphae) or absence (viable hyphae) of septa. The hyphae without septa were further divided as SDH-active or non-active. The percentage of hyphal length with SDH activity was determined as described by Olsson and colleagues (2002) for viable hyphae.

Radiocesium transfer between plants Two-week-old M. truncatula plantlets facing each other were placed in mono-compartmental Petri plates on MSR1 medium supplemented with 100 p.p.m. of AFCF (Fig. 1). The treatments consisted of either a pair of two premycorrhized plants or two non-mycorrhizal plants as control. In each pair (six replicates per treatment), one plant was considered as the donor and the other as the receiver. Leaves of the donor plants were labelled twice with 4000 ⫾ 75 Bq of 134Cs, as described above. Plant shoots were wrapped in individual Sun-bags (Sigma-Aldrich, Bornem, Belgium) to allow separation of the fallen leaves of the donor and receiver plants. Petri plates were covered in black plastic bags to maintain roots and AM fungi in the dark. The culture systems were incubated in a controlled growth chamber under the same conditions as described above. Four weeks after labelling, the hyphal length was measured and the number of spores counted as described above. Shoots and roots of the donor and receiver plants as well as the medium and ERM were collected separately. Fresh weight of plant parts was measured. Roots of both donor and receiver plants as well as hyphae were rinsed in citrate buffer to remove all residues of medium and the activity of the buffer was later added to the activity of the medium. Samples were subjected to gamma-counting on a Wallac 1480 Gamma Counter (Wallac, Turku, Finland). After counting, the roots and shoots were dried at 70°C to a constant weight and their dry weight was determined. Root colonization was measured thereafter, using the method described earlier.

Statistical analyses Data normally distributed and having homogeneous variances were subjected to ANOVA. One-way ANOVA was used to compare different treatments. Pearson’s correlation test was performed between the activity of medium and roots.

Acknowledgements This research project has been supported by a Marie Curie Early stage Research Training Fellowship of the European Community’s Sixth framework Programme under contract number MEST-CT-2005-020387. H.D.D.B. acknowledges receipt of a grant of Chargé de Recherches FRS-FNRS (Belgium). We would like to thank Ingrid Van Aarle for the help with the analysis of metabolic activity and Anne Bol for the assistance with gamma-radiation measurements.

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