Ecological Indicators 39 (2014) 44–53
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Effects of nicosulfuron on the abundance and diversity of arbuscular mycorrhizal fungi used as indicators of pesticide soil microbial toxicity D.G. Karpouzas a,∗ , E. Papadopoulou a,b , I. Ipsilantis c , I. Friedel d , I. Petric e , N. Udikovic-Kolic e , S. Djuric f , E. Kandeler g , U. Menkissoglu-Spiroudi b , F. Martin-Laurent d a
University of Thessaly, Department of Biochemistry and Biotechnology, Ploutonos 26 and Aeolou Street, 41221 Larisa, Greece Aristotle University of Thessaloniki, School of Agriculture Laboratory of Pesticide Science, 54124 Thessaloniki, Greece c Aristotle University of Thessaloniki, School of Agriculture, Laboratory of Soil Science, 54124 Thessaloniki, Greece d INRA, UMR 1347 Agroécologie, BP 86510, F-21065 Dijon Cedex, France e Rudjer Boskovic Institute, Division for Marine and Environmental Research, HR-10002 Zagreb, Croatia f University of NoviSad, Faculty of Agriculture, Sr-Novi-Sad, Serbia g Institute of Soil Science and Land Evaluation, Soil Biology Section, University of Hohenheim, Emil-Wolff-Str. 27, D-70593 Stuttgart, Germany b
a r t i c l e
i n f o
Article history: Received 10 June 2013 Received in revised form 29 November 2013 Accepted 2 December 2013 Keywords: Arbuscular mycorrhizal fungi Pesticides Soil microbial ecotoxicity DGGE Colonization
a b s t r a c t The key role of arbuscular mycorrhizal (AM) fungi in maintaining soil fertility and ecosystem functioning and their general sensitivity to pesticides make them good candidate bioindicators in pesticide soil microbial toxicity assessment. We investigated the impact of the herbicide nicosulfuron on mycorrhizal colonization and community structure of AM fungi via a pot-to-ﬁeld experimental approach. This allowed the assessment of nicosulfuron toxicity (i) at extreme exposure schemes (pot experiment, Tier I) invoked by the repeated application of a range of dose rates (x0, x10, x100, x1000 the recommended dose) and (ii) under realistic exposure scenarios (x0, x1, x2, x5 the recommended dose) in the ﬁeld (Tier II). In the pot experiment, the x100 and x1000 dose rates signiﬁcantly reduced plant biomass, mycorrhizal colonization and AM fungal richness as determined by DGGE. This coincided with the progressive accumulation of herbicide concentrations in soil. In contrast, no effects on AM fungi were observed at the nicosulfuron dose rates tested in the ﬁeld. Clone libraries showed that the majority of AM fungi belonged to the Glomus group and were sensitive to the high levels of nicosulfuron accumulated in soil at the latter culture cycles. In contrast, a Paraglomeraceae and a Glomus etunicatum ribotype were present in maize roots in all cycles and dose rates implying a tolerance to nicosulfuron-induced stress. Overall, the deleterious effects of nicosulfuron on AM fungi induced by the highest dose rates in the pot experiment could be attributed either to fungal-driven toxicity or to plant-driven effects which have subsequent implications for mycorrhizal symbiosis. We suggest that the tiered pot-to-ﬁeld experimental approach followed in our study combined with classic and standardized molecular tools could provide a realistic assessment of the toxicity of pesticides onto AM fungi as potential bioindicators. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Arbuscular mycorrhizal (AM) fungi are obligate symbiotic microorganisms living in association with the vast majority of higher plant species providing increasing supply of P and other minerals, drought tolerance and resistance to pests and diseases
Abbreviations: AM fungi, arbuscular mycorrhizal fungi; ALS, acetolactate synthase; HPLC, high performance liquid chromatography; DGGE, denaturating gradient gel electrophoresis; FOK, ﬁrst order kinetics. ∗ Corresponding author. Tel.: +30 2410 565294; fax: +30 2410 565290. E-mail addresses: [email protected]
, [email protected]
(D.G. Karpouzas). 1470-160X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ecolind.2013.12.004
(Kiers et al., 2011). In agricultural systems AM fungi are exposed to diverse inputs including fertilizers and pesticides. The generally negative effect of phosphorus fertilizers on AM fungi is well documented (Smith et al., 2011). However, less is known regarding their interactions with pesticides. Previous studies showed that AM fungi respond in various ways to pesticide exposure. Fungicides like fenpropimorph and fenhexamid had a clear inhibitory effect on AM fungi (Zocco et al., 2011). In contrast, insecticides like aldicarb, fenamiphos and dimethoate had no inhibitory effect (Nemec, 1981; Schweiger and Jakobsen, 1998) or even stimulated AM fungal colonization and P uptake (Spokes et al., 1981). Finally herbicides could impact AM fungi either directly (Pasaribu et al., 2011; Li et al., 2013) or indirectly by exerting phytotoxicity to their plant hosts (Druille et al., 2012). All the above suggests that AM fungi are generally
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responsive to pesticide exposure. This combined with their key role on plant diversity and functioning of above ground ecosystems (Van der Heijden et al., 1998) makes them good candidate bioindicators for assessing the soil microbial toxicity of pesticides (Wan and Rahe, 1998; Giovannetti et al., 2006). This is in line with the establishment by the International Standard Organization of the ISO10832 standard ‘Effects of pollutants on mycorrhizal fungi’. Despite that the utilization of appropriate experimental protocols and well-standardized methods are necessary for a comprehensive assessment of the toxicity of pesticides onto non-target soil microbes like AM fungi, especially in view of the revision of the relevant regulatory framework at EU level (Martin-Laurent et al., 2013). In the past most studies have used plant growth, AM fungal colonization and/or P uptake to demonstrate potential toxicity of pesticides on AM fungi (Schweiger and Jakobsen, 1998; Druille et al., 2012). In vitro tests in Ri T-DNA-transferred carrot roots have been also used to assess the toxicity of pesticides on AM fungi (Wan and Rahe, 1998; Li et al., 2013). However, these tests do not provide any information regarding the impact of pesticides on the diversity and community structure of AM fungi. The fast development of molecular tools during the past 10 years (Rosendahl, 2008) allowed us to go beyond the simple observation of the indirect effects of pesticides on the performance of mycorrhizal plants, and further explore the direct effect of pesticides on abundance, diversity and function of the micro-symbiotic partner. Nicosulfuron is a low-dose, high-potency herbicide of the sulfonylureas group which is considered the most important group of new era herbicides currently available on the global market (Sarmah and Sabadie, 2002). It is used for the post-emergence control of annual grass and broad-leaf weeds in maize (Hinz and Owen, 1996). In plants, nicosulfuron acts by inhibiting acetolactate synthase (ALS) which catalyzes the ﬁrst common step in the biosynthesis of the branched-chain amino acids leucine, valine, and isoleucine (Babczinski and Zelinski, 1991). This enzyme can be also found in bacteria and fungi. Previous in vitro studies have showed that ALS-inhibitors like nicosulfuron could inhibit this enzyme in microbes (Falco and Dumas, 1985; LaRossa and Schloss, 1984). Despite that, little is known regarding the effect of nicosulfuron on non-target soil microbes. In the only study currently available, Seghers et al. (2005) showed that nicosulfuron did not induce signiﬁcant changes on the abundance and function of soil methanotrophs, whereas transient changes on community structure were evident. The main aim of the current study was to assess the soil microbial ecotoxicity of nicosulfuron using AM fungi as bio-indicators. This was done through a pot-to-ﬁeld tiered approach which allowed us to monitor the potential side effects of the herbicide on the abundance and diversity of AM fungi at both extreme (pot, Tier I) and realistic (ﬁeld, Tier II) exposure scenarios. Possible effects of nicosulfuron on plant growth, colonization capacity and intraradical AM fungal community were investigated with a combination of classical and molecular methods allowing the identiﬁcation of members of the AM fungal community that are either sensitive or tolerant to nicosulfuron exposure. In parallel, the dissipation of nicosulfuron in soil was determined to identify possible correlations between pesticide residues and toxicity.
2. Materials and methods 2.1. Experimental setup – pot experiment A pot experiment was established to assess the impact of nicosulfuron on AM fungi under extreme exposure conditions. The soil used was obtained from the ﬁeld site in Serbia, Novi Sad where the
ﬁeld experiment described below was established. The ﬁeld site did not have a recent history of previous nicosulfuron use. The soil was characterized as loamy clay (36.8% sand; 35.8% silt; 27.4% clay) with pH 6.75, organic carbon content 2.59%, total N 1.92 g kg−1 , CaCO3 1.4 g kg−1 , P2 O5 0.084 g kg−1 and K2 O 0.273 g kg−1 . The soil was initially sieved to pass through a 2 mm mesh and was distributed into 20 plastic pots (4.5 kg of soil dry weight per pot). The soil in the pots were watered to adjust the moisture content of the soil to 70% of its maximum water holding capacity and was preincubated at room temperature for a week. Each pot was seeded with 10 maize seeds which after emergence were thinned to four seedlings per pot. When maize plants reached the 3–4 leaves stage (at approximately 10 days after sowing) the pots were separated into four groups comprising ﬁve pots each. The pots in the ﬁrst three groups were treated uniformly with different dose rates of nicosulfuron corresponding to x10, x100 and x1000 the recommended dose (60 g a.i. ha−1 corresponding to 0.047 mg kg−1 soil dry weight). These dose rates are particularly high compared to the recommended dose and they were chosen to evaluate toxicity under extreme exposure schemes simulating a tier I toxicity assessment. In all cases, the commercial formulation of the herbicide ACCENT® (750 mg kg−1 ) provided by DuPontTM was used. The ﬁnal set of pots received the same amount of sterile water without pesticide to serve as non-treated controls. Plants were grown under controlled temperature (23–25 ◦ C), at 11-h day period and 60% relative humidity. Soil water content was maintained by daily watering (w/v). The plants were harvested after 6 wks by cutting at the base of the stem and the root system was carefully removed, washed free of soil and the shoot and root dry weight (60 ◦ C oven for 2 days) was determined. Root samples were taken for estimation of AM fungal root colonization, while root fragments were also stored at −20 ◦ C for subsequent molecular analysis. In addition, soil subsamples were collected from each pot for analysis of nicosulfuron residues in the soil at the end of each culture cycle. Upon harvest the soil in each pot was re-treated with the same dose rate of nicosulfuron and handled as described above. In total ﬁve culture cycles were performed. The repeated application scheme followed in the pot experiment allowed us to determine the potential toxicity of nicosulfuron on AM fungi on a long-term exposure basis. This is in line with previous studies which suggested that single application approaches do not provide a realistic picture of the potential soil microbial toxicity of pesticides and long-term studies are required to establish a real pesticide-stress condition for the terrestrial microbiota (Zabaloy et al., 2012). 2.2. Experimental set up – ﬁeld experiment A ﬁeld experiment was conducted in a site in the area of Rimski Sancevi (19◦ 51,321 to 45◦ 19,928) Novi Sad, Serbia to assess the impact of nicosulfuron on AM fungi under realistic exposure conditions. A randomized complete block design was followed with four replicate micro-plots (6 m × 5 m) per nicosulfuron dose rate. The soil of the ﬁeld site was on maize cultivation for the last few years. The ﬁeld was seeded with maize (Zea mays variety NS640) in 28 April 2011 at distances of 25 cm within rows and 75 cm between rows using a pneumatic seeder. A 75 cm-wide margin was left between plots to minimize any cross contamination between treatments. Maize emerged a week later and nicosulfuron (ACCENT® , 750 mg kg−1 ) was applied to the different plots at three dose levels (x1, x2 and x5 the recommended dose) in 4 June 2011 using a backpack sprayer. These dose rates represent a realistic exposure scenario simulating a tier II toxicity assessment. For comparison purposes, a treatment where no herbicide was applied was included. Before treatment and at 2, 7, 14, 28, 56 and 116 days post application soil samples were collected from each plot (ﬁve samples collected from the top 10 cm of each plot were homogenized
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providing a composite sample per plot) to assess pesticide dissipation. Further, at 28 days post application ﬁve plants per plot were uprooted with a spade and cut at soil level. Roots were removed, washed free of soil and bulked to one per block for each treatment for AMF root colonization estimation. In addition, fragments of the roots where stored at −20 ◦ C for molecular analysis. 2.3. Determination of nicosulfuron residues Extraction of nicosulfuron from soil was performed using an accelerated solvent extraction system (ASE 200; Dionex, Sunnyvale, CA, USA). Soil samples (5–30 g) were extracted three times with ultrapure water (Millipore, Bedford, MA, USA) as previously described by Degenhardt et al. (2010). The extracts were cleaned up via solid phase extraction as previously described by Said-Pullicino et al. (2004) with slight modiﬁcations. Thus, extracts were initially passed under vacuum (ﬂow rate 5 mL min−1 ) through a Strata-X cartridge (200 mg, 6 mL) (Phenomenex, Torrance, USA) which had been preconditioned with 5 mL of methanol and 5 mL of ultrapure water. After percolation cartridges were washed with 5 mL of ultrapure water and dried under a nitrogen stream for 15 min. Nicosulfuron was eluted with 6 mL of methanol. The eluate was acidiﬁed with 50 L of formic acid and was passed through a second StrataNH2 cartridge (500 mg, 6 mL) which had been pre-conditioned with 5 mL of 1% formic acid in methanol. The clean sample was evaporated to dryness under a gentle nitrogen stream and the residues were re-dissolved in 1 mL of an acetonitrile–water (1:4) mixture and ﬁltered through a 0.2-m membrane before High Performance Liquid Chromatography (HPLC) analysis. Samples were analyzed on a Varian ProStar HPLC system equipped with a diode array detector system (Varian, Walnut Creek, CA, USA). Nicosulfuron was separated on a Zorbax Eclipse® XDB-C18 (5 m, 250 mm × 4.6 mm, Agilent, USA) column with C18 guard column (4 mm × 3 mm i.d.) (Phenomenex, UK) using a mobile phase of acetonitrile (A) and 0.2% acetic acid in water (B) at a ﬂow rate of 1.0 mL min−1 and using a gradient elution previously described by Guibiao et al. (2006). Nicosulfuron detection was achieved at 245 nm and its retention time was 7.9 min. Tests at four concentration levels (0.05, 0.25, 0.50 and 5 mg kg−1 ) showed recoveries > 85% at all fortiﬁcation levels. Analytical grade nicosulfuron was used in all analytical tests (Fluka Buchs, Switzerland). 2.4. AM fungal colonization Roots were cleared in 10% KOH for 45 min at 80 ◦ C, rinsed in tap water and immersed in 2.5% HCl for 30 min. Subsequently roots were stained with Trypan blue (Sylvia, 1994). Mycorrhizal colonization was estimated on slides according to the improved method described by McGonigle et al. (1990). 2.5. DNA extraction, PCR and DGGE DNA was isolated from 0.1 to 0.2 g roots wet weight by grinding with mortar and pestle in liquid N and subsequently using the Nucleospin® Plant kit (Macherey-Nagel, Germany). A semi-nested PCR approach was followed as previously described by Ipsilantis et al. (2012). In the ﬁrst step the primer pair AML1–AML2 (Lee et al., 2008) was utilized leading to the ampliﬁcation of an 800 bp fragment of the 18S rRNA gene. The resulting product was reampliﬁed with primers NS31-GC (Kowalchuk et al., 2002) and Glo1 (Cornejo et al., 2004). Thermocycling conditions were as described by Lee et al. (2008) and Liang et al. (2008) for the ﬁrst and second reaction, respectively. Denaturating gradient gel electrophoresis (DGGE) analysis was carried out on an INGENYphorU-2x2 system (Ingeny International BV, The Netherlands) as previously described by Ipsilantis et al. (2012). Gel electrophoresis images were captured
by a digital camera and subsequent analysis of all DGGE gel pictures was done with the Cross-Checker software (available from http:// www.plantbreeding.wur.nl/UK/software crosschecker.html). 2.6. Clone libraries Clone libraries of the AM fungal community were established for selected samples from the pot experiment. The selection of the root samples which were processed for cloning was based on the presence of bands in their DGGE ﬁngerprint that either disappear or remain unaffected in response to nicosulfuron treatment. This strategy allowed us to identify the members of the AM fungal community which were either sensitive or tolerant to nicosulfuron applications. Products of all three replicates from the ﬁrst PCR carried out with primers AML1-AML2 were combined to provide enough DNA for cloning. PCR products were puriﬁed using the Nucleospin Extract II kit (Macherey-Nagel, Germany). Cloning into the pGEM-T vector (Promega, Madison, USA) was performed following standard procedures (Sambrook et al., 1989). Screening of the clone libraries by PCR and DGGE was carried out as described by Liang et al. (2008). For sequencing, plasmid DNA was extracted by the NucleoSpin plasmid kit (Macharey-Nagel, Germany) and sent for sequencing analysis. Sequences were deposited in the European Molecular Biology Laboratory database under accession numbers HF054003–HF954099. Similarity comparisons to known 18S rRNA gene sequences in the database, were performed using the online program Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/BLAST). 2.7. Data analysis Mycorrhizal colonization data were arcsine transformed to normalize their distributions. Mycorrhizal colonization, plant growth data and nicosulfuron concentrations in soil were subjected to mixed effects model analysis with dose level and cycle as ﬁxed effects and pot identity as a random effect. In cases where signiﬁcant interactions between the two ﬁxed effects were observed one way ANOVA was applied to identify signiﬁcant effects of dose rates in each culture cycle. Data for the presence and absence of bands in the DGGE ﬁngerprints obtained after processing with the CrossChecker software were subjected to hierarchical cluster analysis with Jaccards distance matrix using the Multivariate Statistical Analysis Software 3.13v (http://www.kovacomp.com). The nicosulfuron dissipation pattern in the ﬁeld experiment was ﬁtted to the ﬁrst order kinetics (FOK) or the hockey-stick model and half-life (t1/2 ) values were calculated. The 2 test was used to evaluate the quality of the ﬁt of each model to the dissipation data, with values below 15% indicating that the 2 test is fulﬁlled at a given degrees of freedom (at 5% signiﬁcance level). Parameters of the kinetic models were estimated by least square regression using SPSS 16.0 statistical program. 3. Results 3.1. Dissipation of nicosulfuron in soil 3.1.1. Pot experiment In the pot experiment, nicosulfuron showed a similar behavior regardless of the dose rate applied with an initial effective reduction of its residues until cycle 2 and a progressive and signiﬁcant (p < 0.001) build up of nicosulfuron residues in soil in the following cycles and until the end of the pot experiment (Fig. 1a). 3.1.2. Field experiment A relatively rapid dissipation of nicosulfuron was evident in the ﬁeld experiment regardless of the dose rate used (Fig. 1b). None
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Fig. 1. The concentration of nicosulfuron quantiﬁed in soil at the end of culture/treatment cycles 2–5 (presented as % difference from the corresponding concentration at cycle 1) in the pot experiment; the measured concentrations of nicosulfuron in soil at the same culture/treatment cycles are given in the insert graph (a), and the dissipation of nicosulfuron in the ﬁeld (b). Each value is the mean of ﬁve (pot) or four (ﬁeld) replicates with error bars representing the standard deviation of the mean. In the pot experiment, bars within each dose rate designated with same letters are not signiﬁcantly different (p = 0.05).
Table 1 Half life (t1/2 ), correlation coefﬁcient (R2 ) and 2 error estimated by ﬁtting the nicosulfuron dissipation curves observed in the ﬁeld experiment with either the ﬁrst order kinetics (FOK) or the hockey-stick model. Model
Nicosulfuron dose rates x1
First order kinetics
t1/2 (days) r2 2 (%)
4.7 1.000 25.1
4.0 0.956 19.8
4.5 0.944 21
t1/2 (days) r2 2 (%)
3.6 0.999 29.9
3.5 0.998 26.4
3.7 0.998 29.1
of the two kinetic models tested adequately ﬁtted the dissipation patterns of nicosulfuron in the ﬁeld experiment (2 values > 15%, Table 1). Nevertheless, the t1/2 values calculated with the FOK model showed that the dissipation of the different dose rates of nicosulfuron were similar and ranged from 4 days in the x2 dose rate to 4.7 days in the x1 dose rate (Table 1). 3.2. Plant growth and mycorrhizal colonization in the pot experiment Shoot and root biomass were signiﬁcantly affected by both cycle number and nicosulfuron dose rates (p < 0.001). Further analysis per cycle showed that the application of the x1000 dose rate significantly decreased (p < 0.001) the shoot and root biomass of maize plants at cycle 1 (Fig. 2a) and entirely prevented growth of maize from cycle 2 and onwards (Fig. 2b–e). On the other hand, the application of the x100 dose rate did not have a signiﬁcant effect
(p > 0.05) on plant biomass at cycle 1 but signiﬁcantly impaired (p < 0.001) maize growth from cycle 2 and onwards. Finally the application of the x10 dose rate did not induce signiﬁcant effects (p > 0.05) on plant growth with the only exception of cycle 4 where marginally signiﬁcant decreases in shoot and root biomass were observed (Fig. 2d). Overall, maize plants were successfully colonized by AM fungi in the pot experiment with hyphal mycorrhizal colonization ranging from 20 to 80%. Hyphal colonization was signiﬁcantly affected by the dose rates (p < 0.05) and the culture cycle (p < 0.001). Further analysis of dose effects per cycle showed that the x1000 dose rate induced a signiﬁcant reduction (p < 0.001) in hyphal colonization at the end of cycle 1 (Fig. 3a), however the phytotoxicity observed in subsequent cycles did not allow any further measurements. The x100 dose rate of nicosulfuron induced a signiﬁcant increase (p < 0.001) in hyphal colonization compared to the x10 dose rate and the control at cycle 2 (Fig. 3b), whereas the same dose rate induced a signiﬁcant decrease (p < 0.05) in hyphal colonization in subsequent cycles (Fig. 3c and e). In contrast, the x10 dose rate did not induce any signiﬁcant effects on hyphal colonization compared to the untreated samples with the exception of cycle 4 where a signiﬁcant reduction (p < 0.05) was observed (Fig. 3d). Arbuscular colonization was signiﬁcantly affected by culture cycle (p < 0.001) but not by the different dose rates of nicosulfuron (p > 0.05), whereas signiﬁcant interactions between these two main effects were observed (p < 0.01). Further analysis of the effects of dose rates per cycle showed that arbuscular colonization was significantly affected (p < 0.05) by the x100 dose rates at cycle 3 (Fig. 3c) but even by the x10 dose rate at cycle 4 (Fig. 3d), whereas no arbuscules were found in the plants at cycle 5. Vesicular colonization was limited throughout the experiment and was not signiﬁcantly affected (p > 0.05) by the dose rates or the culture cycle and no signiﬁcant interactions were observed. 3.3. Mycorrhizal colonization on maize plants in the ﬁeld experiment Maize plants showed relatively high levels of hyphal colonization (30–40%) at 28 days post application (Supplementary Data Fig. S1). No signiﬁcant effect (p > 0.05) of nicosulfuron on hyphal, arbuscular and vesicular colonization was found. 3.4. The impact of nicosulfuron on the AM fungal community 3.4.1. Pot experiment Results of DGGE ﬁngerprints revealed that nicosulfuron changed markedly the structure of the AM fungal community (Fig. 4). Thus, in cycle 1 the x1000-treated samples showed an altered community compared to the x10 and x100-treated samples which clustered together and distantly from the untreated samples. In subsequent cycles, the x100 dose rate induced a gradual eradication of the AM fungal community with those samples clustering away from the other treatments (Figs. 4b, c and e). In contrast, the samples treated with the x10 dose rate clustered close to the control samples in all subsequent cycles (Figs. 4b, c and e) with the exception of cycle 4 where the x100 dose had a phytotoxic effect on maize plants (Fig. 4d). Clone libraries obtained from the different cycles showed that in most cases bands from different treatments and/or cycles showing identical electrophoretic mobility represented the same fungal ribotype. Based on this all DGGE bands where given a two-digit number where the ﬁrst digit indicated the culture cycle number and the second digit their relative electrophoretic mobility. Thus bands with the same ﬁrst digit were obtained from treatments of the same culture cycle, while a common second digit suggested identical electrophoretic mobility regardless of the culture cycle.
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Fig. 2. The shoot and root biomass of the maize plants collected at the end of culture/treatment cycles 1 (a), 2 (b), 3 (c), 4 (d) and 5 (e) in the pot experiment. Each value is the mean of ﬁve replicates with error bars representing the standard deviation of the mean. The levels of signiﬁcance obtained after statistical analysis are also shown. Bars designated by the same letter indicate no statistical difference.
Clone libraries suggested that the intraradical AM fungal community was dominated by Glomus ribotypes (bands x.2, x.3, x.4, x.7, and x.9) with the exception of a Paraglomeraceae (band x.8) and a Diversispora (band x.2B) ribotype which were present in maize roots in most culture cycles (Table 2). Overall, bands x.2, which showed highest sequence identity to an uncultured Glomus clone (Table 2), were present in the x10 treated samples but vanished from the ﬁngerprints of the x1000- (cycle 1) and the x100-treated samples (cycles 2, 3 and 5). Band x.3 (highest homology to Glomus mosseae isolate), which constituted the most dominant band in the x0- and x10-treated samples in all cycles, was sensitive to the higher dose rates and disappeared from their respective ﬁngerprints from cycle 2 onwards. In contrast, bands 2.7, 2.5 and 2.6 (highest homology to uncultured Glomus ribotypes) became dominant in the x100 dose rate treatment at cycle 2 but disappeared in subsequent cycles of the same treatment. Finally bands x.8 and x.9 were present in all treatments and cycles (Fig. 4). Clones associated with band x.8 shared highest identity to different uncultured Glomus (1.8, 4.8, 5.8) or Paraglomeraceae clone (2.8). Clones retrieved from band x.9 showed highest identity to a Glomus etunicatum isolate. 3.4.2. Field experiment DGGE analysis revealed a well established community of AM fungi in maize roots (Supplementary Data Fig. S2). Cluster analysis of the ﬁngerprints obtained at 28 days post application did not show a clear herbicide effect. At 0.3 Jaccard similarity index ﬁngerprints were grouped in two main clusters (Fig. 5). The ﬁrst
cluster includes (i) a sub-cluster comprising untreated control samples (x0), the samples treated with the x2 dose rate and a replicate of the x5 dose rate and (ii) a second sub-cluster comprising all the x1 dose rate samples. The remaining three replicates of the x5 dose rate formed a second major cluster. 4. Discussion Pesticides remain one of the pillars of modern agriculture ensuring high yields and quality of crops. Despite that agricultural practices including pesticides have been identiﬁed as a major threat for soil biodiversity and functioning (Van-Camp et al., 2004). In this context, pesticide interactions with non-target soil microorganisms like AM fungi known to play a key role in plant diversity and ecosystem functioning should be explored. We investigated the impact of nicosulfuron, a low-dose herbicide, on the colonization capacity and community structure of AM fungi in maize under two exposure schemes: a worst-case exposure scenario realized in a pot experiment and a realistic exposure scenario tested under ﬁeld conditions. Concurrently the impact of nicosulfuron on the plant host was also explored to decipher whether possible effects of the herbicide on AM fungal community are either direct or indirect appearing as a result of phytotoxicity induced to the host plant. 4.1. Pot experiment In the pot experiment, the repeated applications of nicosulfuron at rates substantially higher than the rates used in the ﬁeld
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Fig. 3. The colonization of maize plants by AM fungi (hyphae, arbuscules and vesicles) at the end of culture/treatment cycles 1 (a), 2 (b), 3 (c), 4 (d) and 5 (e) in the pot experiment. Each value is the mean of ﬁve replicates with error bars representing the standard deviation of the mean. The levels of signiﬁcance obtained after statistical analysis are also shown. Bars designated by the same letter indicate no statistical difference.
experiment induced drastic changes in plant growth, establishment of symbiosis and the diversity and community structure of AM fungi. We initially hypothesized that along the ﬁve culture/treatment cycles repeated applications of nicosulfuron at different dose levels will either result in a (1) gradual adaptation of the soil microbial community to the herbicide, rapid degradation and eventually lack of any adverse effects on plant and AM fungi or (2) gradual build up of pesticide residues with the potential of adverse effects on AM fungi. The soil concentration of nicosulfuron measured at the end of each culture cycle veriﬁed our second hypothesis resulting in a gradual accumulation of nicosulfuron residues from cycle 3 and onwards. The repeated applications of nicosulfuron impaired (x100) or entirely halted maize growth (x1000) from cycle 2 onwards. In contrast, the application of the x10 dose rate induced a decrease in the biomass of plants shoots and roots only at the last two cycles, when the concentration of the herbicide in soil had increased to levels signiﬁcantly higher than the initial dose rate. Although maize is tolerant to post-emergence application of nicosulfuron (Hinz and Owen, 1996), the levels of the herbicide accumulating in soil during the ﬁve culture/treatment cycles were probably too high to be tolerated by the crop. Mycorrhizal colonization showed a similar response as plant biomass with the x100 dose rate drastically limiting the establishment of AM fungi in maize roots. This could be the result of either direct toxicity of the herbicide on the capacity of AM fungal propagules to grow and colonize plant roots or a result of an indirect effect on maize whose tolerance to nicosulfuron is based on its capacity to rapidly detoxify the herbicide (Carey et al., 1997).
As ALS-inhibiting compounds like nicosulfuron have been found to induce inhibitory effects on different fungal strains including soil-borne plant pathogens (Belai and Oros, 1996), the possibility of direct toxicity of nicosulfuron on AM fungi residing in soil cannot be ruled out. Recent in vitro tests with the herbicides prometryn and acetochlor showed a direct negative effect on the physiology and metabolic activity of a G. etunicatum isolate (Li et al., 2013). On the other hand, the accumulation of extremely high concentrations of nicosulfuron in soil might have overwhelmed the detoxiﬁcation mechanism of maize which in turn led to an inhibition of the synthesis of branched-chain amino acids and limited growth (Babczinski and Zelinski, 1991). Indeed, Trappe et al. (1984) have suggested that herbicides could signiﬁcantly alter the metabolism of plants resulting in reduced photosynthates production thereby limiting the establishment of AM symbiosis. An exception to the general reduction pattern was the signiﬁcant increase in the hyphal colonization observed in the plants treated with the x100 dose rate at cycle 2. A possible explanation for this is the selective exclusion of certain members of the AM fungal community by the high nicosulfuron levels. This may result in the establishment of a lowcompetition environment for less abundant nicosulfuron-tolerant AM fungi to proliferate and colonize plants in the absence of other proliﬁc AM fungal colonizers. A similar observation was reported by Ipsilantis et al. (2012) who noted that azadirachtin application induced an increase in overall mycorrhizal colonization of pepper roots but, at the same time, selectively inhibited one of the members of the AM fungal community thus reducing the competition for the other two members to rapidly proliferate in plant roots. Overall,
D.G. Karpouzas et al. / Ecological Indicators 39 (2014) 44–53
Fig. 4. 18S rRNA DGGE ﬁngerprints and corresponding cluster analysis (Jaccards similarity index) of the AM fungal community in maize roots at the end of each culture/treatment cycle in the pot experiment. Dose rates (x100 and x1000) not appearing in the different cycles had a phytotoxicity effect on maize plants. M: marker comprised of a mixture of PCR products of AM fungal isolates presented with the sequence they appear in the gel from top to bottom (indicated also by arrows): Glomus mosseae A (GmA), G. mosseae B (GmB), G. intraradices (G.i.) and G. etunicatum (G.e). Bands designated by a number were sequenced and the identities of the relevant 18S rRNA ribotypes are given in Table 2. All bands were given a two-digit number where the ﬁrst digit indicated the culture cycle number and the second digit a number relative to their electrophoretic mobility. Thus bands with the same ﬁrst digit were obtained from treatments of the same culture cycle, while a common second digit suggested identical electrophoretic mobility regardless of the culture cycle.
the drastic reduction in plant growth and mycorrhizal colonization with repeated applications of the x100 dose is in line with the accumulation of high herbicide levels in the soil at the later culture cycles. The general inhibitory trend observed on plant growth and mycorrhizal colonization in response to application of the x100 dose rate was further supported by the DGGE ﬁngerprinting analysis of the intraradical mycorrhizal community in maize roots where a decrease in the diversity of AM fungi from cycle 2 onwards was observed. In contrast, the application of the x10 dose rate did not appear to induce large changes in the community of AM fungi compared to the untreated control. Clone libraries revealed that maize
roots were mostly colonized by members of the Glomus group. This is in line with most studies carried out in soils taken from ﬁeld sites with increasing agricultural disturbances (Hijri et al., 2006). Certain members of the AM fungal community, sensitive to elevated exposure to nicosulfuron, failed to colonize plant roots. A particular case was the response of the ribotypes represented by bands 2.5, 2.6 and 2.7, to nicosulfuron treatments in the pot experiment. These ribotypes were found to rapidly colonize maize roots exposed to the x100 dose in cycle 2, but failed to colonize plant roots of the same treatment in subsequent cycles. This colonization pattern could be attributed to the increasing herbicide concentrations recorded in the soil in subsequent culture cycles. It also provides
D.G. Karpouzas et al. / Ecological Indicators 39 (2014) 44–53
Table 2 Identity of AM fungal ribotypes associated with DGGE bands obtained after cloning of 18S rRNA sequences ampliﬁed from DNA extracted from maize roots from the pot experiment. The ﬁrst digit of the band number indicates the number of nicosulfuron treatment cycle (cycles 1–5) and the second digit the number of actual band. Bands with the same second digit are obtained from different cycles but show identical electrophoretic mobility. Band no.
No. of clones sequenced
Closest match from GenBank (% sequence similarity by BLAST)
GenBank accession no.
1.1 1.2 1.3 1.4 1.7 1.8 1.9 2.2 2.2B 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.2 3.2B 3.3 3.4 3.7A 3.7B 3.8 3.9 4.1 4.2 4.2B 4.3 4.3B 4.3C 4.4 4.7A 4.7B 4.8 4.9 5.2 5.2B 5.3 5.4 5.6 5.7 5.8 5.9
1 4 8 1 6 1 2 2 1 1 2 2 2 2 5 2 2 2 3 1 3 4 1 3 1 4 1 6 1 3 1 1 3 3 2 3 1 5 1 1 2 1 3
Uncultured Glomus clone U06-31 (99.7%) Uncultured Glomus isolate AVE clone 1–15 (98.7%) Glomus mosseae isolate MC10 (99.3%) Uncultured Glomus clone G+I9-12 (99.7%) Uncultured Glomus clone G3-7 (99.7%) Uncultured Glomus clone 0168 (97.5%) Glomus etunicatum isolate UFPE06 (99.1%) Uncultured Glomus isolate AVE clone 1–15 (98.6%) Uncultured Diversispora clone 3904 PAF NF31 (100%) Glomus mosseae isolate MC10 (99.9%) Uncultured Glomus clone G+I9-12 (99.6%) Glomus irregulare clone 14 (99.9%) Glomus irregulare clone 14 (99.9%) Uncultured Glomus clone G3-7 (99.7%) Uncultured Paraglomeraceae clone 253AM1 1 (99.6%) Glomus etunicatum isolate UFPE06 (99.9%) Uncultured Glomus isolate AVE clone 1–15 (98.6%) Uncultured Diversispora clone 3904 PAF NF31 (99.2%) Glomus mosseae isolate MC10 (99.5%) Uncultured Glomus clone G+I9-12 (99.6%) Uncultured Glomus clone G3-7 (99.7%) Uncultured Glomus clone G3-7 (99.7%) Uncultured Paraglomeraceae clone 253AM1 1 (99.6%) Glomus etunicatum isolate UFPE06 (100%) Uncultured Glomus clone U06-31 (99.3%) Uncultured Glomus isolate AVE clone 1–15 (98.7%) Uncultured Diversispora clone 3904 PAF NF31 (99.2%) Glomus mosseae isolate MC10 (99.5%) Glomus mosseae strain INVAM UT101 (99.9%) Uncultured Glomus clone Ku 4 2 (99.2%) Uncultured Glomus clone G + I9-12 (100%) Uncultured Glomus clone G3-7 (99.5%) Uncultured Glomus clone G3-7 (99.7%) Uncultured Glomus clone j-18.3r (100%) Glomus etunicatum isolate UFPE06 (99.5%) Uncultured Glomus isolate AVE clone 1–15 (98.1%) Uncultured Diversispora clone 3904 PAF NF31 (99.2%) Glomus mosseae isolate MC10 (99.2%) Uncultured Glomus clone G + I9-12 (99.6%) Glomus irregulare clone 14 (99.9%) Uncultured Glomus clone G3-7 (99.7%) Uncultured Glomus clone j-18.3r (100%) Glomus etunicatum isolate UFPE06 (99.9%)
AB698581 HE615074 FR847093 FR693619 FR693579 AB594850 AJ852598 HE615074 FJ831658 FR847093 FR693619 FJ009618 FJ009618 FR693579 JN644452 AJ852598 HE615074 FJ831658 FR847093 FR693619 FR693579 FR693579 JN644452 AJ852598 AB698581 HE615074 FJ831658 FR847093 NG017178 AB695033 FR693619 FR693579 FR693579 GQ140618 AJ852598 HE615074 FJ831658 FR847093 FR693619 FJ009618 FR693579 GQ140618 AJ852598
evidence for the existence of a threshold inhibition concentration which certainly varies among the members of the AM fungal community in a given soil. This is in agreement with Trappe et al. (1984) who suggested that there is an inherent genetic variation among AM fungi with respect to pesticide tolerance. Subsequent studies further supported this hypothesis suggesting that under pesticide exposure signiﬁcant shifts in the structure and the diversity of the AM fungal community could be found with signiﬁcant consequences for plant growth, plant diversity and soil stability (Schreiner and Bethlenfalvay, 1997). Other AM fungal ribotypes (i.e. x.8, uncultured Glomus or Paraglomeraceae clones; x.9, G. etunicatum isolate) were not affected by nicosulfuron and colonized plant roots under all exposure scenarios tested. The appeared tolerance of the G. etunicatum ribotype (band x.9) to nicosulfuron observed in our study is in accordance with previous observations. It is noteworthy that Ipsilantis et al. (2012) found a G. etunicatum ribotype with increasing tolerance to azadirachtin. Similarly Schreiner and Bethlenfalvay (1997) reported an elevated tolerance of G. etunicatum to high concentrations of different fungicides compared to other Glomus species tested. Although these results provide a ﬁrst indication of a possible species-dependent tolerance to pesticides more studies are needed to verify if this attribute for G. etunicatum
is an traits.
4.2. Field experiment Nicosulfuron application at levels x5 the recommended dose did not induce signiﬁcant changes neither on the colonization ability nor on the community structure of AM fungi in the ﬁeld experiment. Previous studies looking at pesticide effects on mycorrhizal establishment have shown contrasting results. Thus, Ramos-Zappata et al. (2012) reported that application of the recommended dose of the herbicide paraquat resulted in a signiﬁcant decrease in mycorrhizal colonization, while Mujica et al. (1999) reported that chlorsulfuron and glyphosate signiﬁcantly inhibited mycorrhizal colonization only when used at application rates higher than the recommended. However, others have observed no effects of herbicides on mycorrhizal colonization (Smith et al., 1981). On the other hand, no information is available regarding the impact of ﬁeld applications of herbicides on the intraradical community of AM fungi. Field dissipation of the different dose rates of nicosulfuron proceeded at similar rates with half-life values which are within the range reported in the literature (Poppell et al., 2002). The similar dissipation patterns of nicosulfuron regardless of the dose rate
D.G. Karpouzas et al. / Ecological Indicators 39 (2014) 44–53
Aerospace Center (DLR, Bonn, Germany) for their helpful and efﬁcient management of SEE-ERA-NET-PLUS program.
x0 NS x0 NS x0 NS
Appendix A. Supplementary data
x2 NS x2 NS x2 NS x0 NS
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ecolind. 2013.12.004.
x2 NS x5 NS
x1 NS x1 NS x1 NS x1 NS x5 NS x5 NS x5 NS
Jaccards similarity index Fig. 5. Cluster analysis (Jaccards similarity index) using the binary data matrix obtained by the 18S rRNA DGGE ﬁngerprinting of the AM fungal community in the roots of maize plants treated with nicosulfuron at rates x0, x1, x2 and x5 the recommended.
applied is in line with the lack of any effects on mycorrhizal establishment at the dose rates tested. 5. Conclusions An up-scaling approach was used to evaluate the toxicity of nicosulfuron on the capacity of AM fungi to colonize maize rots and on the structure of the AM fungal community. The herbicide induced a signiﬁcant reduction in the colonization capacity and in the richness of the AM fungal community only when repeatedly applied at dose rates x100 higher than the recommended. It is noteworthy that application of nicosulfuron at lower dose rates (x10 higher than the recommended or lower) did not induce alterations on AM fungal colonization and on the structure of the AM fungal community. The effects observed coincided with phytotoxicity of nicosulfuron on maize indicating that the effects on mycorrhization could be plant-driven. Molecular ﬁngerprinting analyses, cloning and sequencing of all main AM fungal ribotypes led to the identiﬁcation of key members of the mycorrhizal community which were able to colonize plant roots despite the high soil concentrations of nicosulfuron and the physiological stress of the host plant. Our study has major practical implications regarding the forthcoming revision of the regulatory framework for the assessment of the soil microbial toxicity of pesticides. The tiered pot-to-ﬁeld approach followed in our study coupled with classic and advanced molecular tools could provide a clear assessment of the soil toxicity of pesticides to AM fungi used as microbial indicators. Acknowledgements This study was ﬁnanced by the European Community within the FP7 through the SEE-ERA-NET PLUS project (Project No. 216/1) ‘ECOFUN-MICROBIODIV Development and evaluation of innovative tools to estimate the ecotoxicological impact of low dose pesticide application in agriculture on soil functional microbial biodiversity’ (http://www4.inra.fr/ecofun microbiodiv/). The authors would like to thank Prof. Ralf Hanatscheck and Christian Schache from the International Bureau of the Federal Ministry of Education and Research at the Project Management Agency c/o German
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