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Application of leaves to induce earthworms to reduce phenolic compounds released by decomposing plants
European Journal of Soil Biology 75 (2016) 31e37
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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Application of leaves to induce earthworms to reduce phenolic compounds released by decomposing plants Yan-Meng Bi a, 1, Gei-Lin Tian b, c, 1, Chong Wang a, Cheng-Li Feng a, Yi Zhang a, Lu-Sheng Zhang b, **, Zhen-Jun Sun a, * a b c
College of Resources and Environmental Science, China Agricultural University, Beijing 100193, PR China College of Horticulture, China Agricultural University, Beijing 100193, PR China Department of Botanical Garden Engineering, Heze University, Shandong Province, Heze 274000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 October 2015 Received in revised form 15 April 2016 Accepted 18 April 2016
The negative impacts (allelopathic effects) of phenolic compounds (PCs) in soil on plant growth and microbial communities have recently received considerable attention. Similarly, there have been several recent studies on the effects of microbes on the degradation of PCs. Because of the profound effects of their feeding and burrowing activity, earthworms should have significant effects on PCs degradation; however, few studies have examined this potential effect, and particularly, factors that may affect the course of degradation. We tested different earthworm species and density, different conditions (sterilization or not) of mixture of soil and plant residual, and different source of PCs to evaluate the capacity of earthworms to accelerate PCs degradation. In addition, earthworm behavior experiments were set up to test whether adding plant leaves can stimulate earthworm feeding activity. The results showed that native earthworms exhibited a higher capacity than compost earthworms for degrading PCs; furthermore, when the number of Metaphire guillemi reached 300 individuals m2 in our experimental units, the PCs decreased most quickly, and the residual PCs concentration was 105 mg (p-coumaric acid)g1 (dry soil) less than that of control group. The source of PCs also affected their degradation rate, as PCs derived from leaves seemed to degrade more quickly. The results of our experiment suggested that earthworms avoid feeding on phenolic acids, but can be induced to do so by adding leaves to the substrate. These results indicate that earthworm activity can accelerate the degradation of total PCs, and that this may be further facilitated by incorporation of organic matter, which may be used to alleviate allelopathic effects of PCs in soil. © 2016 Elsevier Masson SAS. All rights reserved.
Handling Editor: C.C. Tebbe Keywords: Earthworm Phenolic compounds Allelochemical Trophic behavior
1. Introduction Phenolic compounds (PCs) are the most important and common plant allelochemicals in soil ecosystems. They are chemical compounds consisting of a hydroxyl group bound directly to an aromatic hydrocarbon group [1], which are the most widespread secondary plant metabolites known [2,3]. PCs are released into the environment through foliar leachate, root exudation, residue decomposition [4,5], among which aboveground and belowground
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (L.-S. Zhang), (Z.-J. Sun). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejsobi.2016.04.007 1164-5563/© 2016 Elsevier Masson SAS. All rights reserved.
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litter are the dominant pathways [3,6]. Some PCs are phytotoxic, and can inhibit plant growth through interaction with the mitochondrial membrane and impairment of dark respiration and ATP synthesis [4,5]. Similarly, phytotoxicity can disrupt the microbial community balance by modifying the population and community structure in the rhizosphere [7,8]. According to Blum and Gerig (2005), phenolic acid treatment of seedlings inhibits transpiration, water utilization, leaf area, and the absolute and relative rates of leaf expansion [9]. Several management approaches have been taken to alleviate allelopathic stress caused by PCs on plants. For example, activated charcoal has been used to adsorb accumulated phytotoxic chemicals with the result of improved growth and yield in strawberry [10], several leafy vegetables, and some ornamentals [11,12]. Similarly, several studies have focused on the application of microorganisms that can biodegrade phenolic acids and colonize the
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rhizosphere of plants to alleviate the stress of autotoxic allelochemicals exuded by crop roots or left by plant residues [13e16]. Although several measures to degrade PCs have been tested and shown to be successful, a major component of natural and agricultural ecosystems, earthworms [17], has received little attention. Earthworms form a major part of the soil decomposer macrofauna, and play an important role in temperate ecosystems [18] and some tropical ecosystems. They can alter soil structure, accelerate the nutrient cycling processes in soil, and significantly impact microbial communities [19e23]. In addition, as an ecosystem engineer, earthworms can promote the degradation of many compounds in soil. Several studies have examined the direct or indirect relationship between earthworms and some toxic substances. For example, studies have examined the effect of earthworms on altering mineralization and biodegrading atrazine [24,25], as well as earthworm mediated enhancement of the removal of dichloro-diphenyl-tricgloroethane or polycyclic aromatic hydrocarbons [22,26]. Thus, earthworms may have the potential to degrade single cyclic molecules such as phenolic acid or other simple PCs with simpler structures than the complex substances mentioned above. Although Butenschoen et al. (2009) studied the fate of catechol affected by earthworms, they did not examine the influence of earthworms on total PCs, nor did they focus on the basic conditions for earthworms to degrade PCs [18]. Furthermore, they only used endogeic earthworms in their experiments, and thus is it unclear whether the different feeding and burrowing activities of epigeic or anecic earthworms might be more (or less) efficient at degradation of PCs. So, it is important to evaluate whether epigeic earthworms and anecic earthworms can accelerate the decomposing of PCs. The objectives of this paper are: first, to evaluate the ability of earthworms to accelerate the degradation of PCs released by decomposing plants residue (strawberry was used as our model plant); second, to investigate influence factors for earthworms to biodegrade phenolics; and finally, to examine the trophic behavior of earthworms in relation to phenolic acid.
15 cm depth) with a small hole in the bottom were filled with 3 kg MSRL and 500 mL deionized water. A plastic net (±1 mm) was placed in the bottom of every pot to prevent earthworms from escaping. All pots were placed in an artificial climate incubator with temperature maintained between 15 and 20 C. All pots were irrigated every 5 days with 100 mL deionized water. Four experiments were conducted to test the influence of earthworm species, density of M. guillemi, sterilization and the source of PCs on degradation rate of PCs (Table 1). In the first experiment, there were 3 treatments: one with 6 individuals of M. guillemi (M), 30 individuals of E. fetida (E) (this resulted in approximately equal biomass for each species) and a control without earthworms (CK). In the second experiment, there were six densities of M. guillemi added to the pots: treatments consisted of 0, 3, 6, 9, 12, and 15 individuals per pot (marked as Z, T, SI, N, TW and F respectively). In the third experiment, there were four treatments: sterilized (autoclaved at 101 kpa, 121 C, 30 min, twice) MSRL with M. guillemi (SM), sterilized MSRL without earthworms (S), ordinary MSRL with M. guillemi (OM), and ordinary MSRL without earthworms (O). In order to verify whether most of the microorganisms in sterilized MSRL have been eliminated, we used dilution-plate method on LB (lysogeny broth) (10 g MSRL and 100 mL sterile normal saline to obtain extraction) to test the effect of sterilization (Fig. 1). The last experiment included six treatments: mixture of soil and ground leaves with M. guillemi (ML), E. fetida (EL) and without earthworm (L), and mixture of soil and ground roots with earthworm M. guillemi (MR), E. fetida (ER), and without earthworm (R). Each treatment in each experiment mentioned above had 4 replicates. All of the experiments lasted for 30 days. The avoidance/selection behavior of earthworms was studied in
Table 1 Earthworms added to pots (Initial) and recovered after 30-day experiments (Final). Experiment
Treatment
E1
M E CK Z T SI N TW F SM S OM O ML EL L MR ER R
2. Materials and methods 2.1. Soil and earthworms Soil was obtained from a continuously cropped experimental field at China Agricultural University in Beijing, where the strawberry cultivar ‘Benihoppe’ (Fragaria ananassa Duch) had been planted for 3 years. The soil was air-dried, sieved (2 mm), and thoroughly mixed with smashed roots and strawberry leaves, creating an experimental mixture of soil (96% mass ratio), root (2% mass ratio) and leaf (2% mass ratio) (MSRL) with the following properties: pH (H2O:soil, 2.5:1) of 7.82, 2.25% soil organic matter, 0.12% total nitrogen, 50.38 mg kg1 available phosphorus (Olsen-P), 255.23 mg kg1 available potassium (NH4OAc-K). Earthworms Metaphire guillemi [27] (anecic) native to the experimental fields of China Agricultural University were collected at the same location where the soil was obtained. Compost earthworms Eisenia fetida (epigeic) were purchased from Beijing Lvhuan Kemao Company. Both species of earthworm were placed in MSRL for 1 week to acclimate to the matrix and replace their gut contents with experimental soil. Adult earthworms with fresh weight of 2.4e2.5 g for M. guillemi, and 0.4e0.5 g for E. fetida were chosen to be used as test animals. Prior to fresh weight determinations, the earthworms were washed and blotted with filter paper [23]. 2.2. Experimental procedure Plastic pots (top diameter 19 cm, bottom diameter 18 cm and
E2
E3
E4
Earthworm abundance (Individuals per pot) Initial
Final
6 30 0 0 3 6 9 12 15 6 0 6 0 6 30 0 6 30 0
6 31.00 0 0 3 6 9 12 14.50 6 0 6 0 6 31.00 0 6 31.75 0
± 1.41
± 0.58
± 0.82
± 1.71
Earthworm survival rates (%)
100 103.3 ± 4.7 e e 100 100 100 100 96.7 ± 3.8 100 e 100 e 100 103.3 ± 2.7 e 100 105.8 ± 5.7 e
Values are mean (standard deviation is zero) or mean ± standard deviation of 4 replicates. Abbreviations: E1: M: treatment with M. guillemi; E: treatment with E. fetida (E); CK: Control group. E2: Z: treatment without earthworm; T: treatment with three individuals of earthworm; SI: treatment with six individuals of earthworm; N: treatment with nine individuals of earthworm; TW: treatment with twelve individuals of earthworm; F: treatment with Fifteen individuals of earthworm. E3: SM: sterilized matrix added with M. guillemi; S: only sterilized matrix; OM: ordinary matrix added with M. guillemi; O: only ordinary matrix. E4: ML: matrix composed of leaves and soil with M. guillemi; EL: matrix composed of leaves and soil with E. fetida; L: matrix composed of leaves and soil; MR: matrix composed of roots and soil with M. guillemi; EL: matrix composed of roots and soil with E. fetida; R: matrix composed of roots and soil.
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Fig. 1. Picture of plates to test the effect of sterilization. The plates of the first row were spread with un-sterile MSRL exaction and the plates of the second row were spread with sterile MSRL exaction. The picture was taken three days after inoculation.
a 6-chamber earthworm-behavior device (Fig. 2). M. guillemi were chosen as the experimental subject for this work based on results of the previous experiments and indication that M. guillemi has high efficiency for decomposing PCs. This experiment was modified from work performed by Schaefer [28], and two experiments were included to test earthworm behavior. The first experiment examined whether earthworms tend to approach or avoid phenolic acids, with p-coumaric acid (PA) chosen as a representative phenolic acid because of its dominant concentration in strawberry cropped soil [29]. 1.8 g PA was weighed and dissolved in 10 mL methanol, and the solution was added to 500 mL deionized water and mixed thoroughly. The mixed solution was added to a watering can and sprayed on 9 kg soil equally (the concentration of PA was approximately 200 mg g1 in the soil). Soil mixed with PA was added to chambers 1, 3, and 5 of the device. The same soil without
PA (same volume methanol and water were added to standardize) was added to chambers 2, 4, and 6 of the device. In total, 48 individuals of M. guillemi were placed in the center of the device, and all of the dividers were removed to allow the earthworms to crawl freely. The number of earthworms in each chamber was counted on the 1st, 3rd, 5th, 7th, and 9th day, and 20 g soil samples were obtained to analyze the concentration of total PCs. Three parallel trials were conducted at the same time to reduce variability in results. Because the first experiment showed M. guillemi to avoid PA, the second experiment was set to test whether additional organic matter in the form of plant leaves could overcome the earthworm's avoidance of PA. In this experiment, three treatments were set: soil without PA or leaf (CK), soil with PA (200 mg dwg1) (P), and soil with PA and leaf (PL). These mixtures were added to 3 chambers of the device, and 24 earthworms were added to the center of the device. The dividers were removed to allow earthworms to crawl freely, and after 24 h, we counted the earthworms in every chamber. The PA was added to the soil using the same method as that in the first experiment. Ten parallel trials were conducted at the same time to reduce variability in results. 2.3. PCs extraction and analysis
Fig. 2. Earthworm-behavior device. The device was designed based on Schaefer (2004) [28], and has been slightly modified. In this device, the boards and the center column can be raised. Before the experiment, all boards and the center column were inserted in the device, forming 7 chambers. Earthworms were placed in the chamber and different substrates were put into the remaining chambers. We then raised all the boards and center column so earthworms could select their preferred environment. (The picture was done with Adobe Illustrator CS5).
PCs were extracted using the modified method of Martens [30]. First, 25 g MSRL was extracted with 25 mL 1 M NaOH and kept at room temperature for 24 h. Then the suspension was centrifuged at 8000 g for 10 min, and the liquid supernatant was separated. The pH of the supernatant was adjusted to 2.5 with 12 M hydrochloric acid (HCl) and left to stand for 2 h. The suspension was centrifuged at 8000 g for 10 min. The supernatant was stored at 4 C and prepared for analysis. Total PCs concentrations were measured using the FolinCiocalteu assay [31]. Total PCs content was standardized against PA and expressed as milligrams per liter of PA. The linearity range for this assay was determined as 0e10 mg mL1 PA (R2 ¼ 0.9990), giving an absorbance range of 0e0.628 AU. The solution of extracts in the last experiment of PCs degradation was filtered through a 0.22 mm microporous membrane filter for subsequent highperformance liquid chromatography (HPLC) analysis (model 1260,
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Agilent, Waldbronn, Germany). Phenolic acid separation was performed by gradient elution high-performance liquid chromatography (GEHPLC) with an HPLC quaternary pump model G1311A, a UV detector model G1314A, and a chromatographic column: XDBC18 (4.6 mm 250 mm). The detector was set to 280 nm, and the flow rate to 1 mL/min. The injection volume was 20 mL, and the column temperature was maintained at 25 C. Methanol (A) and acetic acid solution (pH ¼ 2.80) (B) were used as mobile phases with a gradient elution B: 70% (0 min)/50% (15 min)/30% (16 min)/0 (30 min)/end (30 min) [29]. 2.4. Statistical analysis The means and standard deviations of PCs concentrations and earthworm number were calculated, and analysis of variance (ANOVA) was performed on the data using SPSS 19.0. The interaction effect between PCs source (derive from leaves or roots) and earthworm species on the degradation of PCs was also processed with this software and double factor analysis was chosen here. Mean separations were performed by Student-Newman-Keuls multiple range tests. Differences at P < 0.05 were considered significant. The regression line between earthworm number and PA concentration was fitted by SigmaPlot 12.0. 3. Results
Fig. 3. Residual total phenolic compounds after one month experiment. Error bars represent standard deviations of 4 replicates. CK stands for control group, E stands for treatment added E. fetida and M stands for treatment added M. guillemi. Different letters denote significant differences (p < 0.05).
the number of earthworms continued to increase, and there were no significant differences among N, TW and F.
3.1. Earthworm survival At the end of the experiments, we counted earthworms in every pot (Table 1). All of the treatments containing E. fetida had more earthworms at the end of the experiment, compared to those before, but the increased earthworms were much smaller than the earthworms added. The number of earthworms in F of experiment 2 decreased to 14.50 ± 0.58, and the survival rate was 96.7 ± 3.8%. The earthworm survival rates in the rest of the experiments were 100%. 3.2. Effect of earthworms on phenolic acid degradation Residual PCs content was analyzed after 30 days of incubation of earthworms in the MSRL. The residual total PCs concentration varied among the different treatments (Fig. 3), and was highest in the control group at 508 mg (PA) g1 (dry soil). The concentrations of PCs were 457 mg (PA) g1 (dry soil) and 422 mg (PA) g1 (dry soil) in the E and M group. PCs decreased faster in both of the two experiment groups compared to the control group. Conversely, we also noted that there were no significant differences between the E and M in PCs concentration, although the PCs concentration of M was slightly less than E. 3.3. Effect of M. guillemi density on PC degradation The results demonstrated that the M. guillemi had higher capacity for degrading PCs than the E. fetida when the PCs source was leaves only (without roots) (Table 2). As such, M. guillemi was selected for the remaining experiments. After 30-day incubation, PCs concentrations were tested, and decreased with increasing numbers of earthworms in each pot (Fig. 4). The points of data can be represented by an overall regression line (y ¼ 525.5e14.10x þ 0.39x2, R ¼ 0.9697). When the number of earthworms reached 6 in one pot, the PCs concentration was significantly lower than that of Z. Similarly, when the number of earthworms reached 9, the PCs concentration decreased to 413 ± 22 mg (PA) g1 (dry soil), which was significantly lower than that of T and SI. However, the residual total PCs did not decrease as
3.4. Role of microbes in the earthworm-PCs degradation system To examine the role of microbes in the earthworm-PCs degradation system, we selected sterilized soil as a control, and the concentration of PCs was measured after 30 days (Fig. 5). In the sterilized MSRL without earthworms (S), the concentration of residual PCs was 474 ± 70 mg (PA) g1 (dry soil) and 441 ± 44 mg (PA) g1 (dry soil) in the sterilized matrix containing M. guillemi (SM). There were no significant differences between the S and MS groups. The concentration was 407 ± 41 mg (PA) g1 (dry soil) in the unsterilized soil (O), and this was significantly lower than the S treatment. Residual PCs was lowest (338 ± 43 mg (PA) g1 (dry soil)) in the unsterilized soil with M. guillemi (OM) treatment and this was significantly lower than all of the other experiments.
3.5. Effect of PCs source and earthworm species on PC degradation The concentrations of residual PCs derived from different plant parts were examined, and the degradation rate of two species of earthworms was calculated after 30 days. Using single-factor analysis of variance, we found that M. guillemi degraded PCs to a greater degree than a similar biomass of E. fetida in the leaf-added treatment (Table 2). However, results were different for the rootadded treatment groups. When roots were added as a source of PCs, we found that both species of earthworm stimulated the decomposition of PCs to the same degree (Table 2). When considering both PCs' source and earthworm species, a double-factor analysis of variance showed that earthworm species had a markedly significant effect (P < 0.01) on the degradation rate. In addition, the earthworm species and PCs source had a markedly significant interaction effect on degradation (Table 2). In addition, we found that the properties of individual phenolic acids change during leaf decomposition after earthworm inoculation. Most individual phenolic acids decreased faster, except cinnamic acid, which increased in the M. guillemi group (Fig. 6) compared to the control treatment.
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Table 2 Effects of Earthworm Species and Phenolics Source on the degradation of Phenolics. Earthworm species
Phenolics sources
Control (mg g1(PA))
Metaphire guillemi Metaphire guillemi Eisenia fetida Eisenia fetida Significance due to Earthworm Species Phenolics Source ES PS interreaction
Leaf Root Leaf Root
377.9 269.2 377.9 269.2
± ± ± ±
Treatment (mg g1(PA))
37.0 38.5 37.0 38.5
256.4 200.2 323.5 202.9
± ± ± ±
15.6 11.4 20.3 18.8
Degradation mass (mg g1(PA))
Degradation rate
121.5 ± 22.4 69.0 ± 28.6 54.4 ± 18.4 66.3 ± 21.8
0.320 0.249 0.142 0.241
± ± ± ±
0.029 0.074 0.034 0.054
a a b a
** NS **
Values with different letters for a measure significantly differed (confirmed by Student-Newman-Keulsa multiple range test; p < 0.05). NS means not significant different; **means P < 0.01(confirmed by general linear model).
100%
600
Total Phenolics (µg PA/g dry soil)
90% 550
80%
a
trans-cinnamic acid
70%
ab 500
b
450
ferulic acid
50%
p-cumaric acid
40%
syringic acid
30%
p-hydroxybenzoic acid
20%
chlorogenic acid
10%
c
c
c
60%
0% CK
E
M
Fig. 6. Percentages of standardized residual individual phenolic acids from different experimental groups. CK stands for control group, E stands for treatment added E. fetida and M stands for treatment added M. guillemi.
400
350 0
2
4
6
8
10
12
14
16
3.6. Avoidance behavior of earthworms related to phenolic acids
Earthworm Number Fig. 4. Residual total phenolic compounds after different numbers of M. guillemi placed under experimental conditions for one month. Error bars represent standard deviations of 4 replicates. The treatment of the 6 groups are marked as Z (no earthworms), T (3 individuals of M. guillemi), SI (6 individuals of M. guillemi), N (9 individuals of M. guillemi), TW (12 individuals of M. guillemi) and F (15 individuals of M. guillemi). Different letters denote significant differences (p < 0.05).
We designed earthworm behavior experiments to determine whether earthworms avoid or select soils containing phenolic acids. The earthworms in the behavior chamber were counted every day, and the number of earthworms in the compartment without PA was greater than that with PA; however, the number of earthworms in the two compartments tended to be the same over time since the concentration of PA decreased each day. The number of earthworms was not significantly different in the two treatment groups by the seventh day of the experiment (Fig. 7). Similarly, when the PA concentration decreased to 120 mg g1 (dry soil), the number of earthworms in the two chambers showed no significant
Fig. 5. The effect of earthworms and microbes on the degradation of total phenolic compounds. S stands for sterilized MSRL, SM stands for sterilized MSRL þ M. guillemi, O stands for MSRL without sterilization, and OM stands for MSRL þ M. guillemi. Error bars represent standard deviations of 4 replicates. Different letters denote significant differences (p < 0.05). Fig. 7. Number of earthworms and concentration of p-coumaric acid in compartments of different treatments Error bars represent standard deviations of 4 replicates. Different letters denote significant differences (p < 0.05).
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difference, which implied that earthworms were not sensitive to low concentrations of PA (120 mg g1 (dry soil) or less). Fig. 8 shows that the quantity of earthworms in the PA-added group was significantly less than that in the CK group. However, when leaves were added to soil, the number of earthworms did not significantly differ in the CK group, indicating that leaves can neutralize the negative effects of PA on earthworms.
4. Discussion 4.1. Effects of earthworm on the degradation of total PCs The density of earthworms in each experimental pot did not change significantly, indicating that our experimental conditions were near optimal (Table 1). The number of E. fetida earthworms increased slightly, and the increased earthworms were smaller, so it may be because that a very few of earthworms got produced during the experiment, or that E. fetida cocoons have been introduced with soil which was not excessively processed prior to use in the experiments. Yields of many crops typically decrease if they were planted at the same site year after year [31,32]. There are several possible reasons for this phenomenon, such as soil acidification, nutrient imbalance [33], and changes in biological environment and phytotoxic effects [13]. Among these, phytotoxic effects caused by allelochemicals play an important role; thus it may be critical to reduce concentrations of allelochemicals for many plants, especially for crops grown in greenhouses. In the present study, we used M. guillemi and E. fetida as models to investigate the effects of earthworms on the degradation of total PCs derived from plant (strawberry) residue, with an overall goal of reducing the concentration of allelochemicals in soil. The results showed that both M. guillemi and E. fetida effectively promote the degradation of total PCs (Fig. 3). Although these results are similar to those reported by Butenschoen et al., who concluded that earthworms increase microbial activity, and thus, mineralization of PCs, they used endogeic earthworms in their experiment [18]. We found that both M. guillemi (anecic) and E. fetida (epigeic) can accelerate the biodegradation of PCs. In our pot experiments, degradation efficiency reached its limit when the number of earthworms in the chamber reached nine
(Fig. 4). The surface area of our chambers was 0.03 m2, which means the earthworm density was approximately 300 per m2. Adding more earthworms to the same size chamber did not increase degradation efficiency, possibly because crowding increases intra-specific competition due to resource depletion [34,35]. According to a previous study, earthworm density can reach 300 per m2 in a well-managed vegetable field, however, not all of them are M. guillemi [36]; thus, it is possible to apply earthworms to a density of 300 per m2 to degrade PCs in agricultural soil, but the degradation efficiency may not reach its limit. One consideration is that the present study only examined the degradation effects of PCs in experimental pots. There are some differences between the field conditions and pot experiments, so analogous experiments should be designed in the field to assess the accuracy of earthworm density. Other than the quantity of earthworms, the effect of earthworms in accelerating PCs degradation was dependent on the species of earthworms (Table 2). Although the biomass of the two species of earthworms added to the experiment was uniform, the degradation rate of PCs differed between the two species. Both species of earthworms accelerated the degradation of PCs, and the earthworm M. guillemi showed stronger effects than earthworms E. fetida with PCs derived from leaves. However, if the soil was mixed with roots as the source of PCs, there was no difference in degradation rate between the two species of earthworms. The twofactor analysis demonstrated that earthworm species and the PCs source had a significant interaction. In addition to the species and density of earthworms, we also examined whether PCs degradation by earthworms was associated with microbial activity; our results showed that the presence of both earthworms and microbes showed the highest degradation rate (Fig. 5) among all of the treatments. This implies that earthworms can promote microbes to degrade PCs, which is consistent with the study by Natal-da-Luz et al., who examined the influence of earthworm activity on microbial communities related to the degradation of persistent pollutants [22]. It is widely acknowledged that microbes play a very important role in the degradation of PCs, but the underlying mechanisms of the interaction effect of earthworms and microbes on PCs degradation still need to be studied further.
4.2. Behavior of earthworms associated with PCs
Fig. 8. Number of earthworms in compartments of different treatments. Error bars represent standard deviations of 10 replicates. Different letters denote significant differences (p < 0.05).
Since earthworms have the ability to accelerate the degradation of PCs, it is critical to study the behavior of earthworms related to PCs. Many researchers have found that earthworms are able to select their habitat and have food preferences [37e39]. Here, we wanted to determine whether earthworms prefer PCs, and whether this preference can be induced by adding leaves to soil. Our results suggest that earthworms do not prefer PCs, especially phenolic acids, such as PA (Fig. 7); this may be because earthworms have chemoreceptors that detect the presence of unfavorable substances such as chemicals [17]. This result is similar with the study of Hendriksen et al., who concluded that high polyphenol content in matrix makes it less palatable to earthworms [40]. Moreover, our study showed that this avoidance of earthworms on phenolic acid changed when leaves were added to their habitat (Fig. 8); though leaves contain phenolic compounds, leaves can offer earthworms additional food resources, which may offset the unfavorable conditions created by phenolic acids. Many researchers have suggested that behavior of earthworms can be modified by altering habitat conditions [41,42]. Combined with our results, we conclude that earthworms can be induced to increase PCs degradation by adding a food source.
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5. Conclusions The present study clearly demonstrates that both M. guillemi (anecic) and E. fetida (epigeic) have the ability to accelerate the degradation of PCs, but that these effects differ depending on the density and species of earthworms. Conversely, although earthworms show avoidance to PCs such as phenolic acid, adding leaves as a food source can ameliorate this. Acknowledgments This work was supported by College PhD Subject Science & Technology Programme of the Education Ministry (20110008130003) and National Natural Science Foundation of China (41371305). We thank our American friend Mac A. Callaham of Center for Forest Disturbance Science, Southern Research Station for his help to review and edit the English in this manuscript and Professor Kong Chuihua of China Agricultural University for his valuable suggestions. References [1] Z.H. Li, Q. Wang, X. Ruan, C.D. Pan, D.A. Jiang, Phenolics and plant allelopathy, Molecules 15 (2010) 8933e8952. [2] J.O. Siqueira, M.G. Nair, R. Hammerschmidt, G.R. Safir, Significance of phenoliccompounds in plant-soil-microbial systems, Crit. Rev. Plant Sci. 10 (1991) 63e121. [3] S. Hattenschwiler, P.M. Vitousek, The role of polyphenols in terrestrial ecosystem nutrient cycling, Trends Ecol. Evol. 15 (2000) 238e243. [4] T.L. Weir, S.W. Park, J.M. Vivanco, Biochemical and physiological mechanisms mediated by allelochemicals, Curr. Opin. Plant Biol. 7 (2004) 472e479. [5] G. Jilani, S. Mahmood, A.N. Chaudhry, I. Hassan, M. Akram, Allelochemicals: sources, toxicity and microbial transformation in soil - a review, Ann. Microbiol. 58 (2008) 351e357. [6] C.L. Meier, K.N. Suding, W.D. Bowman, Carbon flux from plants to soil: roots are a below-ground source of phenolic secondary compounds in an alpine ecosystem, J. Ecol. 96 (2008) 421e430. [7] C.H. Kong, F. Hu, P. Wang, J.L. Wu, Effect of allelopathic rice varieties combined with cultural management options on paddy field weeds, Pest Manag. Sci. 64 (2008) 276e282. [8] X.G. Zhou, F.Z. Wu, Artificially applied vanillic acid changed soil microbial communities in the rhizosphere of cucumber (Cucumis sativus L.), Can. J. Soil Sci. 93 (2013) 13e21. [9] U. Blum, T.M. Gerig, Relationships between phenolic acid concentrations, transpiration, water utilization, leaf area expansion, and uptake of phenolic acids: nutrient culture studies, J. Chem. Ecol. 31 (2005) 1907e1932. [10] H. Kitazawa, T. Asao, T. Ban, M.H.R. Pramanik, T. Hosoki, Autotoxicity of root exudates from strawberry in hydroponic culture, J. Hortic. Sci. Biotechnol. 80 (2005) 677e680. [11] T. Asao, H. Kitazawa, T. Ban, M.H.R. Pramanik, Y. Matsui, T. Hosoki, Search of autotoxic substances in some leaf vegetables, J. Jpn. Soc. Hortic. Sci. 73 (2004) 247e249. [12] T. Asao, H. Kitazawa, K. Ushio, Y. Sueda, T. Ban, M.H.R. Pramanik, Autotoxicity in some ornamentals with the means to overcome it, Hortscience 42 (2007) 1346e1350. [13] L.H. Chen, X.M. Yang, W. Raza, J.H. Li, Y.X. Liu, M.H. Qiu, F.G. Zhang, Q.R. Shen, Trichoderma harzianum SQR-T037 rapidly degrades allelochemicals in rhizospheres of continuously cropped cucumbers, Appl. Microbiol. Biotechnol. 89 (2011) 1653e1663. [14] Y. Chen, Y. Peng, C.C. Dai, Q. Ju, Biodegradation of 4-hydroxybenzoic acid by Phomopsis liquidambari, Appl. Soil Ecol. 51 (2011) 102e110. [15] S.M. Park, H.H. Park, W.K. Lim, H.J. Shin, A new variant activator involved in the degradation of phenolic compounds from a strain of Pseudomonas putida, J. Biotechnol. 103 (2003) 227e236. [16] Z.Y. Zhang, L.P. Pan, H.H. Li, Isolation, identification and characterization of soil microbes which degrade phenolic allelochemicals, J. Appl. Microbiol. 108 (2010) 1839e1849. [17] C.A. Edwards, P.J. Bohlen, Biology and Ecology of Earthworms, 1996. [18] O. Butenschoen, R. Ji, A. Schaffer, S. Scheu, The fate of catechol in soil as
37
affected by earthworms and clay, Soil Biol. Biochem. 41 (2009) 330e339. [19] I. Akca, N.D.K. Yilmaz, R. Kizilkaya, Evaluation of suppression of rhizomania disease by earthworm (Lumbricus terrestris L.) and its effects on soil microbial activity in different sugar beet cultivars, Archives Agron. Soil Sci. 60 (2014) 1565e1575. [20] C.A. Edwards, K.E. Fletcher, Interactions between earthworms and microorganisms in organic-matter breakdown, Agric. Ecosyst. Environ. 24 (1988) 235e247. [21] H. Li, C. Wang, X. Li, D. Xiang, Inoculating maize fields with earthworms (Aporrectodea trapezoides) and an arbuscular mycorrhizal fungus (Rhizophagus intraradices) improves mycorrhizal community structure and increases plant nutrient uptake, Biol. Fertil. Soils 49 (2013) 1167e1178. [22] T. Natal-da-Luz, I. Lee, R.A. Verweij, P.V. Morais, M.J. Van Velzen, J.P. Sousa, C.A. Van Gestel, Influence of earthworm activity on microbial communities related with the degradation of persistent pollutants, Environ. Toxicol. Chem./ SETAC 31 (2012) 794e803. [23] B.G. Zhang, G.T. Li, T.S. Shen, J.K. Wang, Z. Sun, Changes in microbial biomass c, n, and p and enzyme activities in soil incubated with the earthworms Metaphire guillelmi or Eisenia fetida, Soil Biol. Biochem. 32 (2000) 2055e2062. [24] T. Alekseeva, P. Besse, F. Binet, A.M. Delort, C. Forano, N. Josselin, M. Sancelme, C. Tixier, Effect of earthworm activity (Aporrectodea giardi) on atrazine adsorption and biodegradation, Eur. J. Soil Sci. 57 (2006) 295e307. [25] F. Binet, A. Kersante, C. Munier-Lamy, R.C. Le Bayon, M.J. Belgy, M.J. Shipitalo, Lumbricid macrofauna alter atrazine mineralization and sorption in a silt loam soil, Soil Biol. Biochem. 38 (2006) 1255e1263. [26] Z. Lin, X.M. Li, Y.T. Li, D.Y. Huang, J. Dong, F.B. Li, Enhancement effect of two ecological earthworm species (Eisenia foetida and Amynthas robustus E. Perrier) on removal and degradation processes of soil ddt, J. Environ. Monit. 14 (2012) 1551e1558. [27] R.W. Sims, E.G. Easton, A numerical revision of the earthworm genus Pheretima auct. (Megascolecidae: Oligochaeta ) with the recognition of new genera and an appendix on the earthworms collected by the royal society north borneo expedition, Biol. J. Linn. Soc. 4 (1972) 169e268. [28] M. Schaefer, Assessing 2,4,6-trinitrotoluene (TNT)-contaminated soil using three different earthworm test methods, Ecotoxicol. Environ. Saf. 57 (2004) 74e80. [29] G. Tian, Y. Bi, Z. Sun, L. Zhang, Phenolic acids in the plow layer soil of strawberry fields and their effects on the occurrence of strawberry anthracnose, Eur. J. Plant Pathology 143 (3) (2015) 581e594. [30] D.A. Martens, Identification of phenolic acid composition of alkali-extracted plants and soils, Soil Sci. Soc. Am. J. 66 (2002) 1240e1248. [31] J.D. Box, Investigation of the Folin-Ciocalteau phenol reagent for the determination of polyphenolic substances in natural-waters, Water Res. 17 (1983) 511e525. [32] R.S. Pitcher, D.W. Way, B.M. Savory, Specific replant diseases of apple and cherry and their control by soil fumigation, J. Hortic. Sci. Biotechnol. 41 (1966) 379e396. [33] X.B. Liu, S.J. Herbert, Fifteen years of research examining cultivation of continuous soybean in northeast china: a review, Field Crops Res. 79 (2002) 1e7. [34] P.M. Waser, Does competition drive dispersal, Ecology 66 (1985) 1170e1175. [35] J. Mathieu, S. Barot, M. Blouin, G. Caro, T. Decaens, F. Dubs, L. Dupont, P. Jouquet, P. Nai, Habitat quality, conspecific density, and habitat pre-use affect the dispersal behaviour of two earthworm species, Aporrectodea icterica and Dendrobaena veneta, in a mesocosm experiment, Soil Biol. Biochem. 42 (2010) 203e209. [36] N. Zhang, Y. Liao, F. Sun, C. Wang, Z. Sun, Earthworm population characteristics in soils different in land use and their relationships with biological fertility of the soils, Acta Pedol. Sin. 49 (2012) 364e372. [37] J.P. Curry, O. Schmidt, The feeding ecology of earthworms - a review, Pedobiologia 50 (2006) 463e477. [38] E. Westernacher, O. Graff, Orientation behavior of earthworms (Lumbricidae) towards different crops, Biol. Fertil. Soils 3 (1987) 131e132. [39] E.G. Sanchez, B. Munoz, M.H. Garvin, J.B. Jesus, D.J.D. Cosin, Ecological preferences of some earthworm species in southwest spain, Soil Biol. Biochem. 29 (1997) 313e316. [40] N.B. Hendriksen, Leaf litter selection by detritivore and geophagous earthworms, Biol. Fertil. Soils 10 (1990) 17e21. [41] V. Nuutinen, K.R. Butt, L. Jauhiainen, M.J. Shipitalo, T. Siren, Dew-worms in white nights: high-latitude light constrains earthworm (Lumbricus terrestris) behaviour at the soil surface, Soil Biol. Biochem. 72 (2014) 66e74. [42] R.C. Bicho, S.I.L. Gomes, A. Soares, M.J.B. Amorim, Non-avoidance behaviour in enchytraeids to boric acid is related to the gabaergic mechanism, Environ. Sci. Pollut. Res. 22 (2015) 6898e6903.
Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Application of leaves to induce earthworms to reduce phenolic compounds released by decomposing plants Yan-Meng Bi a, 1, Gei-Lin Tian b, c, 1, Chong Wang a, Cheng-Li Feng a, Yi Zhang a, Lu-Sheng Zhang b, **, Zhen-Jun Sun a, * a b c
College of Resources and Environmental Science, China Agricultural University, Beijing 100193, PR China College of Horticulture, China Agricultural University, Beijing 100193, PR China Department of Botanical Garden Engineering, Heze University, Shandong Province, Heze 274000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 October 2015 Received in revised form 15 April 2016 Accepted 18 April 2016
The negative impacts (allelopathic effects) of phenolic compounds (PCs) in soil on plant growth and microbial communities have recently received considerable attention. Similarly, there have been several recent studies on the effects of microbes on the degradation of PCs. Because of the profound effects of their feeding and burrowing activity, earthworms should have significant effects on PCs degradation; however, few studies have examined this potential effect, and particularly, factors that may affect the course of degradation. We tested different earthworm species and density, different conditions (sterilization or not) of mixture of soil and plant residual, and different source of PCs to evaluate the capacity of earthworms to accelerate PCs degradation. In addition, earthworm behavior experiments were set up to test whether adding plant leaves can stimulate earthworm feeding activity. The results showed that native earthworms exhibited a higher capacity than compost earthworms for degrading PCs; furthermore, when the number of Metaphire guillemi reached 300 individuals m2 in our experimental units, the PCs decreased most quickly, and the residual PCs concentration was 105 mg (p-coumaric acid)g1 (dry soil) less than that of control group. The source of PCs also affected their degradation rate, as PCs derived from leaves seemed to degrade more quickly. The results of our experiment suggested that earthworms avoid feeding on phenolic acids, but can be induced to do so by adding leaves to the substrate. These results indicate that earthworm activity can accelerate the degradation of total PCs, and that this may be further facilitated by incorporation of organic matter, which may be used to alleviate allelopathic effects of PCs in soil. © 2016 Elsevier Masson SAS. All rights reserved.
Handling Editor: C.C. Tebbe Keywords: Earthworm Phenolic compounds Allelochemical Trophic behavior
1. Introduction Phenolic compounds (PCs) are the most important and common plant allelochemicals in soil ecosystems. They are chemical compounds consisting of a hydroxyl group bound directly to an aromatic hydrocarbon group [1], which are the most widespread secondary plant metabolites known [2,3]. PCs are released into the environment through foliar leachate, root exudation, residue decomposition [4,5], among which aboveground and belowground
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (L.-S. Zhang), (Z.-J. Sun). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejsobi.2016.04.007 1164-5563/© 2016 Elsevier Masson SAS. All rights reserved.
[email protected]
litter are the dominant pathways [3,6]. Some PCs are phytotoxic, and can inhibit plant growth through interaction with the mitochondrial membrane and impairment of dark respiration and ATP synthesis [4,5]. Similarly, phytotoxicity can disrupt the microbial community balance by modifying the population and community structure in the rhizosphere [7,8]. According to Blum and Gerig (2005), phenolic acid treatment of seedlings inhibits transpiration, water utilization, leaf area, and the absolute and relative rates of leaf expansion [9]. Several management approaches have been taken to alleviate allelopathic stress caused by PCs on plants. For example, activated charcoal has been used to adsorb accumulated phytotoxic chemicals with the result of improved growth and yield in strawberry [10], several leafy vegetables, and some ornamentals [11,12]. Similarly, several studies have focused on the application of microorganisms that can biodegrade phenolic acids and colonize the
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rhizosphere of plants to alleviate the stress of autotoxic allelochemicals exuded by crop roots or left by plant residues [13e16]. Although several measures to degrade PCs have been tested and shown to be successful, a major component of natural and agricultural ecosystems, earthworms [17], has received little attention. Earthworms form a major part of the soil decomposer macrofauna, and play an important role in temperate ecosystems [18] and some tropical ecosystems. They can alter soil structure, accelerate the nutrient cycling processes in soil, and significantly impact microbial communities [19e23]. In addition, as an ecosystem engineer, earthworms can promote the degradation of many compounds in soil. Several studies have examined the direct or indirect relationship between earthworms and some toxic substances. For example, studies have examined the effect of earthworms on altering mineralization and biodegrading atrazine [24,25], as well as earthworm mediated enhancement of the removal of dichloro-diphenyl-tricgloroethane or polycyclic aromatic hydrocarbons [22,26]. Thus, earthworms may have the potential to degrade single cyclic molecules such as phenolic acid or other simple PCs with simpler structures than the complex substances mentioned above. Although Butenschoen et al. (2009) studied the fate of catechol affected by earthworms, they did not examine the influence of earthworms on total PCs, nor did they focus on the basic conditions for earthworms to degrade PCs [18]. Furthermore, they only used endogeic earthworms in their experiments, and thus is it unclear whether the different feeding and burrowing activities of epigeic or anecic earthworms might be more (or less) efficient at degradation of PCs. So, it is important to evaluate whether epigeic earthworms and anecic earthworms can accelerate the decomposing of PCs. The objectives of this paper are: first, to evaluate the ability of earthworms to accelerate the degradation of PCs released by decomposing plants residue (strawberry was used as our model plant); second, to investigate influence factors for earthworms to biodegrade phenolics; and finally, to examine the trophic behavior of earthworms in relation to phenolic acid.
15 cm depth) with a small hole in the bottom were filled with 3 kg MSRL and 500 mL deionized water. A plastic net (±1 mm) was placed in the bottom of every pot to prevent earthworms from escaping. All pots were placed in an artificial climate incubator with temperature maintained between 15 and 20 C. All pots were irrigated every 5 days with 100 mL deionized water. Four experiments were conducted to test the influence of earthworm species, density of M. guillemi, sterilization and the source of PCs on degradation rate of PCs (Table 1). In the first experiment, there were 3 treatments: one with 6 individuals of M. guillemi (M), 30 individuals of E. fetida (E) (this resulted in approximately equal biomass for each species) and a control without earthworms (CK). In the second experiment, there were six densities of M. guillemi added to the pots: treatments consisted of 0, 3, 6, 9, 12, and 15 individuals per pot (marked as Z, T, SI, N, TW and F respectively). In the third experiment, there were four treatments: sterilized (autoclaved at 101 kpa, 121 C, 30 min, twice) MSRL with M. guillemi (SM), sterilized MSRL without earthworms (S), ordinary MSRL with M. guillemi (OM), and ordinary MSRL without earthworms (O). In order to verify whether most of the microorganisms in sterilized MSRL have been eliminated, we used dilution-plate method on LB (lysogeny broth) (10 g MSRL and 100 mL sterile normal saline to obtain extraction) to test the effect of sterilization (Fig. 1). The last experiment included six treatments: mixture of soil and ground leaves with M. guillemi (ML), E. fetida (EL) and without earthworm (L), and mixture of soil and ground roots with earthworm M. guillemi (MR), E. fetida (ER), and without earthworm (R). Each treatment in each experiment mentioned above had 4 replicates. All of the experiments lasted for 30 days. The avoidance/selection behavior of earthworms was studied in
Table 1 Earthworms added to pots (Initial) and recovered after 30-day experiments (Final). Experiment
Treatment
E1
M E CK Z T SI N TW F SM S OM O ML EL L MR ER R
2. Materials and methods 2.1. Soil and earthworms Soil was obtained from a continuously cropped experimental field at China Agricultural University in Beijing, where the strawberry cultivar ‘Benihoppe’ (Fragaria ananassa Duch) had been planted for 3 years. The soil was air-dried, sieved (2 mm), and thoroughly mixed with smashed roots and strawberry leaves, creating an experimental mixture of soil (96% mass ratio), root (2% mass ratio) and leaf (2% mass ratio) (MSRL) with the following properties: pH (H2O:soil, 2.5:1) of 7.82, 2.25% soil organic matter, 0.12% total nitrogen, 50.38 mg kg1 available phosphorus (Olsen-P), 255.23 mg kg1 available potassium (NH4OAc-K). Earthworms Metaphire guillemi [27] (anecic) native to the experimental fields of China Agricultural University were collected at the same location where the soil was obtained. Compost earthworms Eisenia fetida (epigeic) were purchased from Beijing Lvhuan Kemao Company. Both species of earthworm were placed in MSRL for 1 week to acclimate to the matrix and replace their gut contents with experimental soil. Adult earthworms with fresh weight of 2.4e2.5 g for M. guillemi, and 0.4e0.5 g for E. fetida were chosen to be used as test animals. Prior to fresh weight determinations, the earthworms were washed and blotted with filter paper [23]. 2.2. Experimental procedure Plastic pots (top diameter 19 cm, bottom diameter 18 cm and
E2
E3
E4
Earthworm abundance (Individuals per pot) Initial
Final
6 30 0 0 3 6 9 12 15 6 0 6 0 6 30 0 6 30 0
6 31.00 0 0 3 6 9 12 14.50 6 0 6 0 6 31.00 0 6 31.75 0
± 1.41
± 0.58
± 0.82
± 1.71
Earthworm survival rates (%)
100 103.3 ± 4.7 e e 100 100 100 100 96.7 ± 3.8 100 e 100 e 100 103.3 ± 2.7 e 100 105.8 ± 5.7 e
Values are mean (standard deviation is zero) or mean ± standard deviation of 4 replicates. Abbreviations: E1: M: treatment with M. guillemi; E: treatment with E. fetida (E); CK: Control group. E2: Z: treatment without earthworm; T: treatment with three individuals of earthworm; SI: treatment with six individuals of earthworm; N: treatment with nine individuals of earthworm; TW: treatment with twelve individuals of earthworm; F: treatment with Fifteen individuals of earthworm. E3: SM: sterilized matrix added with M. guillemi; S: only sterilized matrix; OM: ordinary matrix added with M. guillemi; O: only ordinary matrix. E4: ML: matrix composed of leaves and soil with M. guillemi; EL: matrix composed of leaves and soil with E. fetida; L: matrix composed of leaves and soil; MR: matrix composed of roots and soil with M. guillemi; EL: matrix composed of roots and soil with E. fetida; R: matrix composed of roots and soil.
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Fig. 1. Picture of plates to test the effect of sterilization. The plates of the first row were spread with un-sterile MSRL exaction and the plates of the second row were spread with sterile MSRL exaction. The picture was taken three days after inoculation.
a 6-chamber earthworm-behavior device (Fig. 2). M. guillemi were chosen as the experimental subject for this work based on results of the previous experiments and indication that M. guillemi has high efficiency for decomposing PCs. This experiment was modified from work performed by Schaefer [28], and two experiments were included to test earthworm behavior. The first experiment examined whether earthworms tend to approach or avoid phenolic acids, with p-coumaric acid (PA) chosen as a representative phenolic acid because of its dominant concentration in strawberry cropped soil [29]. 1.8 g PA was weighed and dissolved in 10 mL methanol, and the solution was added to 500 mL deionized water and mixed thoroughly. The mixed solution was added to a watering can and sprayed on 9 kg soil equally (the concentration of PA was approximately 200 mg g1 in the soil). Soil mixed with PA was added to chambers 1, 3, and 5 of the device. The same soil without
PA (same volume methanol and water were added to standardize) was added to chambers 2, 4, and 6 of the device. In total, 48 individuals of M. guillemi were placed in the center of the device, and all of the dividers were removed to allow the earthworms to crawl freely. The number of earthworms in each chamber was counted on the 1st, 3rd, 5th, 7th, and 9th day, and 20 g soil samples were obtained to analyze the concentration of total PCs. Three parallel trials were conducted at the same time to reduce variability in results. Because the first experiment showed M. guillemi to avoid PA, the second experiment was set to test whether additional organic matter in the form of plant leaves could overcome the earthworm's avoidance of PA. In this experiment, three treatments were set: soil without PA or leaf (CK), soil with PA (200 mg dwg1) (P), and soil with PA and leaf (PL). These mixtures were added to 3 chambers of the device, and 24 earthworms were added to the center of the device. The dividers were removed to allow earthworms to crawl freely, and after 24 h, we counted the earthworms in every chamber. The PA was added to the soil using the same method as that in the first experiment. Ten parallel trials were conducted at the same time to reduce variability in results. 2.3. PCs extraction and analysis
Fig. 2. Earthworm-behavior device. The device was designed based on Schaefer (2004) [28], and has been slightly modified. In this device, the boards and the center column can be raised. Before the experiment, all boards and the center column were inserted in the device, forming 7 chambers. Earthworms were placed in the chamber and different substrates were put into the remaining chambers. We then raised all the boards and center column so earthworms could select their preferred environment. (The picture was done with Adobe Illustrator CS5).
PCs were extracted using the modified method of Martens [30]. First, 25 g MSRL was extracted with 25 mL 1 M NaOH and kept at room temperature for 24 h. Then the suspension was centrifuged at 8000 g for 10 min, and the liquid supernatant was separated. The pH of the supernatant was adjusted to 2.5 with 12 M hydrochloric acid (HCl) and left to stand for 2 h. The suspension was centrifuged at 8000 g for 10 min. The supernatant was stored at 4 C and prepared for analysis. Total PCs concentrations were measured using the FolinCiocalteu assay [31]. Total PCs content was standardized against PA and expressed as milligrams per liter of PA. The linearity range for this assay was determined as 0e10 mg mL1 PA (R2 ¼ 0.9990), giving an absorbance range of 0e0.628 AU. The solution of extracts in the last experiment of PCs degradation was filtered through a 0.22 mm microporous membrane filter for subsequent highperformance liquid chromatography (HPLC) analysis (model 1260,
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Agilent, Waldbronn, Germany). Phenolic acid separation was performed by gradient elution high-performance liquid chromatography (GEHPLC) with an HPLC quaternary pump model G1311A, a UV detector model G1314A, and a chromatographic column: XDBC18 (4.6 mm 250 mm). The detector was set to 280 nm, and the flow rate to 1 mL/min. The injection volume was 20 mL, and the column temperature was maintained at 25 C. Methanol (A) and acetic acid solution (pH ¼ 2.80) (B) were used as mobile phases with a gradient elution B: 70% (0 min)/50% (15 min)/30% (16 min)/0 (30 min)/end (30 min) [29]. 2.4. Statistical analysis The means and standard deviations of PCs concentrations and earthworm number were calculated, and analysis of variance (ANOVA) was performed on the data using SPSS 19.0. The interaction effect between PCs source (derive from leaves or roots) and earthworm species on the degradation of PCs was also processed with this software and double factor analysis was chosen here. Mean separations were performed by Student-Newman-Keuls multiple range tests. Differences at P < 0.05 were considered significant. The regression line between earthworm number and PA concentration was fitted by SigmaPlot 12.0. 3. Results
Fig. 3. Residual total phenolic compounds after one month experiment. Error bars represent standard deviations of 4 replicates. CK stands for control group, E stands for treatment added E. fetida and M stands for treatment added M. guillemi. Different letters denote significant differences (p < 0.05).
the number of earthworms continued to increase, and there were no significant differences among N, TW and F.
3.1. Earthworm survival At the end of the experiments, we counted earthworms in every pot (Table 1). All of the treatments containing E. fetida had more earthworms at the end of the experiment, compared to those before, but the increased earthworms were much smaller than the earthworms added. The number of earthworms in F of experiment 2 decreased to 14.50 ± 0.58, and the survival rate was 96.7 ± 3.8%. The earthworm survival rates in the rest of the experiments were 100%. 3.2. Effect of earthworms on phenolic acid degradation Residual PCs content was analyzed after 30 days of incubation of earthworms in the MSRL. The residual total PCs concentration varied among the different treatments (Fig. 3), and was highest in the control group at 508 mg (PA) g1 (dry soil). The concentrations of PCs were 457 mg (PA) g1 (dry soil) and 422 mg (PA) g1 (dry soil) in the E and M group. PCs decreased faster in both of the two experiment groups compared to the control group. Conversely, we also noted that there were no significant differences between the E and M in PCs concentration, although the PCs concentration of M was slightly less than E. 3.3. Effect of M. guillemi density on PC degradation The results demonstrated that the M. guillemi had higher capacity for degrading PCs than the E. fetida when the PCs source was leaves only (without roots) (Table 2). As such, M. guillemi was selected for the remaining experiments. After 30-day incubation, PCs concentrations were tested, and decreased with increasing numbers of earthworms in each pot (Fig. 4). The points of data can be represented by an overall regression line (y ¼ 525.5e14.10x þ 0.39x2, R ¼ 0.9697). When the number of earthworms reached 6 in one pot, the PCs concentration was significantly lower than that of Z. Similarly, when the number of earthworms reached 9, the PCs concentration decreased to 413 ± 22 mg (PA) g1 (dry soil), which was significantly lower than that of T and SI. However, the residual total PCs did not decrease as
3.4. Role of microbes in the earthworm-PCs degradation system To examine the role of microbes in the earthworm-PCs degradation system, we selected sterilized soil as a control, and the concentration of PCs was measured after 30 days (Fig. 5). In the sterilized MSRL without earthworms (S), the concentration of residual PCs was 474 ± 70 mg (PA) g1 (dry soil) and 441 ± 44 mg (PA) g1 (dry soil) in the sterilized matrix containing M. guillemi (SM). There were no significant differences between the S and MS groups. The concentration was 407 ± 41 mg (PA) g1 (dry soil) in the unsterilized soil (O), and this was significantly lower than the S treatment. Residual PCs was lowest (338 ± 43 mg (PA) g1 (dry soil)) in the unsterilized soil with M. guillemi (OM) treatment and this was significantly lower than all of the other experiments.
3.5. Effect of PCs source and earthworm species on PC degradation The concentrations of residual PCs derived from different plant parts were examined, and the degradation rate of two species of earthworms was calculated after 30 days. Using single-factor analysis of variance, we found that M. guillemi degraded PCs to a greater degree than a similar biomass of E. fetida in the leaf-added treatment (Table 2). However, results were different for the rootadded treatment groups. When roots were added as a source of PCs, we found that both species of earthworm stimulated the decomposition of PCs to the same degree (Table 2). When considering both PCs' source and earthworm species, a double-factor analysis of variance showed that earthworm species had a markedly significant effect (P < 0.01) on the degradation rate. In addition, the earthworm species and PCs source had a markedly significant interaction effect on degradation (Table 2). In addition, we found that the properties of individual phenolic acids change during leaf decomposition after earthworm inoculation. Most individual phenolic acids decreased faster, except cinnamic acid, which increased in the M. guillemi group (Fig. 6) compared to the control treatment.
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Table 2 Effects of Earthworm Species and Phenolics Source on the degradation of Phenolics. Earthworm species
Phenolics sources
Control (mg g1(PA))
Metaphire guillemi Metaphire guillemi Eisenia fetida Eisenia fetida Significance due to Earthworm Species Phenolics Source ES PS interreaction
Leaf Root Leaf Root
377.9 269.2 377.9 269.2
± ± ± ±
Treatment (mg g1(PA))
37.0 38.5 37.0 38.5
256.4 200.2 323.5 202.9
± ± ± ±
15.6 11.4 20.3 18.8
Degradation mass (mg g1(PA))
Degradation rate
121.5 ± 22.4 69.0 ± 28.6 54.4 ± 18.4 66.3 ± 21.8
0.320 0.249 0.142 0.241
± ± ± ±
0.029 0.074 0.034 0.054
a a b a
** NS **
Values with different letters for a measure significantly differed (confirmed by Student-Newman-Keulsa multiple range test; p < 0.05). NS means not significant different; **means P < 0.01(confirmed by general linear model).
100%
600
Total Phenolics (µg PA/g dry soil)
90% 550
80%
a
trans-cinnamic acid
70%
ab 500
b
450
ferulic acid
50%
p-cumaric acid
40%
syringic acid
30%
p-hydroxybenzoic acid
20%
chlorogenic acid
10%
c
c
c
60%
0% CK
E
M
Fig. 6. Percentages of standardized residual individual phenolic acids from different experimental groups. CK stands for control group, E stands for treatment added E. fetida and M stands for treatment added M. guillemi.
400
350 0
2
4
6
8
10
12
14
16
3.6. Avoidance behavior of earthworms related to phenolic acids
Earthworm Number Fig. 4. Residual total phenolic compounds after different numbers of M. guillemi placed under experimental conditions for one month. Error bars represent standard deviations of 4 replicates. The treatment of the 6 groups are marked as Z (no earthworms), T (3 individuals of M. guillemi), SI (6 individuals of M. guillemi), N (9 individuals of M. guillemi), TW (12 individuals of M. guillemi) and F (15 individuals of M. guillemi). Different letters denote significant differences (p < 0.05).
We designed earthworm behavior experiments to determine whether earthworms avoid or select soils containing phenolic acids. The earthworms in the behavior chamber were counted every day, and the number of earthworms in the compartment without PA was greater than that with PA; however, the number of earthworms in the two compartments tended to be the same over time since the concentration of PA decreased each day. The number of earthworms was not significantly different in the two treatment groups by the seventh day of the experiment (Fig. 7). Similarly, when the PA concentration decreased to 120 mg g1 (dry soil), the number of earthworms in the two chambers showed no significant
Fig. 5. The effect of earthworms and microbes on the degradation of total phenolic compounds. S stands for sterilized MSRL, SM stands for sterilized MSRL þ M. guillemi, O stands for MSRL without sterilization, and OM stands for MSRL þ M. guillemi. Error bars represent standard deviations of 4 replicates. Different letters denote significant differences (p < 0.05). Fig. 7. Number of earthworms and concentration of p-coumaric acid in compartments of different treatments Error bars represent standard deviations of 4 replicates. Different letters denote significant differences (p < 0.05).
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Y.-M. Bi et al. / European Journal of Soil Biology 75 (2016) 31e37
difference, which implied that earthworms were not sensitive to low concentrations of PA (120 mg g1 (dry soil) or less). Fig. 8 shows that the quantity of earthworms in the PA-added group was significantly less than that in the CK group. However, when leaves were added to soil, the number of earthworms did not significantly differ in the CK group, indicating that leaves can neutralize the negative effects of PA on earthworms.
4. Discussion 4.1. Effects of earthworm on the degradation of total PCs The density of earthworms in each experimental pot did not change significantly, indicating that our experimental conditions were near optimal (Table 1). The number of E. fetida earthworms increased slightly, and the increased earthworms were smaller, so it may be because that a very few of earthworms got produced during the experiment, or that E. fetida cocoons have been introduced with soil which was not excessively processed prior to use in the experiments. Yields of many crops typically decrease if they were planted at the same site year after year [31,32]. There are several possible reasons for this phenomenon, such as soil acidification, nutrient imbalance [33], and changes in biological environment and phytotoxic effects [13]. Among these, phytotoxic effects caused by allelochemicals play an important role; thus it may be critical to reduce concentrations of allelochemicals for many plants, especially for crops grown in greenhouses. In the present study, we used M. guillemi and E. fetida as models to investigate the effects of earthworms on the degradation of total PCs derived from plant (strawberry) residue, with an overall goal of reducing the concentration of allelochemicals in soil. The results showed that both M. guillemi and E. fetida effectively promote the degradation of total PCs (Fig. 3). Although these results are similar to those reported by Butenschoen et al., who concluded that earthworms increase microbial activity, and thus, mineralization of PCs, they used endogeic earthworms in their experiment [18]. We found that both M. guillemi (anecic) and E. fetida (epigeic) can accelerate the biodegradation of PCs. In our pot experiments, degradation efficiency reached its limit when the number of earthworms in the chamber reached nine
(Fig. 4). The surface area of our chambers was 0.03 m2, which means the earthworm density was approximately 300 per m2. Adding more earthworms to the same size chamber did not increase degradation efficiency, possibly because crowding increases intra-specific competition due to resource depletion [34,35]. According to a previous study, earthworm density can reach 300 per m2 in a well-managed vegetable field, however, not all of them are M. guillemi [36]; thus, it is possible to apply earthworms to a density of 300 per m2 to degrade PCs in agricultural soil, but the degradation efficiency may not reach its limit. One consideration is that the present study only examined the degradation effects of PCs in experimental pots. There are some differences between the field conditions and pot experiments, so analogous experiments should be designed in the field to assess the accuracy of earthworm density. Other than the quantity of earthworms, the effect of earthworms in accelerating PCs degradation was dependent on the species of earthworms (Table 2). Although the biomass of the two species of earthworms added to the experiment was uniform, the degradation rate of PCs differed between the two species. Both species of earthworms accelerated the degradation of PCs, and the earthworm M. guillemi showed stronger effects than earthworms E. fetida with PCs derived from leaves. However, if the soil was mixed with roots as the source of PCs, there was no difference in degradation rate between the two species of earthworms. The twofactor analysis demonstrated that earthworm species and the PCs source had a significant interaction. In addition to the species and density of earthworms, we also examined whether PCs degradation by earthworms was associated with microbial activity; our results showed that the presence of both earthworms and microbes showed the highest degradation rate (Fig. 5) among all of the treatments. This implies that earthworms can promote microbes to degrade PCs, which is consistent with the study by Natal-da-Luz et al., who examined the influence of earthworm activity on microbial communities related to the degradation of persistent pollutants [22]. It is widely acknowledged that microbes play a very important role in the degradation of PCs, but the underlying mechanisms of the interaction effect of earthworms and microbes on PCs degradation still need to be studied further.
4.2. Behavior of earthworms associated with PCs
Fig. 8. Number of earthworms in compartments of different treatments. Error bars represent standard deviations of 10 replicates. Different letters denote significant differences (p < 0.05).
Since earthworms have the ability to accelerate the degradation of PCs, it is critical to study the behavior of earthworms related to PCs. Many researchers have found that earthworms are able to select their habitat and have food preferences [37e39]. Here, we wanted to determine whether earthworms prefer PCs, and whether this preference can be induced by adding leaves to soil. Our results suggest that earthworms do not prefer PCs, especially phenolic acids, such as PA (Fig. 7); this may be because earthworms have chemoreceptors that detect the presence of unfavorable substances such as chemicals [17]. This result is similar with the study of Hendriksen et al., who concluded that high polyphenol content in matrix makes it less palatable to earthworms [40]. Moreover, our study showed that this avoidance of earthworms on phenolic acid changed when leaves were added to their habitat (Fig. 8); though leaves contain phenolic compounds, leaves can offer earthworms additional food resources, which may offset the unfavorable conditions created by phenolic acids. Many researchers have suggested that behavior of earthworms can be modified by altering habitat conditions [41,42]. Combined with our results, we conclude that earthworms can be induced to increase PCs degradation by adding a food source.
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5. Conclusions The present study clearly demonstrates that both M. guillemi (anecic) and E. fetida (epigeic) have the ability to accelerate the degradation of PCs, but that these effects differ depending on the density and species of earthworms. Conversely, although earthworms show avoidance to PCs such as phenolic acid, adding leaves as a food source can ameliorate this. Acknowledgments This work was supported by College PhD Subject Science & Technology Programme of the Education Ministry (20110008130003) and National Natural Science Foundation of China (41371305). We thank our American friend Mac A. Callaham of Center for Forest Disturbance Science, Southern Research Station for his help to review and edit the English in this manuscript and Professor Kong Chuihua of China Agricultural University for his valuable suggestions. References [1] Z.H. Li, Q. Wang, X. Ruan, C.D. Pan, D.A. Jiang, Phenolics and plant allelopathy, Molecules 15 (2010) 8933e8952. [2] J.O. Siqueira, M.G. Nair, R. Hammerschmidt, G.R. Safir, Significance of phenoliccompounds in plant-soil-microbial systems, Crit. Rev. Plant Sci. 10 (1991) 63e121. [3] S. Hattenschwiler, P.M. Vitousek, The role of polyphenols in terrestrial ecosystem nutrient cycling, Trends Ecol. Evol. 15 (2000) 238e243. [4] T.L. Weir, S.W. Park, J.M. Vivanco, Biochemical and physiological mechanisms mediated by allelochemicals, Curr. Opin. Plant Biol. 7 (2004) 472e479. [5] G. Jilani, S. Mahmood, A.N. Chaudhry, I. Hassan, M. Akram, Allelochemicals: sources, toxicity and microbial transformation in soil - a review, Ann. Microbiol. 58 (2008) 351e357. [6] C.L. Meier, K.N. Suding, W.D. Bowman, Carbon flux from plants to soil: roots are a below-ground source of phenolic secondary compounds in an alpine ecosystem, J. Ecol. 96 (2008) 421e430. [7] C.H. Kong, F. Hu, P. Wang, J.L. Wu, Effect of allelopathic rice varieties combined with cultural management options on paddy field weeds, Pest Manag. Sci. 64 (2008) 276e282. [8] X.G. Zhou, F.Z. Wu, Artificially applied vanillic acid changed soil microbial communities in the rhizosphere of cucumber (Cucumis sativus L.), Can. J. Soil Sci. 93 (2013) 13e21. [9] U. Blum, T.M. Gerig, Relationships between phenolic acid concentrations, transpiration, water utilization, leaf area expansion, and uptake of phenolic acids: nutrient culture studies, J. Chem. Ecol. 31 (2005) 1907e1932. [10] H. Kitazawa, T. Asao, T. Ban, M.H.R. Pramanik, T. Hosoki, Autotoxicity of root exudates from strawberry in hydroponic culture, J. Hortic. Sci. Biotechnol. 80 (2005) 677e680. [11] T. Asao, H. Kitazawa, T. Ban, M.H.R. Pramanik, Y. Matsui, T. Hosoki, Search of autotoxic substances in some leaf vegetables, J. Jpn. Soc. Hortic. Sci. 73 (2004) 247e249. [12] T. Asao, H. Kitazawa, K. Ushio, Y. Sueda, T. Ban, M.H.R. Pramanik, Autotoxicity in some ornamentals with the means to overcome it, Hortscience 42 (2007) 1346e1350. [13] L.H. Chen, X.M. Yang, W. Raza, J.H. Li, Y.X. Liu, M.H. Qiu, F.G. Zhang, Q.R. Shen, Trichoderma harzianum SQR-T037 rapidly degrades allelochemicals in rhizospheres of continuously cropped cucumbers, Appl. Microbiol. Biotechnol. 89 (2011) 1653e1663. [14] Y. Chen, Y. Peng, C.C. Dai, Q. Ju, Biodegradation of 4-hydroxybenzoic acid by Phomopsis liquidambari, Appl. Soil Ecol. 51 (2011) 102e110. [15] S.M. Park, H.H. Park, W.K. Lim, H.J. Shin, A new variant activator involved in the degradation of phenolic compounds from a strain of Pseudomonas putida, J. Biotechnol. 103 (2003) 227e236. [16] Z.Y. Zhang, L.P. Pan, H.H. Li, Isolation, identification and characterization of soil microbes which degrade phenolic allelochemicals, J. Appl. Microbiol. 108 (2010) 1839e1849. [17] C.A. Edwards, P.J. Bohlen, Biology and Ecology of Earthworms, 1996. [18] O. Butenschoen, R. Ji, A. Schaffer, S. Scheu, The fate of catechol in soil as
37
affected by earthworms and clay, Soil Biol. Biochem. 41 (2009) 330e339. [19] I. Akca, N.D.K. Yilmaz, R. Kizilkaya, Evaluation of suppression of rhizomania disease by earthworm (Lumbricus terrestris L.) and its effects on soil microbial activity in different sugar beet cultivars, Archives Agron. Soil Sci. 60 (2014) 1565e1575. [20] C.A. Edwards, K.E. Fletcher, Interactions between earthworms and microorganisms in organic-matter breakdown, Agric. Ecosyst. Environ. 24 (1988) 235e247. [21] H. Li, C. Wang, X. Li, D. Xiang, Inoculating maize fields with earthworms (Aporrectodea trapezoides) and an arbuscular mycorrhizal fungus (Rhizophagus intraradices) improves mycorrhizal community structure and increases plant nutrient uptake, Biol. Fertil. Soils 49 (2013) 1167e1178. [22] T. Natal-da-Luz, I. Lee, R.A. Verweij, P.V. Morais, M.J. Van Velzen, J.P. Sousa, C.A. Van Gestel, Influence of earthworm activity on microbial communities related with the degradation of persistent pollutants, Environ. Toxicol. Chem./ SETAC 31 (2012) 794e803. [23] B.G. Zhang, G.T. Li, T.S. Shen, J.K. Wang, Z. Sun, Changes in microbial biomass c, n, and p and enzyme activities in soil incubated with the earthworms Metaphire guillelmi or Eisenia fetida, Soil Biol. Biochem. 32 (2000) 2055e2062. [24] T. Alekseeva, P. Besse, F. Binet, A.M. Delort, C. Forano, N. Josselin, M. Sancelme, C. Tixier, Effect of earthworm activity (Aporrectodea giardi) on atrazine adsorption and biodegradation, Eur. J. Soil Sci. 57 (2006) 295e307. [25] F. Binet, A. Kersante, C. Munier-Lamy, R.C. Le Bayon, M.J. Belgy, M.J. Shipitalo, Lumbricid macrofauna alter atrazine mineralization and sorption in a silt loam soil, Soil Biol. Biochem. 38 (2006) 1255e1263. [26] Z. Lin, X.M. Li, Y.T. Li, D.Y. Huang, J. Dong, F.B. Li, Enhancement effect of two ecological earthworm species (Eisenia foetida and Amynthas robustus E. Perrier) on removal and degradation processes of soil ddt, J. Environ. Monit. 14 (2012) 1551e1558. [27] R.W. Sims, E.G. Easton, A numerical revision of the earthworm genus Pheretima auct. (Megascolecidae: Oligochaeta ) with the recognition of new genera and an appendix on the earthworms collected by the royal society north borneo expedition, Biol. J. Linn. Soc. 4 (1972) 169e268. [28] M. Schaefer, Assessing 2,4,6-trinitrotoluene (TNT)-contaminated soil using three different earthworm test methods, Ecotoxicol. Environ. Saf. 57 (2004) 74e80. [29] G. Tian, Y. Bi, Z. Sun, L. Zhang, Phenolic acids in the plow layer soil of strawberry fields and their effects on the occurrence of strawberry anthracnose, Eur. J. Plant Pathology 143 (3) (2015) 581e594. [30] D.A. Martens, Identification of phenolic acid composition of alkali-extracted plants and soils, Soil Sci. Soc. Am. J. 66 (2002) 1240e1248. [31] J.D. Box, Investigation of the Folin-Ciocalteau phenol reagent for the determination of polyphenolic substances in natural-waters, Water Res. 17 (1983) 511e525. [32] R.S. Pitcher, D.W. Way, B.M. Savory, Specific replant diseases of apple and cherry and their control by soil fumigation, J. Hortic. Sci. Biotechnol. 41 (1966) 379e396. [33] X.B. Liu, S.J. Herbert, Fifteen years of research examining cultivation of continuous soybean in northeast china: a review, Field Crops Res. 79 (2002) 1e7. [34] P.M. Waser, Does competition drive dispersal, Ecology 66 (1985) 1170e1175. [35] J. Mathieu, S. Barot, M. Blouin, G. Caro, T. Decaens, F. Dubs, L. Dupont, P. Jouquet, P. Nai, Habitat quality, conspecific density, and habitat pre-use affect the dispersal behaviour of two earthworm species, Aporrectodea icterica and Dendrobaena veneta, in a mesocosm experiment, Soil Biol. Biochem. 42 (2010) 203e209. [36] N. Zhang, Y. Liao, F. Sun, C. Wang, Z. Sun, Earthworm population characteristics in soils different in land use and their relationships with biological fertility of the soils, Acta Pedol. Sin. 49 (2012) 364e372. [37] J.P. Curry, O. Schmidt, The feeding ecology of earthworms - a review, Pedobiologia 50 (2006) 463e477. [38] E. Westernacher, O. Graff, Orientation behavior of earthworms (Lumbricidae) towards different crops, Biol. Fertil. Soils 3 (1987) 131e132. [39] E.G. Sanchez, B. Munoz, M.H. Garvin, J.B. Jesus, D.J.D. Cosin, Ecological preferences of some earthworm species in southwest spain, Soil Biol. Biochem. 29 (1997) 313e316. [40] N.B. Hendriksen, Leaf litter selection by detritivore and geophagous earthworms, Biol. Fertil. Soils 10 (1990) 17e21. [41] V. Nuutinen, K.R. Butt, L. Jauhiainen, M.J. Shipitalo, T. Siren, Dew-worms in white nights: high-latitude light constrains earthworm (Lumbricus terrestris) behaviour at the soil surface, Soil Biol. Biochem. 72 (2014) 66e74. [42] R.C. Bicho, S.I.L. Gomes, A. Soares, M.J.B. Amorim, Non-avoidance behaviour in enchytraeids to boric acid is related to the gabaergic mechanism, Environ. Sci. Pollut. Res. 22 (2015) 6898e6903.