The earthworm (Aporrectodea caliginosa) primes the release of mobile and available micronutrients in soil

Pedobiologia 55 (2012) 93–99

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Pedobiologia - International Journal of Soil Biology journal homepage: www.elsevier.de/pedobi

The earthworm (Aporrectodea caliginosa) primes the release of mobile and available micronutrients in soil Nikolai P. Bityutskii ∗ , Polina I. Kaidun, Kirill L. Yakkonen Department of Agricultural Chemistry, Saint Petersburg State University, 16th-linia 29, V.O. Saint-Petersburg 199178, Russia

a r t i c l e

i n f o

Article history: Received 28 June 2011 Received in revised form 25 November 2011 Accepted 26 November 2011 Keywords: Earthworm Micronutrient Mobility Availability Plant

a b s t r a c t The objectives of our study were to quantify the impact of endogeic earthworms Aporrectodea caliginosa (Savigny) on iron (Fe), manganese (Mn) and zinc (Zn) mobility and availability in soil. Dried rye straw (Cecale cereale L.), clover aboveground parts (Trifolium pratense L.) or calcium carbonate were added to determine the effects on soil micronutrient mobility. To test the importance of soil–water saturation mediated by earthworms, soil samples were modified to 60% (control) and 100% (as in casts) water holding capacity (WHC). To assess availability of micronutrients, a cucumber plant (Cucumis sativus L.) bioassay were used. Earthworm casts had generally higher amounts of water-soluble micronutrients compared with bulk soils regardless of their moisture contents. The increased micronutrient mobility was more pronounced in casts from soil samples amended with plant residues (especially with straw) and was significantly higher than mobility in control soil for at least 1 week after the casts were deposited. Pre-incubation of soils amended with clover or straw with living earthworms for 4 weeks produced an increase in both shoot biomass and translocation rate of micronutrients (Mn, Zn) into xylem sap of cucumber compared to soils not worked by earthworms. The earthworm-mediated plant performances were determined 4 weeks after the earthworms were removed. The results demonstrated that earthworms can significantly impact the formation of mobile and available micronutrients in a soil. The relationship between micronutrient availability to cucumber plants and earthworm contribution to nitrogen (N) mineralization and micronutrient mobility are discussed. © 2012 Elsevier GmbH. All rights reserved.

Introduction Earthworms generally are assumed to be a major and beneficial component of the soil fauna of many terrestrial ecosystems (Lee 1985; Edwards and Bohlen 1995). The most distinguishing feature of earthworms is their propensity to consume soil and associated material. Due to their high consumption rates and burrowing activity they affect soil structure, aggregate stability, aeration, microbial turnover, decomposition processes and nutrient dynamics (Edwards and Bohlen 1995; Tiunov and Scheu 1999; Parmelee et al. 1998; Eisenhauer et al. 2007) with important consequences for plant growth and ecology (Scheu 2003; Partsch et al. 2006). While it is commonly accepted that earthworms benefit plant growth and productivity, the mechanisms of earthworm-mediated soil fertility and plant growth are not well understood. Most studies have concentrated on the earthworm effects related to availability of plant macronutrients especially of nitrogen (N) (Amador and Görres 2005; Eisenhauer and Scheu

∗ Corresponding author. Tel.: +7 812 3213358; fax: +7 812 3213358. E-mail address: [email protected] (N.P. Bityutskii). 0031-4056/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.pedobi.2011.11.003

2008; Laossi et al. 2011) as well as on metal mobility and availability in metal-contaminated soils (see review: Sizmur and Hodson 2009; Karaca et al. 2010). Unfortunately, earthworm-mediated availability of plant micronutrients is rarely controlled. In addition, contradictory results can be found when determining the impact of earthworms on plant micronutrient availability (Wen et al. 2004; Lukkari et al. 2006). For these reasons, it is crucial to develop quantitative information on the effect of earthworms on micronutrient mobility as well as our understanding of earthworm mechanisms that mediate micronutrient mobility and availability in soils. The aim of this study was to investigate whether earthworms impact the mobility and availability of iron (Fe), manganese (Mn) and zinc (Zn) which are essential for plants. Materials and methods Experimental design Two experiments were conducted where the bulk soil and earthworm cast material (experiment I), and soils worked and not worked by earthworms (experiment II) were compared. To

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vary initial micronutrient mobility in soil, the following treatments were applied: control soil without additions, soil amended with different plant residues or soil amended with calcium carbonate (CaCO3 ). Additionally, to test whether the moistening of soil during its passage through the digestive tract of earthworms influences micronutrient mobility, soil samples were subjected to different soil moisture levels: 60% (control) and 100% (average moisture of casts) of their water holding capacity (WHC). To examine any earthworm-mediated priming effect, micronutrient mobility in both casts and bulk soils was monitored over 8 days. Experiment II was conducted in two stages. The first stage involved a pre-incubation period (for 4 weeks) in the presence of living earthworms during which earthworm-induced formation of soil water-soluble micronutrients and available nitrogen affecting plant growth was expected. During the second stage of experiment II, after removal of earthworms, plants (cucumber) were grown for 4 weeks. This experimental design allowed us to assess the importance of earthworm-triggered soil alterations in relation to plant growth and micronutrient availability to plants. In both experiment I and II there were four replicates of each treatment. To assess availability of micronutrients both a chemical method and plant bioassay were used. The chemical method was focused on characterizing the water-soluble pool of micronutrients (use of water as a metal extracting agent). As stated by Sizmur and Hodson (2009), “changes in the water extractable metal fraction of earthworm-worked soil will provide stronger evidence for the impact of earthworms on metal mobility and availability than the use of more involved chemical extractions”. The bioassay focused on the collection and analysis of xylem sap. Sap composition reflects both behavior of nutrients in the soil and the manner of their absorption by plant roots (Noguchi et al. 2001). Sampling and processing Treatment of soil and residue samples Soil samples were collected twice in spring (experiment I) and in autumn (experiment II) near The Biological Research Institute of Saint-Petersburg State University, Peterhof, Russia. The soil is mapped as an umbric albeluvisol. The samples (the humus horizon) had a pH of 5.0 (determined in a 1:2.5 soil–water solution) and contained 2.30% Corg , 0.24% total N, 22 mg kg−1 available P (determined in a 1:5 soil – 0.2 N HCl solution), and 21 mg kg−1 of dry soil available K (determined in a 1:10 soil – 1 N ammonium acetate solution) (Arinushkina 1970). The field moist soil was air-dried at room temperature and then passed through a 1 mm sieve. Plant residues for incubation included dried rye straw (Cecale cereale L.) and clover shoots (Trifolium pratense L.). Clover residues had a C-to-N ratio of 12:1 and contained 156 ± 16 (mean ± SD, ␮g g−1 DW) total Fe, 67 ± 3 total Mn, and 20 ± 2 total Zn, whereas rye straw residues had a C-to-N ratio of 39:1 and contained 51 ± 11, 12 ± 2, and 16 ± 4, total Fe, Mn and Zn, respectively. The plant residues were dried at 60 ◦ C to a constant weight and ground with a kitchen mill. To allow ingestion of plant residues by earthworms, relatively small litter fragments were obtained. For this reason, the ground litter was sieved through a 1 mm sieve. Prior to the experiments, dry soil was thoroughly mixed with the litter species (20 g kg−1 dry soil) or with CaCO3 (50 g kg−1 dry soil) and pre-incubated for 5 days by adding distilled water to reach 60% WHC. After the pre-incubation, the samples amended with CaCO3 had a pH of 7.4. Earthworms, their casts and excreta Endogeic earthworms Aporrectodea caliginosa (Savigny) were collected as described for soil collection. Mature earthworms with

an individual biomass of 0.4 ± 0.1 g were selected. The earthworms were rinsed with distilled water at least four times. The gut content was removed by storing adult specimens on moist filter paper over 4 days at 6 ◦ C in the dark. The evacuation of the gut was determined visually, as the absence of dark soil particles. Then earthworms with empty guts were rinsed with distilled water and placed into the experimental soils. In experiment I, the earthworms were first placed (12 specimens per pot) into plastic pots (inner diameter 11 cm, height 10 cm) filled with soil and kept in the dark at room temperature for 1 day. The pots were covered by a 1 mm mesh to prevent earthworms from escaping. Soil samples without earthworms were kept under the same conditions. During the pre-incubation, soil moisture was maintained at 60% WHC. The WHC of the soil or soil mixture with plant residues was determined by the amount of water held in a soil sample after its saturation versus the dry weight of the sample. After that, the earthworms were removed from the pots, rinsed with distilled water and placed into Petri dishes (12 specimens per dish) in the dark for 24 h. The soil samples without earthworms were also placed into Petri dishes in amounts equivalent to expected amounts of earthworm casts. At the same time, some of the soil samples were adjusted to 100% WHC by adding distilled water, because previously we found that the fresh casts of A. caliginosa had on average this moisture content. Thus, experiment I was started with addition of earthworm individuals or soil samples to Petri dishes. After 24 h, the earthworms were removed, whereas earthworm casts as well as soil samples with different moisture contents were kept for another 1 week. To prevent rapid water evaporation from surfaces of earthworms, casts or soils during incubation, the Petri dishes with the samples were kept at 6 ◦ C. However, throughout the experiment the moisture contents of soil and casts in dishes were controlled every day and adjusted by distilled water when necessary. During the pre-incubation stage of experiment II—the experiment focused on testing micronutrient availability to plants—the earthworms were kept in black plastic pots 1 l (three individuals per pot) each filled with 1 kg soil (dry weight) at room temperature in the dark for 4 weeks. In the experiment, the soil consisted of three treatments: soil without additions, soil amended with rye straw residues, and soil amended with clover residues. The earthworms were present in soils mixed with plant residues only. The moisture of soil samples was maintained at 60% WHC every second day. For incubation with plants, earthworms were removed from pre-incubated soils and the soils thoroughly mixed in each pot (irrespective of earthworm presence) prior to seed planting. For excreta collection, earthworms with empty guts were rinsed with distilled water and placed into four Petri dishes with 20 ml distilled water (3 specimens per dish) for 24 h at 6 ◦ C in the dark. After that, the earthworms were removed from the Petri dishes. The excreta from Petri dishes were collected for further micronutrient analysis and calculation of micronutrient loss from one earthworm specimen over 24 h.

Plant material and growth conditions Cucumber seeds (Cucumis sativus L. cv. Semcross) were used in our experiments. Surface-sterilized seeds were germinated between two sheets of cellulose paper moistened with distilled water for one day in the dark at 28 ◦ C, and then transferred to the pre-incubated soils (experiment II). Three plants were grown in each 1 l black plastic pot. The soil cultures were maintained with a day/night regime of 16/8 h under light intensity 7000 lx at the plant level provided by DRLP 400 W fluorescent bulbs (Lisma, Russia). The temperature was 18–20 ◦ C in the dark and 24–26 ◦ C in the light. During the incubation the soils were adjusted by distilled water to 60% WHC every day.

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Shoot biomass After sampling the cucumber shoots were dried to a constant weight at 65 ◦ C, and the dry weight determined. Xylem sap collection and analyses The xylem sap of cucumber plants was taken using a pipette 2 h after the stalks were cut at a height of 1 cm from the soil surface as described by Noguchi et al. (2001), Liang et al. (2006). The sap was sampled close to the time of micronutrient measurements. The collected samples of known volume were transferred to plastic bottles, thoroughly mixed and their micronutrient concentrations measured using atomic absorption spectroscopy as described below. Xylem sap translocation rates of micronutrients were calculated (the sap micronutrient concentration × exudate rate) to determine actual micronutrient availability in soil.

Table 1 ANOVA table for the effect of earthworms (Aporrectodea caliginosa), plant species addition (Cecale cereale, Trifolium pretense), CaCO3 addition, soil moisture (60% and 100% WHC), and time of exposure (samplings after 0, 1 and 8 days) on concentrations of water-soluble Fe, Mn, and Zn in soil with error d.f. 42. Dependent variable

Independent variable

d.f.

F-value

P-value

Fe concentration

Earthworms (E) Plant species (PS) CaCO3 (Ca) Soil moisture (SM) Sampling (SA) E × SA PS × SA Ca × SA SM × SA

1 2 1 1 2 2 4 2 2

154.36 20.14 9.32 2.61 38.97 102.02 3.32 11.50 1.62

<0.001 <0.001 0.004 0.114 <0.001 <0.001 0.014 <0.001 0.204

Mn concentration

Earthworms (E) Plant species (PS) CaCO3 (Ca) Soil moisture (SM) Sampling (SA) E × SA PS × SA Ca × SA SM × SA

1 2 1 1 2 2 4 2 2

5.55 20.86 107.56 0.62 5.37 5.76 10.36 0.13 0.04

0.023 <0.001 <0.001 0.804 0.006 0.005 <0.001 0.881 0.964

Zn concentration

Earthworms (E) Plant species (PS) CaCO3 (Ca) Soil moisture (SM) Sampling (SA) E × SA PS × SS Ca × SA SM × SA

1 2 1 1 2 2 4 2 2

22.62 7.30 225.09 3.09 8.32 14.21 8.42 3.52 15.06

<0.001 0.002 <0.001 0.086 0.001 <0.001 <0.001 0.034 <0.001

Chemical analyses To prevent a decrease in micronutrient mobility induced by soil drying, which was observed in previous experiments, fresh cast or soil samples were used for the water extraction. The watersoluble forms of micronutrients were extracted using distilled water (1:10 m/v, shaken for an hour) around the same time as micronutrient measurements. Then, the samples were centrifuged at 3800 × g for 10 min resulting in a supernatant solution containing water-soluble micronutrients. In soil solutions and fresh xylem sap, micronutrients (Fe, Mn, and Zn) were directly quantified using electrothermal atomic absorption spectrometry (Bings et al. 2010). An atomic absorption spectrometer model MGA 915 (Lumex, Russia) with Zeeman background correction system was used for all measurements. Specifically, a Fe and Mn hollow cathode lamp (Cortec, Russia), operating at 17 and 13 mA, respectively, and a Zn high frequency lamp (Lumex, Russia) operating at 26 V were employed. All determinations were accomplished using the Fe 371.2 nm, Mn 278.5 nm and Zn 213.9 nm resonance lines. Graphite tubes (Schunk, Germany) were fitted. Pure argon (Lentehgaz, Russia) was used as a protective gas throughout. In dry plant residues, total micronutrients were also determined by atomic absorption spectroscopy after a dry digestion procedure. Nitrate ions were extracted from the fresh soil samples with a 0.05% solution of K2 SO4 ; ammonium ions, with a 0.01 N KCl solution, followed by filtration and measurement of NH4 + and NO3 − colourimetrically (Arinushkina 1970). Soil micronutrient, NH4 + and NO3 − concentrations were expressed on a dry soil weight basis. Statistical analyses All statistical analyses were performed using IBM SPSS Statistics, version 19. Data were subjected to analysis of variance procedures (ANOVA, type III). For experiment I, ANOVA incomplete factorial design (without contrasts) was performed to test significance of main treatment effects, with treatments (earthworm influence “earthworm”, plant species addition “plant species”, CaCO3 addition “Ca”, soil moisture, time of exposure “sampling”) and the interaction between the treatments and time. Statistical analyses were conducted on the average values of the three sampling dates, which were calculated separately for the four replicates of each treatment. For experiment II, an ANOVA incomplete factorial design (without contrasts) was applied to test the significance of main treatment effects, with the factors “earthworms” and plant species addition “plant species”. The experiment consisted of four independent measurements of the parameter investigated. The residuals of each model were analysed to test for normality of variance. The data were inspected for homogeneity of variance (Levene test).

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Significant effects are given in bold.

Means were separated, using Student–Newman–Keuls (SNK) test at P < 0.05. To test whether available soil micronutrients and nitrogen, xylem sap micronutrients, and shoot DW were correlated, Pearson coefficient (r) was determined. Results Experiment I The earthworms, addition of plant residues or CaCO3 significantly affected soil water-solubility of Fe, Mn and Zn, whereas soil moisture did not significantly affect the mobility of the micronutrients (Table 1 and Fig. 1). In addition, treatment effects on metal mobility depended on the interaction of the factors with sampling time (Table 1). The cast water-solubility of the micronutrients was generally higher compared with bulk soils. Overall, in one-day-old casts, water-soluble Fe and Zn increased by 3–13 and 1.3–3 times, respectively, in comparison with one-day-old bulk soils depending on treatment combination and soil moisture (Fig. 1a and c). The earthworm-mediated increase in water soluble Mn was found in one-day-old casts from soil samples amended with clover (on average by 2 times) and straw residues (on average by 5 times) although no increase was found in one-day-old casts from untreated soil (Table 1 and Fig. 1b). The daily loss of Fe, Mn and Zn due to excretion from the body surface and the digestive tract of one A. caliginosa individual was determined to be 64 ± 4, 23 ± 4, and 7 ± 2 (mean ± SD): (ng) of Fe, Mn and Zn, respectively. Hence, the daily water-soluble Fe, Mn and Zn input of 12 specimens placed in a Petri dish for cast sampling was calculated as 768, 276 and 84 (ng g−1 soil), respectively. Thus, the amounts were many times less than the earthworm-mediated increase in water-soluble micronutrients detected in one-day-old casts. No earthworm mortality was observed after first sampling.

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Fig. 1. The concentrations (␮g g−1 dry soil) of water-soluble Fe (a), Mn (b) and Zn (c) in soils and casts of Aporrectodea caliginosa. Data are expressed as mean ± SD. Values sharing the same letter are not significantly different (Student–Newman–Keuls test, P < 0.05, n = 4).

After 8 days’ incubation, the concentrations of Fe in casts remained significantly higher (by 2–7 times) relative to the bulk soil samples from which they were produced and subsequently moistened both to either 60% or 100% WHC (Fig. 1a). However, compared to the one-day-old cast, the Fe water solubility of eight-day-old casts from untreated soil and from soil with clover decreased by 1.3–2 times, however there was no change in these casts for soils with added CaCO3 or straw. Water-soluble Mn in one-dayold casts from straw-treated soil did not change compared to the first sampling and remained 6.8 times higher than that in bulk soils with both 60 and 100% WHC (Fig. 1b). Throughout the experiment, the cast water-soluble Mn from soil with clover

decreased by an average of 3.2 times. At the end of the experiment, amounts of cast water-soluble Mn from soil with clover were somewhat higher than the bulk soil moistened to 60% WHC and were less compared to the bulk soil moistened to 100% WHC (Fig. 1b). Amounts of water-soluble Zn in casts with straw and with clover were on average 3 times higher than that in bulk soils at 60% WHC (Fig. 1c). On the eighth day, zinc mobility in casts from soil amended with straw remained higher compared to the bulk soil at 100% WHC. In addition, no differences were found in Zn mobility in the treatment with clover for casts and bulk soil at 100% WHC. For the water-soluble Mn and Zn, the analysis did not show any significant differences in the treatment with CaCO3

N.P. Bityutskii et al. / Pedobiologia 55 (2012) 93–99 Table 2 ANOVA table for the effect of earthworm (Aporrectodea caliginosa) worked soil and plant species addition (Cecale cereale, Trifolium pratense) on the soil properties (extractable Fe, Mn, Zn, NO3 –N, NH4 –N) and on the plant (Cucumis sativus) properties (xylem sap Fe, Mn and Zn, xylem exudate rate, shoot DW) with error d.f. 16. Dependent variable Soil properties Fe concentration Mn concentration Zn concentration NO3 –N concentration NH4 –N concentration Plant properties Sap Fe content Sap Mn content Sap Zn content Xylem exudate rate Shoot DW

Independent variable

d.f.

F-value

P-value

Earthworms (E) Plant species (PS) Earthworms (E) Plant species (PS) Earthworms (E) Plant species (PS) Earthworms (E) Plant species (PS) Earthworms (E) Plant species (PS)

1 2 1 2 1 2 1 2 1 2

9.23 29.33 12.61 6.27 0.833 1.42 2.96 268.84 105.49 11.58

0.008 <0.001 0.003 0.010 0.375 0.269 0.105 <0.001 <0.001 0.001

Earthworms (E) Plant species (PS) Earthworms (E) Plant species (PS) Earthworms (E) Plant species (PS) Earthworms (E) Plant species (PS) Earthworms (E) Plant species (PS)

1 2 1 2 1 2 1 2 1 2

16.46 1.43 26.02 47.81 0.160 33.12 0.004 22.95 28.90 97.88

0.001 0.268 <0.001 <0.001 0.695 <0.001 0.952 <0.001 <0.001 <0.001

Significant effects are given in bold.

for bulk soils and casts at the end of the experiment (Fig. 1b and c). Experiment II Soil analysis In soils not worked by earthworms, the addition of straw residues and especially of clover residues increased water-soluble Fe and Mn by 1.5–2.9 times compared to the control without additions, while water-soluble Zn remained not affected by plant residue addition (Tables 2 and 3). The presence of earthworms for 4 weeks increased water-soluble Fe and Mn in straw amended soil (+43 and +111%, respectively), and did not affect mobility of the micronutrients for soil amended with clover residues. Addition of clover residues alone induced the greatest increase in water-soluble Mn. Water-soluble Zn showed no significant difference among soils worked and not worked by earthworms (Tables 2 and 3). At the end of the pre-incubation, most of the mineral nitrogen in untreated soil was in the form of NO3 − rather than

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NH4 + (Table 3). The amount of soil NO3 –N decreased (i.e. Nimmobilization occurred) in soils treated with clover and especially with straw (Tables 2 and 3). The effect was consistent regardless of the presence of earthworms. On the other hand, the amount of NH4 –N was highest in the earthworm treatments (especially with clover) and lowest in the soils without earthworm treatments (Table 3). No earthworm mortality was observed at the end of the pre-incubation. Plant performance Earthworms and the addition of plant residue type significantly affected shoot dry weight of cucumber (Tables 2 and 3). Four weeks after pre-incubation with or without earthworms, shoot DW of cucumber decreased 3.8 times in soil with straw but not worked by earthworms and 1.8 times in earthworm worked soil with straw compared to the untreated control (Table 3). Clover addition did not affect cucumber shoot dry weight. However, significant enhancement in cucumber shoot biomass was observed in microcosms with clover after pre-incubation with earthworms compared to both the untreated control and clover-amended soil without earthworms: +22% and +35%, respectively (Table 3). Generally, addition of plant residues significantly affected xylem exudate rate and xylem sap contents of all micronutrients, excluding Fe (Table 2). The earthworms significantly affected Fe and Mn transport in xylem sap and did not affect xylem sap exudation rate (Table 2). For xylem sap Zn, the analysis did not show any significant differences between soils pre-incubated with or without earthworms (Tables 2 and 3). The depression in growth of cucumber seedlings by straw addition was accompanied by a decrease in xylem sap Fe, Mn and Zn (Table 3). However, after the straw amended soil was pre-incubated with earthworms, the sap Mn and Fe contents increased by 2.4 and 9.7 times, respectively, with respect to soil with straw but not worked by earthworms. Clover addition generally did not affect xylem sap contents of micronutrients with the exception of manganese, the content of which decreased compared with untreated soil (Table 3). However, this decrease was eliminated upon pre-incubation with earthworms. Shoot DW strongly correlated (r = 0.710, P < 0.001) with extracted mineral N (NH4 + + NO3 − ) detected in pre-incubated soils. In addition, extracted mineral N correlated with both sap Mn (r = 0.815, P < 0.001) and Zn (r = 0.456, P = 0.04). Discussion Species of the Aporrectodea genus have demonstrated the ability to both increase (Lukkari et al. 2006) and decrease metal mobility and availability in amended soils (Zorn et al. 2005; Lukkari et al. 2006). We found that A. caliginosa generally made iron, manganese

Table 3 Concentrations of extractable Fe, Mn, Zn, NH4 –N, and NO3 –N in soils with different treatment combinations after pre-incubation with or without earthworms (Aporrectodea caliginosa) for 4 weeks. Shoot DW and Fe, Mn, Zn translocation with xylem exudates in Cucumis sativus 4 weeks later after the end of the pre-incubation. Data are expressed as mean ± SD. Values sharing the same letter are not significantly different (Student–Newman–Keuls test, P < 0.05, n = 4). Treatment property

Soil

Soil property after pre-incubation for 4 weeks 1.3 ± 0.1a Fe, ␮g g−1 35 ± 3a Mn, ng g−1 67 ± 7a Zn 10.5 ± 0.7b NH4 –N, ␮g g−1 32.0 ± 4.4d NO3 –N Plant property 4 week later after the end of the pre-incubation −1 −1 Micronutrient input with xylem exudates, ng plant h Fe 3.0 ± 0.4b Mn 9.8 ± 0.9c 1.5 ± 0.4b Zn 52 ± 10bc Xylem exudate rate, ␮l plant−1 h−1 226 ± 38c Shoot dry weight, mg plant−1

Soil + clover

Soil + straw

Soil + clover treated with earthworms

Soil + straw treated with earthworms

4.0 88 60 9.4 6.3

± ± ± ± ±

0.3b 8c 7a 0.7ab 0.3bc

3.5 54 56 8.0 2.0

± ± ± ± ±

0.7b 9b 15a 0.8a 0.1a

4.1 89 50 26.6 8.4

± ± ± ± ±

0.5b 9c 6a 1.6d 1.2c

5.0 114 58 16.4 3.3

± ± ± ± ±

0.2c 10d 3a 1.4c 0.5ab

2.7 6.4 1.9 97 204

± ± ± ± ±

1.0b 1.6b 0.5b 19d 29c

0.7 1.9 0.6 18 60

± ± ± ± ±

0.1a 0.7a 0.2a 6a 14a

3.0 11.2 2.0 74 275

± ± ± ± ±

0.2b 2.2c 0.2b 10cd 13d

6.8 4.6 0.7 42 119

± ± ± ± ±

0.9c 1.2b 0.2a 3b 20b

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or zinc in its casts more water soluble compared to the bulk soil. Moreover, the effects were observed in soils with different initial micronutrient mobility induced by plant residues or CaCO3 addition (Table 1 and Fig. 1). The earthworm-mediated increase in metal mobility was especially pronounced in one-day-old casts produced from soil with straw residues as well as in earthworm-worked soils also amended with straw residues (Tables 2 and 3). Straw contained either less micronutrient (see section ‘Materials and methods’) or induced more immobilization during incubation due to its lower C-to-N ratio compared to clover residues (Fig. 1). Perhaps, this is why the earthworm effect on metal mobility (especially on Fe and Mn mobility) in straw amended soil was more marked than in soil amended with clover. Increases in mobile metal species due to earthworm activity are known to mainly relate to the high organic matter and carbon contents of earthworm excreta. A significant correlation has been found between the effect of E. fetida in increasing dissolved organic carbon (DOC) and the concentration of water extractable metals (Wen et al. 2004). DOC can form complexes with various metal ions, and these complexes are more soluble and readily taken up than free metal ions (Norvell 1972; Prasad et al. 1976). The saturated moisture regime of cast material is another factor which could affect changes in mobility of Fe, Mn and Zn in the presence of earthworms. In contrast to aerated soils, the earthworm gut is relatively moist and free of detectable O2 (Drake and Horn 2007). In reducing environments, such as waterlogged soils, Fe (II) species and oxides dominate and are more mobile compared with Fe (III) species and oxides (Lindsay 1995). Manganese availability to plants can be increased by subjecting a soil to reducing conditions by high water saturation or temporary water logging (Piper 1931), whereas extractable zinc can be decreased (Haldar and Mandal 1979). However, in one-day-old casts with plant residues, a significant increase in both Fe and Zn mobility was observed even in comparison with a saturated soil moisture regime (Fig. 1a and c). This suggests that a large increase in micronutrient mobility caused by the passage of soil through the earthworm digestive tract is probably not related to an earthworm-mediated increase in cast moisture. Pre-incubation of soils amended with clover or straw with living earthworms induced an increase in both cucumber shoot biomass and translocation rate of micronutrients (Mn, Zn) in xylem sap compared to the soils not worked by earthworms (Tables 2 and 3), suggesting that earthworms can impact micronutrient availability to plants. Although earthworms induced an increase in soil mobile Fe and Mn (Tables 2 and 3), the increase in micronutrient input into the xylem as well as the increased growth of cucumber may be related to factors other than earthworm-induced changes in water-solubility of soil micronutrients. Extracted soil mineral N (NH4 + + NO3 − ) has been shown to be linearly related to the shoot DW as well as both Mn- and Zn-input with cucumber xylem exudates, indicating that earthworms can also indirectly prime changes in micronutrient nutrition of plants by affecting N-mineralization in soil. Mineral nitrogen can increase root growth and therefore micronutrient uptake. The earthworm-mediated effects were more pronounced in the presence of straw—a plant residue with low C-to-N ratio. The straw addition to soil without earthworms induced the greatest N-immobilization in soil and decrease in plant growth (Table 3). Both the N-immobilization and the decreased shoot growth were alleviated in the presence of earthworms (Table 3). According to Brown et al. (2004), the positive effect of an earthworm species on plant growth is mainly due to an increase in mineralization. Eisenhauer and Scheu (2008) found increased uptake of mineral N in soil and enhanced plant growth, in particular that of grasses, in the presence of earthworms. Previously we showed that earthworm excreta alone, without soil passage through earthworm intestines, can exert a stimulating effect on nitrogen mineralization and produce long-term, cumulative

effects (Bityutskii et al. 2007). However, other potentially influential mechanisms must also be considered (Laossi et al. 2010). Excreta analysis has shown that daily total metal excretion of A. caliginosa, through both the body surface and the digestive tract, was much less than the amount of metal released from casts over 24 h, suggesting that earthworms can prime micronutrient turnover resulting in the appearance of mobile and available micronutrients in soil. Until now, most studies have described priming effects as interactions with the organic pools of various availabilities on the one hand, and living and dead soil organic matter on the other (Kuzyakov 2010). So, the priming effects (PEs) are commonly exemplified by intensified SOM turnover. In the case of micronutrients, however, the acceleration in their mobility can be caused by both increased SOM decomposition and increased release of micronutrients originating from the soil mineral phases. Microbial biomass drives PEs (Blagodatskaya and Kuzyakov 2008), and the microbial activity may affect (1) the acceleration of SOM decomposition and therefore a release of SOM-bound micronutrients and (2) a forming of metallofores. The latter can chelate soil metals, influencing their solubility (Whiting et al. 2001). As has been demonstrated (Cheng and Wong 2002), organically bound metals in earthworm excreta can be released into the soil solution and thus become available for plant uptake through the rapid microbial decomposition of organic compounds in earthworm excreta. A declining trend in earthworm-mediated Fe mobility with incubation time was observed for all treatment combinations, excluding soil amended with CaCO3 (Fig. 1a). However, even after eight-day-incubation water-soluble Fe in casts remained much higher than that in bulk soils. The mobility of Mn and Zn was less marked than what was observed for Fe (Fig. 1b and c). Hence, the duration of the earthworm-induced priming effect expressed as an increase of cast metal mobility was prolonged at least for 1 week after the casts were produced. An increase in input of micronutrients (Mn, Zn) into xylem exudates of cucumber seedlings in earthworm-worked soil detected 4 week after removing of adult earthworms (Table 3) is also evidence of a prolonged PE stimulation by earthworms with respect to soil micronutrient availability. Conclusions Earthworms can drive a prolonged acceleration of the watersolubility of micronutrients (Fe, Mn and Zn) found in casts relative to the bulk soils from which they were produced. The duration of an earthworm-induced priming effect expressed as an increase of cast metal mobility was prolonged at least for 1 week after the casts were produced. Earthworm working of soil affected the performance of cucumber seedlings in a predominantly beneficial way. Both earthworm-mediated increase in shoot biomass and translocation of micronutrients (Mn, Zn) into xylem sap were measured 4 weeks later after the earthworms were removed. Acknowledgements We would like to thank Stefan Scheu and three anonymous reviewers for their valuable comments on the manuscript. This work was supported by the Russian Ministry of Education and by the Russian Foundation for Basic Research. References Amador, J.A., Görres, J.H., 2005. Role of the anecic earthworm Lumbricus terrestris L. in the distribution of plant residue nitrogen in a corn (Zea mays)-soil system. Appl. Soil Ecol. 30, 203–214. Arinushkina, E.V., 1970. Handbook on the Chemical Analysis of Soils. Moscow Gos. Univ, Moscow [in Russian]. Bings, N.H., Bogaerts, A., Broekaert, J.A.C., 2010. Atomic spectroscopy: a review. Anal. Chem. 82, 4653–4681.

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