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Effect of arbuscular mycorrhizal (AM) fungi on 137Cs uptake by plants grown on different soils
Journal of Environmental Radioactivity 115 (2013) 151e156
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Effect of arbuscular mycorrhizal (AM) fungi on on different soils
137
Cs uptake by plants grown
M. Vinichuk a, b, *, A. Mårtensson a, T. Ericsson c, K. Rosén a a
Department of Soil and Environment, Swedish University of Agricultural Sciences, P.O. Box 7014, SE-750 07 Uppsala, Sweden Department of Ecology, Zhytomyr State Technological University, 103 Chernyakhovsky Str., 10005 Zhytomyr, Ukraine c Department of Urbanand Rural Development, Swedish University of Agricultural Sciences, SLU, Box 7012, SE-75007 Uppsala, Sweden b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 February 2012 Received in revised form 13 July 2012 Accepted 4 August 2012 Available online 30 August 2012
The potential use of mycorrhiza as a bioremediation agent for soils contaminated by radiocesium was evaluated in a greenhouse experiment. The uptake of 137Cs by cucumber, perennial ryegrass, and sunflower after inoculation with a commercial arbuscular mycorrhizal (AM) product in soils contaminated with 137Cs was investigated, with non-mycorrhizal quinoa included as a “reference” plant. The effect of cucumber and ryegrass inoculation with AM fungi on 137Cs uptake was inconsistent. The effect of AM fungi was most pronounced in sunflower: both plant biomass and 137Cs uptake increased on loamy sand and loamy soils. The total 137Cs activity accumulated within AM host sunflower on loamy sand and loamy soils was 2.4 and 3.2-fold higher than in non-inoculated plants. Although the enhanced uptake of 137 Cs by quinoa plants on loamy soil inoculated by the AM fungi was observed, the infection of the fungi to the plants was not confirmed. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Radiocesium Radionuclides Contaminated soil
1. Introduction Root uptake of radiocesium is the main pathway for the migration of radioactive cesium from soil to humans via the food chain. In soils with high amounts of clay, 137Cs binds or is fixed to clay minerals, whereas, in soils with high organic matter content, 137 Cs can be bound either to organic matter (Rigol et al., 2002) or to biomass (mostly fungal hyphae) (Vinichuk and Johansson, 2003). Although the fraction of radionuclides associated with microorganisms and particularly fungi may be relatively small, these organisms play an important role in the retention of radiocesium in forest soils. For instance, in forest soils, ectomycorrhizal (ECM) fungi accumulate up to 270 times more radiocesium than plants growing in their immediate vicinity (Bakken and Olsen, 1990). Despite these observations, the role of arbuscular mycorrhizal (AM) fungi in uptake and accumulation (“binding”) of 137Cs in agricultural soils has not received much attention. Although data are still limited, Declerck et al. (2003) suggest the extraradical mycelium of the AM fungus Glomus lamellosum can take up, possibly
* Corresponding author. Department of Soil and Environment, Swedish University of Agricultural Sciences, P.O. Box 7014, SE-750 07 Uppsala, Sweden. Tel.: þ46 18 67 14 42; fax: þ46 18 67 28 95. E-mail addresses: [email protected], [email protected], [email protected] (M. Vinichuk). 0265-931X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jenvrad.2012.08.004
accumulate, and unambiguously translocate radiocesium from a 137Cs-labelled synthetic root-free compartment to a root compartment and to within the roots in root-organ culture. Kripka (2005) provides evidence of the direct involvement of AM fungi in radionuclide transport to the plant, and de Boulois et al. (2006) demonstrate the capacity of AM fungi to transport cesium (Cs). However, the results from studies on the capacity of AM fungi to accumulate and transport radiocesium is inconsistent and inconclusive (Berreck and Haselwandter, 2001; Joner et al., 2004; Rosén et al., 2005) or uncertain and even controversial (de Boulois et al., 2006). Apart from the wide variation in changes in 137Cs concentration in different plants, from 2- to 4-fold within cereals to 27-fold for all field crops (Sanzharova et al., 1997), variation in changes in 137Cs concentration can occur in cultivars of the same species (Dushenkov et al., 1999). Thus, there is an indication AM fungi have a possible role in phytoremediation of soils contaminated with radionuclides. However, their ability to participate in phytoremediation strategies remains questionable and requires further investigation. In this study, four plant species, cucumber, ryegrass, sunflower and quinoa, were tested. Cucumber, ryegrass, and sunflower form mycorrhiza, whereas, as quinoa is generally non-mycorrhizal (Wang and Qiu, 2006), it was included as a “reference” plant. All plants are known to accumulate 137Cs efficiently: cucumber (Gouthu et al.,1997); ryegrass (Waegeneers et al., 2005); sunflower (Sorochinsky et al., 1998; Soudek et al., 2006), and quinoa (Broadley and Willey, 1997).
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M. Vinichuk et al. / Journal of Environmental Radioactivity 115 (2013) 151e156
2. Materials and methods
from a field site at Vara in the county of Västergötland, and in 1961, the soil was contaminated with 35.7 MBq m2 137Cs, mixed with a plough layer of soil, and crops grown for many years (Haak and Lönsjö, 1996). After 47 y, 137Cs activity had decreased from ca. 148 kBq kg1 soil to about 47.1 Bq kg1 soil. The loamy soil for this study was collected from the same micro-plots as in 1961. The soil characteristics are presented in Table 1. In the laboratory, the soils were thoroughly mixed, sieved through a 2 mm sieve to separate stones, and aliquots (about 2/3) of each soil type were autoclaved for 3 h at 121 C at 2 bars of pressure. The mixture containing the carrier material (peat, sand, and perlite) was steam-sterilized by the producer.
2.1. Experiment layout
2.3. Plant material and growth conditions
The greenhouse experiment comprised a block design including two treatments and five replicates: AMþ and AM- applied to each soil type. In treatment AMþ, soil was autoclaved before the experiment and then inoculated with a commercial AM fungus G. mosseae. The fungal strain had been isolated from agricultural soil in Finland and contained spores and hyphae of the arbuscular mycorrhizal fungus G. mosseae together with a carrier material consisting of a steam-sterilized mixture of peat, sand, and Perlite. In treatment AM-, soil was autoclaved but non-inoculated.
The plants were grown in a greenhouse in plastic rose pots with raised bottoms and a volume of 4.2 dm3. The pots were placed on saucers (22 cm diameter) and filled with the same amount of each soil type (ca. 4.0 kg). To avoid possible interaction between ions during radiocesium transport by AM fungi (Gyuricza et al., 2008), no mineral fertilizers such as potassium (K) or phosphorus (P) were applied to the soil. The crops used in the pot experiment were cucumber (Cucumis sativus L. cultivar ‘Melen F1’), perennial ryegrass (Lolium multiflorum, variety ‘Corvus’), sunflower (Helianthus spp.), and quinoa (Chenopodium quinoa Willd), and were supplied by Weibulls Horto AB, Sweden. There were 40 pots (5 AMþ, 5 AM ¼ 10 4 crops) for each soil type, which gave a total 120 pots (40 3 soil types). Before seeding, inoculum material containing live arbuscular mycorrhizal fungi (5e10 AMF propagules/g inoculum) was applied to the soil (treatment AMþ) in the proportion 1 part inoculum material to 100 parts soil, according to the manufacturer’s advice. Inoculum material was then mixed with the upper 2e3 cm of soil and the pots were supplied with distilled water as required. Plants were randomly seeded by hand within the pot area (ryegrass and quinoa 10 per pot; cucumber and sunflower 2 seeds per pot), and were weeded and watered as required. The climatic conditions in the greenhouse were a 16 h daytime and 8 h nighttime cycle; a light intensity (photosynthetically active radiation) of 110e140 mmol m2 s1; daytime temperature of 20 C and nighttime temperature of 15 C; and, relative air humidity of 70%. To compensate for an increase in daylight, the light intensity was reduced to between 70 and 110 mmol m2 s1 after about 11 weeks. The plants were grown for 16e20 weeks, depending on the crop: cucumber fruits were ready for harvest after 8e9 weeks from planting; ryegrass after first (1st) and second (2nd) cut; and sunflower and quinoa after ripening of the seeds. From different treatments, the same crop was harvested at the same time in both treatments. After harvest, plants were air-dried to constant weight and aboveground parts divided in seeds and remaining parts. Seeds of sunflower and quinoa were sieved with a 4 and 2 mm sieve, and the remaining parts were milled. Both seeds and remaining parts were analysed for 137Cs activity.
The objective was to investigate the potential use of mycorrhiza as a bioremediation agent for soils contaminated by radiocesium. The effect of AM fungus Glomus mosseae on 137Cs uptake by different crops on silty clay, loamy, and loamy sand soils was investigated. The hypothesis was that plant species/AM fungal strains associations are unique and radiocesium accumulation in mycorrhizal plants may be higher, lower, or equal to the accumulation in non-mycorrhizal plants. Consequently, the desired effect might only be achieved with the right combination of plant species and AM fungal strain.
2.2. Soils The three soil types used (silty clay, loamy and loamy sand soil) were collected in September 2008 from three locations in central Sweden (Table 1) and had been contaminated with 137Cs after the Chernobyl accident in 1986. At each sampling location, the upper 0e15 cm layer of soil (about 400 kg) was collected from 3 to 4 places within an area of about 100 m2. The loamy soil used in the experiment had previously been used in a micro-plot experiment in 1961 (Haak and Lönsjö, 1996). In autumn 1960, soil was collected
Table 1 Main characteristics of the soils used in the experiment. Soil Parameters
Silty clay
Loamy
Loamy sand
Soil sampling locations long, alt.
Skogsvallen 17 100 W, 60 100 N
Vara 12 490 W, 58 130 N
Hille 17 110 W, 60 440 N
Land use
Pasture
Pasture
Cs, kBq kg1dw (mean se) Particle size distribution: % Gravel (>2 mm) Sand (2e0.06 mm) % Silt (0.06e0.002 mm) % Clay (<0.002 mm) Bulk density, g cm3 %C %N pH (0.01 M CaCl) Exchange cations (meq 100 g1 soil) Ca Mg K K, HNO3 KeAL Total, exchange base cations Titratable acidity CEC at pH 7 Base saturation, %
0.53 0.01
Microplot experiment 41.5 0.17
5.2 5.1 13.1 0.78 40.2 2.7 41.5 1.7 0.96 4.5 0.35 0.39 0.03 4.8 0.01
0.80 0.57 42.3 3.6 41.7 4.0 16.2 0.49 1.10 2.6 0.02 0.30 0.01 4.9 0.08
13.4 0.07 76.1 0.57 6.5 0.49 4.1 0.2 1.16 2.6 0.13 0.20 0.01 4.2 0.03
10.9 0.49 0.98 0.03 0.66 0.03 6.2 0.04 0.71 0.05 12.8
1.8 0.25 0.31 0.01 0.11 0.01 1.6 0.11 0.01 2.3
5.5 18.3 69.9
5.8 8.1 28.6
137
15.8 2.03 0.54 13.6 0.47 18.9 7.8 26.7 70.8
0.11 0.02 0.05 0.25 0.02
0.69 0.01
2.4. Quantification of AM fungal colonization For AM fungi colonization, approximately 25e50 g fresh weight of root was randomly dug from selected plants (soils) and stored in a freezer at 40 C before staining. Then, these samples were immersed in 20% potassium hydroxide for 1 d, washed in tap water, and acidified with 1% hydrochloric acid. After this, the roots were stained with 0.05% trypan blue in a 14:1:1 lactic acid:glycerol:water solution for one day, and then rinsed in a de-staining solution (14:1:1 lactic acid:glycerol:water) until no visual blue staining remained. For examination, the roots were spread onto glass slides
M. Vinichuk et al. / Journal of Environmental Radioactivity 115 (2013) 151e156
and the appearance of fungal infections was visually recorded under a binocular microscope. The rate of mycorrhizal infection frequency in the roots was graded as 0 ¼ no infection; 1 ¼ sign of infection; 2 ¼ moderate infection; and, 3 ¼ abundant infection.
Table 3 Effect of soil inoculation with AM fungi on 137Cs concentration ratios (CR) in roots of cucumber, sunflower, and quinoa, and the percentage increase (þ) and decrease () in 137Cs CR when grown on loamy soil. AMþ, soil autoclaved and inoculated with AM fungi; AM, soil autoclaved and non-inoculated (n ¼ 5). Plant, root size
2.5. Radiometry and data treatment
153
CRa
AMþ/AM, %b
AMþ
Soil and whole plant samples were air-dried prior to analyses and plants were cut into <2 mm pieces: cucumber fruits were analysed on fresh weight basis. In addition to harvesting aboveground parts, the roots were dug out for determining radiocesium distribution within the plant. The roots were washed in tap water, air dried, and analysed for 137Cs activity. Gamma-spectrometry with HPGe detectors coupled to a multi-channel analyzer system was used to determine 137Cs activity in the soil and plants. The systems were calibrated with a standard source used for intercomparative studies and in appropriate (60 and 35 ml) standard containers. All results were decay corrected to date of sampling (harvesting) and 137Cs concentration ratios (CR) were calculated as 137 Cs Bq kg1dry weight (dw) in plant divided by 137Cs Bq kg1dw in soil. Data were analysed by a 2-sample t-test with Minitab (Ó 2010 Minitab Inc.) software to evaluate the differences in 137Cs activity between treatments at the level of significance 0.05. 3. Results 3.1.
137
Cs concentration ratios in roots and root infection frequency
3.1.1. Mycorrhizal plants Infection frequency of cucumber plants grown on inoculated soil was only positively correlated with cucumber plant biomass on silty clay soil (r ¼ 0.697; p < 0.05) (Table 2). Roots (<2 mm) of cucumber grown on inoculated loamy soil had about 30% higher 137 Cs CR than plants grown on non-inoculated soil (Table 3). Infection frequency of cucumber plants grown on inoculated soil was not correlated, or negatively correlated, with 137Cs uptake (Table 4). Fungal infection of ryegrass roots was only found in loamy sand soil: neither ryegrass biomass nor 137Cs CR correlated with AM
Table 2 Infection frequency in roots of cucumber, ryegrass, sunflower and quinoa, and correlation coefficients (r) among root infection frequency and plant biomass (g pot1dw) when grown on different soils (n ¼ 5). Soil
Plants/parts, cuts
Cucumber, mycorrhizal Silty clay Fruits Plant Loamy Fruits Plant Loamy sand Fruits Plant Ryegrass, mycorrhizal Silty clay e Loamy e Loamy sand 1st þ 2nd cut Sunflower, mycorrhizal Silty clay Seeds Plant Loamy Seeds Plant Loamy sand Seeds Plant Quinoa, non-mycorrhizal Silty clay e Loamy e Loamy sand e *p < 0.05; **p < 0.01.
Infection frequency
r
1.0 0.7
0.37 0.70* 0.22 0.24 No data 0.21
1.0 1.0 1.8 0.45
0 0 0.6
No infection No infection 0.36
0.6 0.55
0.07 0.04 0.37 0.02 0.80** 0.81**
1.2 1.0 1.6 0.55
0 0 0
No infection No infection No infection
Cucumber, <2 mm Sunflower, <2 mm Sunflower, 2e5 mm Quinoa, <2 mm Quinoa, 2e5 mm
0.30 0.17 0.029 0.10 0.094
AM
0.07 0.04 0.02 0.06 0.04
0.23 0.10 0.022 0.087 0.052
0.08 0.02 0.01 0.01 0.05
31.0 72.7 27.4 14.3 81.1
CR ¼ 137Cs Bq kg1 in plant/137Cs Bq kg1 in soil. Cs CR in AMþ minus 137Cs CR in AM divided by 137Cs in AM multiplied by 100. a
b 137
fungi infection frequency (Tables 2 and 4). In the fine roots (<2 mm) of sunflower, 137Cs CR did not differ between treatments; however, in coarse roots (2e5 mm) of plants grown on inoculated soil, 137Cs CR was 3- to 4-fold higher than in plants grown on non-inoculated soil (Table 3). The infection frequency of sunflower correlated with plant biomass on loamy sand soil (Table 2) and 137Cs CR on loamy and loamy sand soils (Table 4). 3.1.2. Non-mycorrhizal plants In the fine (<2 mm) roots of quinoa, there was no difference in 137 Cs CR; however, in coarse roots (2e5 mm) of plants grown on inoculated soil, 137Cs CR was about 80% higher than in plants grown on non-inoculated soil (Table 3). 3.2. Biomass 3.2.1. Mycorrhizal plants The effect of AM fungi inoculation on cucumber plant biomass was evident in silty clay soil (Fig. 1). In loamy sand soil, there was a negative effect of soil inoculation on cucumber biomass, as plants were weakly developed and only limited numbers of fruits formed: this rendered it difficult to compare and estimate the effect of soil inoculation. Fungi did not affect the biomass of perennial ryegrass, whereas, AMþ plants growing on loamy sand yielded less biomass than plants with AM treatments (Fig. 1): the negative AM effect was not statistically significant. On silty clay soil, fungal inoculation had no effect on aboveground sunflower plant biomass, but on loamy and loamy sand soils fungal inoculation resulted in higher (p < 0.01) sunflower plant biomass (Fig. 1). The effect of loamy soil inoculation on sunflower biomass was positive but not statistically significant. 3.2.2. Non-mycorrhizal plants The AM host quinoa plants grown on inoculated silty clay soil had 50% higher biomass than plants grown on non-inoculated soil. There was no effect of soil inoculation on plant biomass on loamy sand soil (Fig. 1). Table 4 Correlation coefficients among 137Cs concentration ratios (CR ¼ 137Cs, Bq kg1 in plant/137Cs, Bq kg1 in soil) and root infection frequency in cucumber, ryegrass, sunflower, and quinoa when grown on different soils (n ¼ 5). Plant/Soil Cucumber Cucumber Ryegrass Sunflower Sunflower Quinoa *p < 0.05.
fruits plant seeds plant
Silty clay
Loamy
Loamy sand
0.29 0.12 No infection 0.48 0.23 No infection
0.05 0.08 No infection 0.42 0.72* No infection
0.60 0.08 0.33 0.19 0.73* No infection
154
M. Vinichuk et al. / Journal of Environmental Radioactivity 115 (2013) 151e156 Table 5 Effect of soil inoculation with AM fungi on 137Cs concentration ratios (CR) in cucumber, ryegrass, sunflower, and quinoa, and the percentage increase (þ) and decrease () in 137Cs CR when grown on different soils. AMþ, soil autoclaved and inoculated with AM fungi; AM, soil autoclaved and non-inoculated (n ¼ 5).
80.0 70.0 60.0 g pot -1
50.0 40.0
Soil
30.0 20.0 10.0 0.0 Cucumber Silty clay AM+
Silty clay AM-
Ryegrass Loamy AM+
Sunflower Loamy AM-
Loamy sand AM+
Quinoa* Loamy sand AM-
Fig. 1. The effect of soil inoculation on aboveground plant biomass, g dry weight pot1, of cucumber, ryegrass, sunflower and quinoa grown on silty clay, loamy and loamy sand soils. Standard error, n ¼ 5. * Non-mycorrhizal plant.
3.3.
137
Cs concentration ratios in plants
3.3.1. Mycorrhizal plants Uptake of 137Cs increased in cucumber plants grown on inoculated loamy soil, whereas, in plants grown on non-inoculated loamy sand and silty clay soils the uptake of 137Cs decreased. The CR of 137 Cs in plants grown on non-inoculated loamy sand and silty clay soils were generally higher than the CR of 137Cs in plants grown on soils inoculated with fungi. In cucumber fruits 137Cs CR was high and varied between about 2.1e6.9 on loamy sand soil and 1.1e1.5 on loamy soil (Table 5). The application of AM fungi resulted in higher 137Cs uptake by ryegrass plants (2nd cut) grown in loamy soil but lower uptake in plants grown in loamy sand and silty clay soils (Table 5): the 137Cs CR values were in the range 0.01 (lowest, silty clay soil) and 0.83 (highest, loamy sand soil). The AM host sunflower plants had two- to three-fold higher 137Cs uptake on loamy sand and loamy soils than plants grown on non-inoculated soil: this effect was negative on silty clay soil (Table 5). In sunflower plants, 137Cs CR varied between 0.002 (for seeds on loamy soil) and 0.42 (for plants on loamy sand soil) (Table 5). The only statistically significant difference in 137Cs CR values between treatments AMþ and AMe were for ryegrass on loamy sand and sunflower on loamy soils. 3.3.2. Non-mycorrhizal plants The AM host quinoa plants responded positively to 137Cs uptake on loamy sand and loamy soils, although this response was not statistically significant compared to AMe plants (Table 5). However, soil inoculation with AM fungi did not affect 137Cs uptake by quinoa plants grown on silty clay soil. In quinoa plants, 137Cs CR was high, and varied between 0.13 (loamy soil) to about 1.5 (loamy sand soil: Table 2).
Plant parts, cuts
Cucumber, mycorrhizal Silty clay Fruits Plant Loamy Fruits Plant Loamy sand Fruits Plant Ryegrass, mycorrhizal Silty clay 1st cut 2nd cut Loamy 1st cut 2nd cut Loamy sand 1st cut 2nd cut Sunflower, mycorrhizal Silty clay Seeds Plant Loamy Seeds Plant Loamy sand Seeds Plant Quinoa, non-mycorrhizal Silty clay Seeds Plant Loamy Seeds Plant Loamy sand Seeds Plant
CRa
AMþ/AM, %b
AMþ
AM
1.07 0.19 1.77 0.35 2.05 0.26
0.46 0.04 0.70 0.06 0.94 0.11
1.45 0.19 1.35 0.27 6.97 0.26
0.28 0.05 0.66 0.09 4.0 0.13
26 3 31 27 71 e
0.012 0.025 0.015 0.039 0.31 0.42
0.02 0.01 0.01 0.02 0.09 0.26
0.018 0.013 0.013 0.028 0.65 0.83
0.01 0.01 0.003 0.16 0.16 0.19
37 86 16 38 52* 48*
0.0041 0.029 0.0045 0.045 0.10 0.42
0.004 0.01 0.002 0.02 0.02 0.08
0.0091 0.027 0.0024 0.015 0.04 0.29
0.01 0.01 0.001 0.01 0.05 0.08
55 11 103* 195** 156 45
0.090 0.14 0.13 0.37 0.39 1.52
0.06 0.07 0.03 0.07 0.22 0.75
0.096 0.15 0.094 0.35 0.26 0.77
0.07 0.05 0.03 0.07 0.15 0.49
*p < 0.05; **p < 0.01. a CR ¼ 137Cs Bq kg1 in plant/137Cs Bq kg1 in soil. b 137 Cs CR in AMþ minus 137Cs CR in AM divided by by 100.
137
6 12 40 6 48 84
Cs in AM multiplied
The infection frequency in sunflower roots correlated with both biomass and 137Cs CR (Tables 2 and 4). 3.4.2. Non-mycorrhizal plants The uptake of 137Cs by quinoa AM host plants increased by a factor of about two only on loamy sand soil (Fig. 2). In quinoa plants soil inoculation with AM fungi generally did not result in higher uptake of 137Cs across all soil types (Fig. 3a). Inoculation with AMþ fungi did not affect 137Cs CR when comparing the soil types across all plants studied (Fig. 3b). The highest 137Cs ratios were observed on loamy sand. 4. Discussion
3.4. Total
137
Cs activity and % of total
137
Cs uptake by plants
3.4.1. Mycorrhizal plants Uptake of 137Cs by AM host cucumber plants was similar to the uptake of 137Cs in cucumber plants grown on non-inoculated clay and loamy soil but lower in loamy sand (Fig. 2). In terms of combined effect of increased plant biomass and enhanced 137Cs uptake, ryegrass presented inconsistent results: 1.5-fold higher uptake in loamy soils, 2.2-fold lower uptake in loamy sand soil, and no effect in silty clay soil (Fig. 2). The effect of soil inoculation on the percentage of total 137Cs uptake by ryegrass was negative when analysed across all soil types (Fig. 3a). Uptake of 137Cs by AM host sunflower plants was 3.0-fold higher on loamy soil and 2.5-fold higher on loamy sand soil than in non-inoculated plants (Fig. 2). However, the overall effect of AM fungi application on the amount of 137Cs uptake by plants in all soils was not established (Fig. 3a).
The potential use of plants to decontaminate radionuclidepolluted soil is an approach that deserves attention. This approach has been partly tested by Entry et al. (1999), and Dushenkov (2003) and Willey (2007) conclude phytoremediation of radionuclides could become useful in the future. Therefore, the challenge is to develop viable methods for extracting radionuclides from soil. One option for enhancing the potential for phytoextraction is with mycorrhiza. Soil inoculation may result in enhanced radionuclide uptake, have no effect, or even cause lower uptake due to the specific and intricate balance between the plants, the fungi, and the environment. However, this phenomenon deserves attention before widespread use of mycorrhizal inoculation for the bioextraction of radionuclides can be recommended. Cucumber moderately infected by AM fungi on silty clay and loamy soils only (non-statistically significantly) improved 137Cs
M. Vinichuk et al. / Journal of Environmental Radioactivity 115 (2013) 151e156
14.0
Silty clay
AM+
Bq pot-1
10.0
137Cs,
12.0
6.0
a
AM-
155
1.20 AM+
1.00
AM-
8.0 137Cs,
%
0.80
4.0
0.60 0.40
2.0
0.1 0.1
0.0 Cucumber
Ryegrass
Sunflower
Mycorr.
0.20
Quinoa
0.00
Non-mycorr.
Cucumber
0.90
800
0.70
600
0.60
CR
Bq pot-1
b
137Cs,
AM-
1,000
137Cs
400
0.80
0.50 0.40 0.30
12.9 8.9
0
0.20
Cucumber
Ryegrass
Sunflower
Mycorr.
Quinoa
0.10
Non-mycorr.
0.00 Silty clay
AM-
Loamy sand
AM-
5.0
Fig. 3. The effect of soil inoculation on amount of 137Cs uptake by cucumber, ryegrass, sunflower, and quinoa: (a) as % of total 137Cs in soil across soil types; (b) 137Cs concentration ratios of the same plants (CR ¼ 137Cs Bq kg1 in plant/137Cs Bq kg1 in soil) grown on silty clay, loamy and loamy sand soil across plant species.
4.0
137
7.0 Bq pot-1
Loamy AM+
AM+
Loamy sand
8.0
137Cs,
Quinoa Non-mycorr.
AM+
1,200
200
Sunflower
Mycorr.
Loamy
1,400
Ryegrass
6.0
3.0 2.0 1.0 0.0 Cucumber
Ryegrass Mycorr.
Sunflower
Quinoa Non-mycorr.
Fig. 2. The effect of soil inoculation on total 137Cs activity, Bq pot1, in aboveground plant parts of cucumber, ryegrass, sunflower, and quinoa grown on silty clay, loamy and loamy sand soil. Standard error, n ¼ 5.
uptake on loamy soil despite being densely infected in loamy sand soil, where no effect of soil inoculation on 137Cs uptake was detected (Table 5). The frequency of root infection in mycorrhized cucumber did not correlate with 137Cs uptake (Table 4), but positively correlated with plant biomass on silty clay soil (Table 2). Thus, the effect of AM fungi on total 137Cs uptake by cucumber could not be linked to the infection frequency of the mycorrhizal fungi, as abundant mycorrhizal infection of cucumber on loamy sand and moderate infection on silty clay soils had a negative or no effect, whereas, moderate infection on loamy soil caused a non-statistically significant positive effect. The efficiency of symbiotic relationships could not be linked to root infection frequency monitored as fungal infections; instead, the efficiency needs to be monitored by activity caused by partners such as photosynthetic flow and nitrogen uptake. Similar to cucumber, inconsistent effects of ryegrass grown on soil inoculated with AM fungi were observed. Ryegrass inoculation with a mixture of arbuscular mycorrhizae does not affect 137Cs uptake by ryegrass (Rosén et al., 2005), and lower accumulation of
Cs in mycorrhizal ryegrass than in non-mycorrhizal plants is reported by Dighton and Terry (1996). The largest apparent effect of AM fungi on 137Cs uptake was in sunflower plants grown on loamy soil. This supported the results presented by Dubchak et al. (2010), where the uptake of 134Cs in sunflower plants inoculated with Glomus intraradices was five times greater than in non-mycorrhizal plants. Our data indicate that the difference in 137Cs uptake between mycorrhizal and nonmycorrhizal sunflower plants could be linked to the presence of mycorrhiza and effect of root colonization. Sunflower accumulates 137Cs efficiently (Broadley and Willey, 1997), especially in roots (Dushenkov et al., 1997; Sorochinsky et al., 1998). Although, AM host sunflower plants accumulated higher amounts of 137Cs than plants grown on non-inoculated soil, the percentage of total 137Cs uptake was about one order of magnitude lower (ca. 0.06%) than in quinoa (ca. 0.5%). These values were considerably lower than values found in hydroponic medium (Soudek et al., 2006), where accumulation of 137Cs by sunflower, measured 2, 4, 8, 16 and 32 days after cultivation, are about 12%. The positive but non-statistically significant effect of AM fungi application on total 137Cs uptake by non-mycorrhizal plants, such as quinoa on loamy soil, was unclear. In quinoa plants, partially decomposed spores and some hyphae were sometimes present, which indicated the presence of mycorrhiza. As the presence of AM mycorrhiza in quinoa plants could not be verified in this study, it was not possible to assume enhanced 137Cs uptake due to AM inoculation. One plausible explanation could be the presence of fungi in sterilized soil favoured the release and rate of turnover of the nutrient, possibly radiocesium. The potential of ECM fungi to solubilize rocks and liberate potassium and phosphorus is reported (Alves et al., 2010).
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To be able to highlight differences in the uptake of 137Cs between plants, the 137Cs uptake as a percentage of the total 137Cs in soil was calculated, irrespective of soil type and treatments (Fig. 3a). In terms of total 137Cs accumulation per pot as a percentage of total 137Cs activity in soil, the plants could be ranked in the following decreasing order: cucumber > quinoa > perennial ryegrass > sunflower. The effect of soil inoculation on 137Cs uptake by plants varied with soil type. The use of mycorrhiza in loamy soil resulted in plants with higher 137Cs CR and root infection frequencies, whereas, no or negative effects were observed in silty clay and loamy sand soil. The differences in mycorrhizal response between the different soil types could be attributed to the texture of the respective soil. The loamy soil had an improved soil texture and this encouraged more infection, whereas, the clay soil had a compact structure and the lowest infection frequency. The sandy soil had a low organic matter content, which likely hampered mycorrhizal fungal growth. In addition, the nutrient status of the soil may influence mycorrhization. For instance, the loamy sand soil was nutrient poor and no potassium or phosphorus fertilizers were applied; this may have stimulated the infection frequency on loamy sand soil, as it was the highest among the soils. Apart, three experimental soils had marked differences in 137 Cs concentrations, with the concentration being distinctively higher (ca. 70 times) in loamy soil. Even if this soil was artificially contaminated a long time ago (about 50 y), the effect of soil inoculation on 137Cs uptake by plants on loamy soil was positive in all four plant species. Although the effect of arbuscular mycorrhizal fungi on 137 Cs uptake by plants probably depends on the level of soil contamination, this requires further research as it could not be tested in this study. The averaged values for 137Cs CR could be ranked in the following decreasing order: loamy sand > loamy > silty clay soil (Fig. 3b). The average 137Cs CR was four times higher in loamy sand soil than in the loamy and silty clay soils. Even just 4% clay, such as in the loamy sand soil (Table 1), appeared sufficient to selectively and strongly bind much of the radiocesium found in Swedish soils contaminated by the Chernobyl incident. The ability of plants to take up radiocesium is dependent on plant species. Irrespective of soil type and treatment, cucumber plants, which are more a manufactured plant species, had the greatest ability to take up radiocesium. The 137Cs ratio was 2.26 in cucumber fruits and 0.25 in cucumber plants, in comparison, the 137Cs ratio in quinoa plants, which are virtually wild plant species, was 0.50 in plants and 0.17 in seeds. The efficiency of domesticated perennial ryegrass and sunflower to take up cesium was lower: the 137Cs ratio was 0.20 in ryegrass, 0.12 in sunflower plants, and 0.02 in sunflower seeds. 5. Conclusions It is appeared from this study that there is a potential for mycorrhiza to be used on sites contaminated by radiocesium. Among mycorrhizal plants sunflower (Helianthus spp.) grown on soil inoculated with the AM fungus G. mosseae appears a plant species/AM fungal strain association that substantially increases 137Cs uptake on contaminated loamy and loamy sand soils. However, the response of plants to 137Cs uptake after soil inoculation is conditional and requires future studies to determine the role of other factors, such as nutrient status and pH. The beneficial effects of mycorrhizal inoculation raise the awareness of the role of AM fungi in field conditions, where concomitant colonization can be expected. The inoculation experiments with specific “non-native” mycorrhizal fungus on the uptake of 137 Cs need to be tested in field conditions. Acknowledgements The Swedish Radiation Safety Authorities and Swedish University of Agricultural Sciences funded the project. The authors are
grateful to S. Bengston and K. Thored (Department of Soil and Environment, SLU, Sweden) for kind assistance in performing the experiment, sample preparation, and 137Cs measurements. We would also like to thank the Editor and anonymous referees for comments and suggestions that substantially improved the paper. References Alves, L., Oliveira, V.L., Silva Filho, G.N., 2010. Utilization of rocks and ectomycorrhizal fungi to promote growth of eucalypt. Braz. J. Microbiol. 41 (3), 676e684. Bakken, L.R., Olsen, R.A., 1990. Accumulation of radiocaesium in fungi. Can. J. Microbiol. 36, 704e710. Berreck, M., Haselwandter, K., 2001. Effect of the arbuscular mycorrhizal symbiosis upon uptake of cesium and other cations by plants. Mycorrhiza 10, 275e280. Broadley, M., Willey, N.J., 1997. Differences in root uptake of radiocaesium by 30 plant taxa. Environ. Pollut. 1e2, 11e15. Dighton, J., Terry, G.M., 1996. Uptake and immobilization of caesium in UK grassland and forest soils by fungi following the Chernobyl accident. In: Frankland, J.C., Magan, N., Gadd, G.M. (Eds.), Fungi and Environmental Change. Cambridge University Press, Cambridge, UK, pp. 184e200. Dubchak, S., Ogar, D., Mietelski, J.W., Turnau, K., 2010. Influence of silver and titanium nanoparticles on arbuscular mycorrhiza colonization and accumulation of radiocaesium in Helianthus annuus. Span. J. Agric. Res. 8 (1), 103e108. Dushenkov, S., Kapulnik, Y., Blaylock, M., Sorochinsky, B., Raskin, I., Ensley, B., 1997. Phytoremediation: a novel approach to an old problem. In: Wise, D.L. (Ed.), Global Environmental Biotechnology. Elsevier Science B.V, Amsterdam, pp. 563e572. Dushenkov, S., Mikheev, A., Prokhnevsky, A., Ruchko, M., Sorochinsky, B., 1999. Phytoremediation of radiocesium-contaminated soil in the vicinity of Chernobyl, Ukraine. Environ. Sci. Technol. 33, 469e475. Dushenkov, S., 2003. Trends in phytoremediation of radionuclides. Plant Soil 249, 167e175. Declerck, S., Dupré de Boulois, H., Bivort, C., Delvaux, B., 2003. Extraradical mycelium of the arbuscular mycorrhizal fungus Glomus lamellosumcan take up, accumulate and translocate radiocaesium under root-organ culture conditions. Environ. Microbiol. 5, 510e516. de Boulois, H., Voets, L., Delvaux, B., Jakobsen, I., Declerck, S., 2006. Transport of radiocaesium by arbuscular mycorrhizal fungi to Medicago truncatula under in vitro conditions. Environ. Microbiol. 8, 1926e1934. Entry, J.A., Astrud, L.S., Reeves, M., 1999. Accumulation of 137Cs and 90Sr from contaminated soil by three grass species inoculated with mycorrhizal fungi. Environ. Pollut. 104, 449e457. Gouthu, S., Arie, T., Ambe, S., Yamaguchi, I., 1997. Screening of plant species for comparative uptake abilities of radioactive Co, Rb, Sr and Cs from soil. J. Radioanal. Nucl. Chem. 222, 247e251. Gyuricza, V., Dupré de Boulois, H., Declerck, S., 2008. Effect of potassium and phosphorus on the transport of radiocesium by arbuscular mycorrhizal fungi. J. Environ. Radioact. 110 (6), 482e487. Haak, E., Lönsjö, H., 1996. Long-term transfer of 137Cs and 90Sr to grass on contrasting types of Swedish pastures. In: Gerzabek, M. (Ed.), International Symposium on Radioecology 1996: Ten Years Terrestrial Radioecological Research Following the Chernobyl Accident. Austrian Soil Science Society, Vienna, pp. 129e136. Joner, E.J., Roos, P., Jansa, J., Frossard, E., Leyval, C., Jakobsen, I., 2004. No significant contribution of arbuscular mycorrhizal fungi to transfer of radiocesium from soil to plants. Appl. Environ. Microb. 70, 6512e6517. Kripka, G.V., 2005. Using of Arbuscular Mycorrhiza Fungi in Phytoremediation of Soils from Radionuclides. PhD thesis. Institute of Cell Biology and Genetic Engineering, Kiev, Ukraine, (In Ukrainian). Rosén, K., Weiliang, Z., Mårtensson, A., 2005. Arbuscular mycorrhizal fungi mediated uptake of 137Cs in leek and ryegrass. Sci. Total Environ. 338, 283e290. Rigol, A., Vidal, M., Rauret, G., 2002. An overview of the effect of organic matter on soil-radiocaesium interaction: implications in root uptake. J. Environ. Radioact. 58 (2e3), 191e216. Sanzharova, N.I., Fesenko, S.F., Lisyanskii, K.B., Kuznetsov, V.K., Abramova, T.N., Kotik, V.A., 1997. Forms and accumulation dynamics of 137Cs in crops after the accident at the Chernobyl nuclear power plant. Pochvodenie 2, 159e164 (in Russian). Sorochinsky, B.V., Mikheev, A.N., Kuchko, M.V., Prokhrevsky, A.T., 1998. Decontamination of small water reservoirs of the 10-km zone of Chernobyl NPP by rhizofiltration. In: Problems of Chernobyl Exclusion Zone. Naukova Dumka, Kiev, pp. 97e102. Vavríková, Z., Vane k, T., 2006. 137Cs and 90Sr uptake by Soudek, P., Valenová, S., sunflower cultivated under hydroponic conditions. J. Environ. Radioact. 88, 236e250. Vinichuk, M.M., Johansson, K.J., 2003. Accumulation of 137Cs by fungal mycelium in forest ecosystems of Ukraine. J. Environ. Radioact. 64, 27e43. Wang, B., Qiu, Y.-L., 2006. Phylogenetic distribution and evolution of mycorrhizasin land plants. Mycorrhyza 16, 299e363. Waegeneers, N., Smolders, E., Merckx, R., 2005. Modelling 137Cs uptake in plants from undisturbed soil monoliths. J. Environ. Radioact. 81, 187e199. Willey, N., 2007. Soils contaminated with radionuclides. Some insights for phytoextraction of inorganic contaminants. In: Willey, N. (Ed.), Phytoremediation Methods and Reviews. Humana Press Inc., Totowa, New Jersey, pp. 305e317.
Contents lists available at SciVerse ScienceDirect
Journal of Environmental Radioactivity journal homepage: www.elsevier.com/locate/jenvrad
Effect of arbuscular mycorrhizal (AM) fungi on on different soils
137
Cs uptake by plants grown
M. Vinichuk a, b, *, A. Mårtensson a, T. Ericsson c, K. Rosén a a
Department of Soil and Environment, Swedish University of Agricultural Sciences, P.O. Box 7014, SE-750 07 Uppsala, Sweden Department of Ecology, Zhytomyr State Technological University, 103 Chernyakhovsky Str., 10005 Zhytomyr, Ukraine c Department of Urbanand Rural Development, Swedish University of Agricultural Sciences, SLU, Box 7012, SE-75007 Uppsala, Sweden b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 February 2012 Received in revised form 13 July 2012 Accepted 4 August 2012 Available online 30 August 2012
The potential use of mycorrhiza as a bioremediation agent for soils contaminated by radiocesium was evaluated in a greenhouse experiment. The uptake of 137Cs by cucumber, perennial ryegrass, and sunflower after inoculation with a commercial arbuscular mycorrhizal (AM) product in soils contaminated with 137Cs was investigated, with non-mycorrhizal quinoa included as a “reference” plant. The effect of cucumber and ryegrass inoculation with AM fungi on 137Cs uptake was inconsistent. The effect of AM fungi was most pronounced in sunflower: both plant biomass and 137Cs uptake increased on loamy sand and loamy soils. The total 137Cs activity accumulated within AM host sunflower on loamy sand and loamy soils was 2.4 and 3.2-fold higher than in non-inoculated plants. Although the enhanced uptake of 137 Cs by quinoa plants on loamy soil inoculated by the AM fungi was observed, the infection of the fungi to the plants was not confirmed. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Radiocesium Radionuclides Contaminated soil
1. Introduction Root uptake of radiocesium is the main pathway for the migration of radioactive cesium from soil to humans via the food chain. In soils with high amounts of clay, 137Cs binds or is fixed to clay minerals, whereas, in soils with high organic matter content, 137 Cs can be bound either to organic matter (Rigol et al., 2002) or to biomass (mostly fungal hyphae) (Vinichuk and Johansson, 2003). Although the fraction of radionuclides associated with microorganisms and particularly fungi may be relatively small, these organisms play an important role in the retention of radiocesium in forest soils. For instance, in forest soils, ectomycorrhizal (ECM) fungi accumulate up to 270 times more radiocesium than plants growing in their immediate vicinity (Bakken and Olsen, 1990). Despite these observations, the role of arbuscular mycorrhizal (AM) fungi in uptake and accumulation (“binding”) of 137Cs in agricultural soils has not received much attention. Although data are still limited, Declerck et al. (2003) suggest the extraradical mycelium of the AM fungus Glomus lamellosum can take up, possibly
* Corresponding author. Department of Soil and Environment, Swedish University of Agricultural Sciences, P.O. Box 7014, SE-750 07 Uppsala, Sweden. Tel.: þ46 18 67 14 42; fax: þ46 18 67 28 95. E-mail addresses: [email protected], [email protected], [email protected] (M. Vinichuk). 0265-931X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jenvrad.2012.08.004
accumulate, and unambiguously translocate radiocesium from a 137Cs-labelled synthetic root-free compartment to a root compartment and to within the roots in root-organ culture. Kripka (2005) provides evidence of the direct involvement of AM fungi in radionuclide transport to the plant, and de Boulois et al. (2006) demonstrate the capacity of AM fungi to transport cesium (Cs). However, the results from studies on the capacity of AM fungi to accumulate and transport radiocesium is inconsistent and inconclusive (Berreck and Haselwandter, 2001; Joner et al., 2004; Rosén et al., 2005) or uncertain and even controversial (de Boulois et al., 2006). Apart from the wide variation in changes in 137Cs concentration in different plants, from 2- to 4-fold within cereals to 27-fold for all field crops (Sanzharova et al., 1997), variation in changes in 137Cs concentration can occur in cultivars of the same species (Dushenkov et al., 1999). Thus, there is an indication AM fungi have a possible role in phytoremediation of soils contaminated with radionuclides. However, their ability to participate in phytoremediation strategies remains questionable and requires further investigation. In this study, four plant species, cucumber, ryegrass, sunflower and quinoa, were tested. Cucumber, ryegrass, and sunflower form mycorrhiza, whereas, as quinoa is generally non-mycorrhizal (Wang and Qiu, 2006), it was included as a “reference” plant. All plants are known to accumulate 137Cs efficiently: cucumber (Gouthu et al.,1997); ryegrass (Waegeneers et al., 2005); sunflower (Sorochinsky et al., 1998; Soudek et al., 2006), and quinoa (Broadley and Willey, 1997).
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2. Materials and methods
from a field site at Vara in the county of Västergötland, and in 1961, the soil was contaminated with 35.7 MBq m2 137Cs, mixed with a plough layer of soil, and crops grown for many years (Haak and Lönsjö, 1996). After 47 y, 137Cs activity had decreased from ca. 148 kBq kg1 soil to about 47.1 Bq kg1 soil. The loamy soil for this study was collected from the same micro-plots as in 1961. The soil characteristics are presented in Table 1. In the laboratory, the soils were thoroughly mixed, sieved through a 2 mm sieve to separate stones, and aliquots (about 2/3) of each soil type were autoclaved for 3 h at 121 C at 2 bars of pressure. The mixture containing the carrier material (peat, sand, and perlite) was steam-sterilized by the producer.
2.1. Experiment layout
2.3. Plant material and growth conditions
The greenhouse experiment comprised a block design including two treatments and five replicates: AMþ and AM- applied to each soil type. In treatment AMþ, soil was autoclaved before the experiment and then inoculated with a commercial AM fungus G. mosseae. The fungal strain had been isolated from agricultural soil in Finland and contained spores and hyphae of the arbuscular mycorrhizal fungus G. mosseae together with a carrier material consisting of a steam-sterilized mixture of peat, sand, and Perlite. In treatment AM-, soil was autoclaved but non-inoculated.
The plants were grown in a greenhouse in plastic rose pots with raised bottoms and a volume of 4.2 dm3. The pots were placed on saucers (22 cm diameter) and filled with the same amount of each soil type (ca. 4.0 kg). To avoid possible interaction between ions during radiocesium transport by AM fungi (Gyuricza et al., 2008), no mineral fertilizers such as potassium (K) or phosphorus (P) were applied to the soil. The crops used in the pot experiment were cucumber (Cucumis sativus L. cultivar ‘Melen F1’), perennial ryegrass (Lolium multiflorum, variety ‘Corvus’), sunflower (Helianthus spp.), and quinoa (Chenopodium quinoa Willd), and were supplied by Weibulls Horto AB, Sweden. There were 40 pots (5 AMþ, 5 AM ¼ 10 4 crops) for each soil type, which gave a total 120 pots (40 3 soil types). Before seeding, inoculum material containing live arbuscular mycorrhizal fungi (5e10 AMF propagules/g inoculum) was applied to the soil (treatment AMþ) in the proportion 1 part inoculum material to 100 parts soil, according to the manufacturer’s advice. Inoculum material was then mixed with the upper 2e3 cm of soil and the pots were supplied with distilled water as required. Plants were randomly seeded by hand within the pot area (ryegrass and quinoa 10 per pot; cucumber and sunflower 2 seeds per pot), and were weeded and watered as required. The climatic conditions in the greenhouse were a 16 h daytime and 8 h nighttime cycle; a light intensity (photosynthetically active radiation) of 110e140 mmol m2 s1; daytime temperature of 20 C and nighttime temperature of 15 C; and, relative air humidity of 70%. To compensate for an increase in daylight, the light intensity was reduced to between 70 and 110 mmol m2 s1 after about 11 weeks. The plants were grown for 16e20 weeks, depending on the crop: cucumber fruits were ready for harvest after 8e9 weeks from planting; ryegrass after first (1st) and second (2nd) cut; and sunflower and quinoa after ripening of the seeds. From different treatments, the same crop was harvested at the same time in both treatments. After harvest, plants were air-dried to constant weight and aboveground parts divided in seeds and remaining parts. Seeds of sunflower and quinoa were sieved with a 4 and 2 mm sieve, and the remaining parts were milled. Both seeds and remaining parts were analysed for 137Cs activity.
The objective was to investigate the potential use of mycorrhiza as a bioremediation agent for soils contaminated by radiocesium. The effect of AM fungus Glomus mosseae on 137Cs uptake by different crops on silty clay, loamy, and loamy sand soils was investigated. The hypothesis was that plant species/AM fungal strains associations are unique and radiocesium accumulation in mycorrhizal plants may be higher, lower, or equal to the accumulation in non-mycorrhizal plants. Consequently, the desired effect might only be achieved with the right combination of plant species and AM fungal strain.
2.2. Soils The three soil types used (silty clay, loamy and loamy sand soil) were collected in September 2008 from three locations in central Sweden (Table 1) and had been contaminated with 137Cs after the Chernobyl accident in 1986. At each sampling location, the upper 0e15 cm layer of soil (about 400 kg) was collected from 3 to 4 places within an area of about 100 m2. The loamy soil used in the experiment had previously been used in a micro-plot experiment in 1961 (Haak and Lönsjö, 1996). In autumn 1960, soil was collected
Table 1 Main characteristics of the soils used in the experiment. Soil Parameters
Silty clay
Loamy
Loamy sand
Soil sampling locations long, alt.
Skogsvallen 17 100 W, 60 100 N
Vara 12 490 W, 58 130 N
Hille 17 110 W, 60 440 N
Land use
Pasture
Pasture
Cs, kBq kg1dw (mean se) Particle size distribution: % Gravel (>2 mm) Sand (2e0.06 mm) % Silt (0.06e0.002 mm) % Clay (<0.002 mm) Bulk density, g cm3 %C %N pH (0.01 M CaCl) Exchange cations (meq 100 g1 soil) Ca Mg K K, HNO3 KeAL Total, exchange base cations Titratable acidity CEC at pH 7 Base saturation, %
0.53 0.01
Microplot experiment 41.5 0.17
5.2 5.1 13.1 0.78 40.2 2.7 41.5 1.7 0.96 4.5 0.35 0.39 0.03 4.8 0.01
0.80 0.57 42.3 3.6 41.7 4.0 16.2 0.49 1.10 2.6 0.02 0.30 0.01 4.9 0.08
13.4 0.07 76.1 0.57 6.5 0.49 4.1 0.2 1.16 2.6 0.13 0.20 0.01 4.2 0.03
10.9 0.49 0.98 0.03 0.66 0.03 6.2 0.04 0.71 0.05 12.8
1.8 0.25 0.31 0.01 0.11 0.01 1.6 0.11 0.01 2.3
5.5 18.3 69.9
5.8 8.1 28.6
137
15.8 2.03 0.54 13.6 0.47 18.9 7.8 26.7 70.8
0.11 0.02 0.05 0.25 0.02
0.69 0.01
2.4. Quantification of AM fungal colonization For AM fungi colonization, approximately 25e50 g fresh weight of root was randomly dug from selected plants (soils) and stored in a freezer at 40 C before staining. Then, these samples were immersed in 20% potassium hydroxide for 1 d, washed in tap water, and acidified with 1% hydrochloric acid. After this, the roots were stained with 0.05% trypan blue in a 14:1:1 lactic acid:glycerol:water solution for one day, and then rinsed in a de-staining solution (14:1:1 lactic acid:glycerol:water) until no visual blue staining remained. For examination, the roots were spread onto glass slides
M. Vinichuk et al. / Journal of Environmental Radioactivity 115 (2013) 151e156
and the appearance of fungal infections was visually recorded under a binocular microscope. The rate of mycorrhizal infection frequency in the roots was graded as 0 ¼ no infection; 1 ¼ sign of infection; 2 ¼ moderate infection; and, 3 ¼ abundant infection.
Table 3 Effect of soil inoculation with AM fungi on 137Cs concentration ratios (CR) in roots of cucumber, sunflower, and quinoa, and the percentage increase (þ) and decrease () in 137Cs CR when grown on loamy soil. AMþ, soil autoclaved and inoculated with AM fungi; AM, soil autoclaved and non-inoculated (n ¼ 5). Plant, root size
2.5. Radiometry and data treatment
153
CRa
AMþ/AM, %b
AMþ
Soil and whole plant samples were air-dried prior to analyses and plants were cut into <2 mm pieces: cucumber fruits were analysed on fresh weight basis. In addition to harvesting aboveground parts, the roots were dug out for determining radiocesium distribution within the plant. The roots were washed in tap water, air dried, and analysed for 137Cs activity. Gamma-spectrometry with HPGe detectors coupled to a multi-channel analyzer system was used to determine 137Cs activity in the soil and plants. The systems were calibrated with a standard source used for intercomparative studies and in appropriate (60 and 35 ml) standard containers. All results were decay corrected to date of sampling (harvesting) and 137Cs concentration ratios (CR) were calculated as 137 Cs Bq kg1dry weight (dw) in plant divided by 137Cs Bq kg1dw in soil. Data were analysed by a 2-sample t-test with Minitab (Ó 2010 Minitab Inc.) software to evaluate the differences in 137Cs activity between treatments at the level of significance 0.05. 3. Results 3.1.
137
Cs concentration ratios in roots and root infection frequency
3.1.1. Mycorrhizal plants Infection frequency of cucumber plants grown on inoculated soil was only positively correlated with cucumber plant biomass on silty clay soil (r ¼ 0.697; p < 0.05) (Table 2). Roots (<2 mm) of cucumber grown on inoculated loamy soil had about 30% higher 137 Cs CR than plants grown on non-inoculated soil (Table 3). Infection frequency of cucumber plants grown on inoculated soil was not correlated, or negatively correlated, with 137Cs uptake (Table 4). Fungal infection of ryegrass roots was only found in loamy sand soil: neither ryegrass biomass nor 137Cs CR correlated with AM
Table 2 Infection frequency in roots of cucumber, ryegrass, sunflower and quinoa, and correlation coefficients (r) among root infection frequency and plant biomass (g pot1dw) when grown on different soils (n ¼ 5). Soil
Plants/parts, cuts
Cucumber, mycorrhizal Silty clay Fruits Plant Loamy Fruits Plant Loamy sand Fruits Plant Ryegrass, mycorrhizal Silty clay e Loamy e Loamy sand 1st þ 2nd cut Sunflower, mycorrhizal Silty clay Seeds Plant Loamy Seeds Plant Loamy sand Seeds Plant Quinoa, non-mycorrhizal Silty clay e Loamy e Loamy sand e *p < 0.05; **p < 0.01.
Infection frequency
r
1.0 0.7
0.37 0.70* 0.22 0.24 No data 0.21
1.0 1.0 1.8 0.45
0 0 0.6
No infection No infection 0.36
0.6 0.55
0.07 0.04 0.37 0.02 0.80** 0.81**
1.2 1.0 1.6 0.55
0 0 0
No infection No infection No infection
Cucumber, <2 mm Sunflower, <2 mm Sunflower, 2e5 mm Quinoa, <2 mm Quinoa, 2e5 mm
0.30 0.17 0.029 0.10 0.094
AM
0.07 0.04 0.02 0.06 0.04
0.23 0.10 0.022 0.087 0.052
0.08 0.02 0.01 0.01 0.05
31.0 72.7 27.4 14.3 81.1
CR ¼ 137Cs Bq kg1 in plant/137Cs Bq kg1 in soil. Cs CR in AMþ minus 137Cs CR in AM divided by 137Cs in AM multiplied by 100. a
b 137
fungi infection frequency (Tables 2 and 4). In the fine roots (<2 mm) of sunflower, 137Cs CR did not differ between treatments; however, in coarse roots (2e5 mm) of plants grown on inoculated soil, 137Cs CR was 3- to 4-fold higher than in plants grown on non-inoculated soil (Table 3). The infection frequency of sunflower correlated with plant biomass on loamy sand soil (Table 2) and 137Cs CR on loamy and loamy sand soils (Table 4). 3.1.2. Non-mycorrhizal plants In the fine (<2 mm) roots of quinoa, there was no difference in 137 Cs CR; however, in coarse roots (2e5 mm) of plants grown on inoculated soil, 137Cs CR was about 80% higher than in plants grown on non-inoculated soil (Table 3). 3.2. Biomass 3.2.1. Mycorrhizal plants The effect of AM fungi inoculation on cucumber plant biomass was evident in silty clay soil (Fig. 1). In loamy sand soil, there was a negative effect of soil inoculation on cucumber biomass, as plants were weakly developed and only limited numbers of fruits formed: this rendered it difficult to compare and estimate the effect of soil inoculation. Fungi did not affect the biomass of perennial ryegrass, whereas, AMþ plants growing on loamy sand yielded less biomass than plants with AM treatments (Fig. 1): the negative AM effect was not statistically significant. On silty clay soil, fungal inoculation had no effect on aboveground sunflower plant biomass, but on loamy and loamy sand soils fungal inoculation resulted in higher (p < 0.01) sunflower plant biomass (Fig. 1). The effect of loamy soil inoculation on sunflower biomass was positive but not statistically significant. 3.2.2. Non-mycorrhizal plants The AM host quinoa plants grown on inoculated silty clay soil had 50% higher biomass than plants grown on non-inoculated soil. There was no effect of soil inoculation on plant biomass on loamy sand soil (Fig. 1). Table 4 Correlation coefficients among 137Cs concentration ratios (CR ¼ 137Cs, Bq kg1 in plant/137Cs, Bq kg1 in soil) and root infection frequency in cucumber, ryegrass, sunflower, and quinoa when grown on different soils (n ¼ 5). Plant/Soil Cucumber Cucumber Ryegrass Sunflower Sunflower Quinoa *p < 0.05.
fruits plant seeds plant
Silty clay
Loamy
Loamy sand
0.29 0.12 No infection 0.48 0.23 No infection
0.05 0.08 No infection 0.42 0.72* No infection
0.60 0.08 0.33 0.19 0.73* No infection
154
M. Vinichuk et al. / Journal of Environmental Radioactivity 115 (2013) 151e156 Table 5 Effect of soil inoculation with AM fungi on 137Cs concentration ratios (CR) in cucumber, ryegrass, sunflower, and quinoa, and the percentage increase (þ) and decrease () in 137Cs CR when grown on different soils. AMþ, soil autoclaved and inoculated with AM fungi; AM, soil autoclaved and non-inoculated (n ¼ 5).
80.0 70.0 60.0 g pot -1
50.0 40.0
Soil
30.0 20.0 10.0 0.0 Cucumber Silty clay AM+
Silty clay AM-
Ryegrass Loamy AM+
Sunflower Loamy AM-
Loamy sand AM+
Quinoa* Loamy sand AM-
Fig. 1. The effect of soil inoculation on aboveground plant biomass, g dry weight pot1, of cucumber, ryegrass, sunflower and quinoa grown on silty clay, loamy and loamy sand soils. Standard error, n ¼ 5. * Non-mycorrhizal plant.
3.3.
137
Cs concentration ratios in plants
3.3.1. Mycorrhizal plants Uptake of 137Cs increased in cucumber plants grown on inoculated loamy soil, whereas, in plants grown on non-inoculated loamy sand and silty clay soils the uptake of 137Cs decreased. The CR of 137 Cs in plants grown on non-inoculated loamy sand and silty clay soils were generally higher than the CR of 137Cs in plants grown on soils inoculated with fungi. In cucumber fruits 137Cs CR was high and varied between about 2.1e6.9 on loamy sand soil and 1.1e1.5 on loamy soil (Table 5). The application of AM fungi resulted in higher 137Cs uptake by ryegrass plants (2nd cut) grown in loamy soil but lower uptake in plants grown in loamy sand and silty clay soils (Table 5): the 137Cs CR values were in the range 0.01 (lowest, silty clay soil) and 0.83 (highest, loamy sand soil). The AM host sunflower plants had two- to three-fold higher 137Cs uptake on loamy sand and loamy soils than plants grown on non-inoculated soil: this effect was negative on silty clay soil (Table 5). In sunflower plants, 137Cs CR varied between 0.002 (for seeds on loamy soil) and 0.42 (for plants on loamy sand soil) (Table 5). The only statistically significant difference in 137Cs CR values between treatments AMþ and AMe were for ryegrass on loamy sand and sunflower on loamy soils. 3.3.2. Non-mycorrhizal plants The AM host quinoa plants responded positively to 137Cs uptake on loamy sand and loamy soils, although this response was not statistically significant compared to AMe plants (Table 5). However, soil inoculation with AM fungi did not affect 137Cs uptake by quinoa plants grown on silty clay soil. In quinoa plants, 137Cs CR was high, and varied between 0.13 (loamy soil) to about 1.5 (loamy sand soil: Table 2).
Plant parts, cuts
Cucumber, mycorrhizal Silty clay Fruits Plant Loamy Fruits Plant Loamy sand Fruits Plant Ryegrass, mycorrhizal Silty clay 1st cut 2nd cut Loamy 1st cut 2nd cut Loamy sand 1st cut 2nd cut Sunflower, mycorrhizal Silty clay Seeds Plant Loamy Seeds Plant Loamy sand Seeds Plant Quinoa, non-mycorrhizal Silty clay Seeds Plant Loamy Seeds Plant Loamy sand Seeds Plant
CRa
AMþ/AM, %b
AMþ
AM
1.07 0.19 1.77 0.35 2.05 0.26
0.46 0.04 0.70 0.06 0.94 0.11
1.45 0.19 1.35 0.27 6.97 0.26
0.28 0.05 0.66 0.09 4.0 0.13
26 3 31 27 71 e
0.012 0.025 0.015 0.039 0.31 0.42
0.02 0.01 0.01 0.02 0.09 0.26
0.018 0.013 0.013 0.028 0.65 0.83
0.01 0.01 0.003 0.16 0.16 0.19
37 86 16 38 52* 48*
0.0041 0.029 0.0045 0.045 0.10 0.42
0.004 0.01 0.002 0.02 0.02 0.08
0.0091 0.027 0.0024 0.015 0.04 0.29
0.01 0.01 0.001 0.01 0.05 0.08
55 11 103* 195** 156 45
0.090 0.14 0.13 0.37 0.39 1.52
0.06 0.07 0.03 0.07 0.22 0.75
0.096 0.15 0.094 0.35 0.26 0.77
0.07 0.05 0.03 0.07 0.15 0.49
*p < 0.05; **p < 0.01. a CR ¼ 137Cs Bq kg1 in plant/137Cs Bq kg1 in soil. b 137 Cs CR in AMþ minus 137Cs CR in AM divided by by 100.
137
6 12 40 6 48 84
Cs in AM multiplied
The infection frequency in sunflower roots correlated with both biomass and 137Cs CR (Tables 2 and 4). 3.4.2. Non-mycorrhizal plants The uptake of 137Cs by quinoa AM host plants increased by a factor of about two only on loamy sand soil (Fig. 2). In quinoa plants soil inoculation with AM fungi generally did not result in higher uptake of 137Cs across all soil types (Fig. 3a). Inoculation with AMþ fungi did not affect 137Cs CR when comparing the soil types across all plants studied (Fig. 3b). The highest 137Cs ratios were observed on loamy sand. 4. Discussion
3.4. Total
137
Cs activity and % of total
137
Cs uptake by plants
3.4.1. Mycorrhizal plants Uptake of 137Cs by AM host cucumber plants was similar to the uptake of 137Cs in cucumber plants grown on non-inoculated clay and loamy soil but lower in loamy sand (Fig. 2). In terms of combined effect of increased plant biomass and enhanced 137Cs uptake, ryegrass presented inconsistent results: 1.5-fold higher uptake in loamy soils, 2.2-fold lower uptake in loamy sand soil, and no effect in silty clay soil (Fig. 2). The effect of soil inoculation on the percentage of total 137Cs uptake by ryegrass was negative when analysed across all soil types (Fig. 3a). Uptake of 137Cs by AM host sunflower plants was 3.0-fold higher on loamy soil and 2.5-fold higher on loamy sand soil than in non-inoculated plants (Fig. 2). However, the overall effect of AM fungi application on the amount of 137Cs uptake by plants in all soils was not established (Fig. 3a).
The potential use of plants to decontaminate radionuclidepolluted soil is an approach that deserves attention. This approach has been partly tested by Entry et al. (1999), and Dushenkov (2003) and Willey (2007) conclude phytoremediation of radionuclides could become useful in the future. Therefore, the challenge is to develop viable methods for extracting radionuclides from soil. One option for enhancing the potential for phytoextraction is with mycorrhiza. Soil inoculation may result in enhanced radionuclide uptake, have no effect, or even cause lower uptake due to the specific and intricate balance between the plants, the fungi, and the environment. However, this phenomenon deserves attention before widespread use of mycorrhizal inoculation for the bioextraction of radionuclides can be recommended. Cucumber moderately infected by AM fungi on silty clay and loamy soils only (non-statistically significantly) improved 137Cs
M. Vinichuk et al. / Journal of Environmental Radioactivity 115 (2013) 151e156
14.0
Silty clay
AM+
Bq pot-1
10.0
137Cs,
12.0
6.0
a
AM-
155
1.20 AM+
1.00
AM-
8.0 137Cs,
%
0.80
4.0
0.60 0.40
2.0
0.1 0.1
0.0 Cucumber
Ryegrass
Sunflower
Mycorr.
0.20
Quinoa
0.00
Non-mycorr.
Cucumber
0.90
800
0.70
600
0.60
CR
Bq pot-1
b
137Cs,
AM-
1,000
137Cs
400
0.80
0.50 0.40 0.30
12.9 8.9
0
0.20
Cucumber
Ryegrass
Sunflower
Mycorr.
Quinoa
0.10
Non-mycorr.
0.00 Silty clay
AM-
Loamy sand
AM-
5.0
Fig. 3. The effect of soil inoculation on amount of 137Cs uptake by cucumber, ryegrass, sunflower, and quinoa: (a) as % of total 137Cs in soil across soil types; (b) 137Cs concentration ratios of the same plants (CR ¼ 137Cs Bq kg1 in plant/137Cs Bq kg1 in soil) grown on silty clay, loamy and loamy sand soil across plant species.
4.0
137
7.0 Bq pot-1
Loamy AM+
AM+
Loamy sand
8.0
137Cs,
Quinoa Non-mycorr.
AM+
1,200
200
Sunflower
Mycorr.
Loamy
1,400
Ryegrass
6.0
3.0 2.0 1.0 0.0 Cucumber
Ryegrass Mycorr.
Sunflower
Quinoa Non-mycorr.
Fig. 2. The effect of soil inoculation on total 137Cs activity, Bq pot1, in aboveground plant parts of cucumber, ryegrass, sunflower, and quinoa grown on silty clay, loamy and loamy sand soil. Standard error, n ¼ 5.
uptake on loamy soil despite being densely infected in loamy sand soil, where no effect of soil inoculation on 137Cs uptake was detected (Table 5). The frequency of root infection in mycorrhized cucumber did not correlate with 137Cs uptake (Table 4), but positively correlated with plant biomass on silty clay soil (Table 2). Thus, the effect of AM fungi on total 137Cs uptake by cucumber could not be linked to the infection frequency of the mycorrhizal fungi, as abundant mycorrhizal infection of cucumber on loamy sand and moderate infection on silty clay soils had a negative or no effect, whereas, moderate infection on loamy soil caused a non-statistically significant positive effect. The efficiency of symbiotic relationships could not be linked to root infection frequency monitored as fungal infections; instead, the efficiency needs to be monitored by activity caused by partners such as photosynthetic flow and nitrogen uptake. Similar to cucumber, inconsistent effects of ryegrass grown on soil inoculated with AM fungi were observed. Ryegrass inoculation with a mixture of arbuscular mycorrhizae does not affect 137Cs uptake by ryegrass (Rosén et al., 2005), and lower accumulation of
Cs in mycorrhizal ryegrass than in non-mycorrhizal plants is reported by Dighton and Terry (1996). The largest apparent effect of AM fungi on 137Cs uptake was in sunflower plants grown on loamy soil. This supported the results presented by Dubchak et al. (2010), where the uptake of 134Cs in sunflower plants inoculated with Glomus intraradices was five times greater than in non-mycorrhizal plants. Our data indicate that the difference in 137Cs uptake between mycorrhizal and nonmycorrhizal sunflower plants could be linked to the presence of mycorrhiza and effect of root colonization. Sunflower accumulates 137Cs efficiently (Broadley and Willey, 1997), especially in roots (Dushenkov et al., 1997; Sorochinsky et al., 1998). Although, AM host sunflower plants accumulated higher amounts of 137Cs than plants grown on non-inoculated soil, the percentage of total 137Cs uptake was about one order of magnitude lower (ca. 0.06%) than in quinoa (ca. 0.5%). These values were considerably lower than values found in hydroponic medium (Soudek et al., 2006), where accumulation of 137Cs by sunflower, measured 2, 4, 8, 16 and 32 days after cultivation, are about 12%. The positive but non-statistically significant effect of AM fungi application on total 137Cs uptake by non-mycorrhizal plants, such as quinoa on loamy soil, was unclear. In quinoa plants, partially decomposed spores and some hyphae were sometimes present, which indicated the presence of mycorrhiza. As the presence of AM mycorrhiza in quinoa plants could not be verified in this study, it was not possible to assume enhanced 137Cs uptake due to AM inoculation. One plausible explanation could be the presence of fungi in sterilized soil favoured the release and rate of turnover of the nutrient, possibly radiocesium. The potential of ECM fungi to solubilize rocks and liberate potassium and phosphorus is reported (Alves et al., 2010).
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M. Vinichuk et al. / Journal of Environmental Radioactivity 115 (2013) 151e156
To be able to highlight differences in the uptake of 137Cs between plants, the 137Cs uptake as a percentage of the total 137Cs in soil was calculated, irrespective of soil type and treatments (Fig. 3a). In terms of total 137Cs accumulation per pot as a percentage of total 137Cs activity in soil, the plants could be ranked in the following decreasing order: cucumber > quinoa > perennial ryegrass > sunflower. The effect of soil inoculation on 137Cs uptake by plants varied with soil type. The use of mycorrhiza in loamy soil resulted in plants with higher 137Cs CR and root infection frequencies, whereas, no or negative effects were observed in silty clay and loamy sand soil. The differences in mycorrhizal response between the different soil types could be attributed to the texture of the respective soil. The loamy soil had an improved soil texture and this encouraged more infection, whereas, the clay soil had a compact structure and the lowest infection frequency. The sandy soil had a low organic matter content, which likely hampered mycorrhizal fungal growth. In addition, the nutrient status of the soil may influence mycorrhization. For instance, the loamy sand soil was nutrient poor and no potassium or phosphorus fertilizers were applied; this may have stimulated the infection frequency on loamy sand soil, as it was the highest among the soils. Apart, three experimental soils had marked differences in 137 Cs concentrations, with the concentration being distinctively higher (ca. 70 times) in loamy soil. Even if this soil was artificially contaminated a long time ago (about 50 y), the effect of soil inoculation on 137Cs uptake by plants on loamy soil was positive in all four plant species. Although the effect of arbuscular mycorrhizal fungi on 137 Cs uptake by plants probably depends on the level of soil contamination, this requires further research as it could not be tested in this study. The averaged values for 137Cs CR could be ranked in the following decreasing order: loamy sand > loamy > silty clay soil (Fig. 3b). The average 137Cs CR was four times higher in loamy sand soil than in the loamy and silty clay soils. Even just 4% clay, such as in the loamy sand soil (Table 1), appeared sufficient to selectively and strongly bind much of the radiocesium found in Swedish soils contaminated by the Chernobyl incident. The ability of plants to take up radiocesium is dependent on plant species. Irrespective of soil type and treatment, cucumber plants, which are more a manufactured plant species, had the greatest ability to take up radiocesium. The 137Cs ratio was 2.26 in cucumber fruits and 0.25 in cucumber plants, in comparison, the 137Cs ratio in quinoa plants, which are virtually wild plant species, was 0.50 in plants and 0.17 in seeds. The efficiency of domesticated perennial ryegrass and sunflower to take up cesium was lower: the 137Cs ratio was 0.20 in ryegrass, 0.12 in sunflower plants, and 0.02 in sunflower seeds. 5. Conclusions It is appeared from this study that there is a potential for mycorrhiza to be used on sites contaminated by radiocesium. Among mycorrhizal plants sunflower (Helianthus spp.) grown on soil inoculated with the AM fungus G. mosseae appears a plant species/AM fungal strain association that substantially increases 137Cs uptake on contaminated loamy and loamy sand soils. However, the response of plants to 137Cs uptake after soil inoculation is conditional and requires future studies to determine the role of other factors, such as nutrient status and pH. The beneficial effects of mycorrhizal inoculation raise the awareness of the role of AM fungi in field conditions, where concomitant colonization can be expected. The inoculation experiments with specific “non-native” mycorrhizal fungus on the uptake of 137 Cs need to be tested in field conditions. Acknowledgements The Swedish Radiation Safety Authorities and Swedish University of Agricultural Sciences funded the project. The authors are
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