Impact of multi-resistant transgenic Bt maize on straw decomposition and the involved microbial communities

Applied Soil Ecology 73 (2014) 9–18

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Impact of multi-resistant transgenic Bt maize on straw decomposition and the involved microbial communities Regina Becker, Ben Bubner 1 , Rainer Remus, Stephan Wirth, Andreas Ulrich ∗ Leibniz Centre for Agricultural Landscape Research (ZALF), Institute for Landscape Biogeochemistry, Müncheberg, Germany

a r t i c l e

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Article history: Received 7 June 2013 Received in revised form 2 August 2013 Accepted 5 August 2013 Keywords: Residue-colonizing bacteria Bacterial and fungal community structure 16S rRNA gene T-RFLP Real-time PCR Maize straw decomposition

a b s t r a c t A general concern associated with the use of transgenic Bt maize is its possible negative influence on nontarget organisms and ecosystem functions such as organic matter transformation. Our study was carried out to assess the impact of the multi-resistant Bt maize hybrid MON 89034 × MON 88017 on straw decomposition and the residue-colonizing microbial communities in comparison to the near-isogenic control and two conventionally bred varieties. Straw decomposition was analyzed by a substrate-induced respiration method and monitoring of the complete decomposition after 14 C pulse labelling of the maize plants. Both approaches indicated significant differences in the decomposition rate between the conventional varieties, but the transgenic Bt hybrid and its near-isogenic control could not be distinguished. Potential effects on the residue-colonizing bacterial and fungal communities were analyzed by the quantification of metabolically active microbial groups using taxon-specific primers and T-RFLP analyses of the small-subunit ribosomal RNA gene. Results obtained over three years and three sampling dates did not reveal significant differences between the transgenic hybrid and the control. The abundance of the metabolically active microbial groups varied between the varieties only in some cases and temporarily restricted. Similarly, T-RFLP analysis did not show an impact of the plant genotype on the bacterial and fungal community structure. In contrast, seasonal effects given by different sampling dates as well as varying soil properties within the field trial were identified as drivers of the microbial communities colonizing the rotting maize straw. Conclusively, the multi-resistant Bt hybrid MON 89034 × MON 88017 did not indicate an adverse impact on straw decomposition and the involved microbial communities. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The cultivation of Bt maize resistant to economically important pests has been steadily increasing since its introduction in 1996. In 2012 transgenic maize varieties, most of which represented Bt maize hybrids, were grown on 55 Mio ha, that is about 35% of the worldwide maize cultivation area (James, 2012). Maize varieties expressing cry genes for the production of crystal delta endotoxins of Bacillus thuringiensis to control the European corn borer (Ostrinia nubilalis) or the corn rootworm (Diabrotica virgifera) provide stable yields and a reduced need for application of insecticides (Klaphengst et al., 2011) as well as an improved food safety by lower mycotoxin levels (Folcher et al., 2010; Ostry et al., 2010). To extend protection, various Bt hybrids have been developed that harbour two or more cry genes originally present in different

∗ Corresponding author at: Leibniz Centre for Agricultural Landscape Research (ZALF), Institute for Landscape Biogeochemistry, Eberswalder Str. 84, D-15374 Müncheberg, Germany. Tel.: +49 33432 82345; fax: +49 33432 82344. E-mail address: [email protected] (A. Ulrich). 1 Present address: Thünen Insitute – Federal Research Institute for Rural Areas, Forestry and Fisheries, Institute of Forest Genetics, Waldsieversdorf, Germany. 0929-1393/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsoil.2013.08.002

parental varieties. These multi-resistant hybrids, also referred to as “stacked events”, combine several potentially interacting transgenic traits, and thus represent new transgenic plants which need to be evaluated in light of ecological risk assessment. A general concern associated with the cultivation of Bt maize is its possible adverse impact on the agro-ecosystem. Both the production of the insecticidal proteins and their release into the environment were hypothesized to affect non-target organisms and ecosystem functions. In particular, effects might be strengthened through the simultaneous use of various cry genes, as contained in the stacked hybrid MON 89034 × MON 88017. Currently, most of the risk assessment of Bt maize is based on single event hybrids that are equipped with only one insecticidal resistance. Influences on the soil biota and functions have been addressed in several studies dealing with the soil and plant associated microflora under Bt maize cultivation and the decomposition of the voluminous crop residues as an important ecological factor in nutrient cycling and carbon sequestration in soils (Mocali, 2011; Yanni et al., 2010). Analyses of the soil and root-associated microbial communities in Bt and non-Bt maize fields resulted in no specific or temporary effects of the transgenic plants (Barriuso et al., 2012; Miethling-Graff et al., 2010; Prischl et al., 2012). The structure of the communities was shown to be less affected by the transgenic

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hybrid and the released cry protein as compared to environmental factors such as plant age or site characteristics (Baumgarte and Tebbe, 2005; Tan et al., 2010). On the other hand, conflicting results have been reported on the crop chemistry and decomposition rate of the plant residues after harvest. While a range of previous studies displayed increased lignin contents (Masoero et al., 1999; Poerschmann et al., 2005; Saxena and Stotzky, 2001) and a slower decomposition rate of Bt maize (Castaldini et al., 2005; Dinel et al., 2003; Flores et al., 2005), several other studies did not reveal such changes of Bt maize under climate chamber and field conditions (Jung and Sheaffer, 2004; Mungai et al., 2005; Zurbrügg et al., 2010). It remains open if these contrasting results are due to the different plant material, growth conditions and methodological approaches. The transformation of organic matter is essentially mediated by the activities and interactions of a multitude of soil microorganisms living in close relation to the plant residues. At present, relatively little research has been done in investigating the microbial communities involved in decomposition of residues from transgenic plants. Studies analysing cultivable soil bacteria and fungi displayed no significant or only short-term effects of the Bt-plant residues (Flores et al., 2005; Mulder et al., 2006). Minor, not lasting or not significant changes within the soil and residue colonizing microflora were also found in polyphasic approaches involving DNA-based DGGE (denaturating gradient gel electrophoresis) and T-RFLP (terminal restriction fragment length polyphormism) analyses targeting ribosomal RNA genes (Tan et al., 2010; Xue et al., 2011). Both, culture techniques combined with complementary methods and the different fingerprint techniques are suitable instruments to detect changes in the microbial communities (Smalla et al., 2007; van Elsas et al., 1998). However, analyses based on extracted sample DNA describe the total microbial community, which includes metabolically active groups as well as dormant and dead cells. Since soil microorganisms are thought to be in large part inactive (Olsen and Bakken, 1987) and DNA can persist in dead cells and as extracellular DNA in soils (Willerslev et al., 2004), these approaches do not necessarily reflect the microbial groups currently performing metabolic functions. Recently, RNA-based methods have been increasingly used to analyze metabolically active members of the microbial communities. The amount of rRNA per cell roughly correlates with the growth activity of bacteria (Molin and Givskov, 1999; Wagner, 1994) and allows the detection of living microorganisms constituting most of the metabolic activity. Though this approach has a couple of pitfalls, such as the extraction of RNA from soil (Sessitsch et al., 2002; Wang et al., 2012), varying ribosome contents per cell and the occurrence of RNA reserves in dormant cells (Sukenik et al., 2012), RNA based surveys represent a suitable strategy for revealing metabolically active microbial communities and detecting links to functional parameters such as organic matter decomposition. Assuming that some of the plant compounds and their ratio are changed by the transgenic modification, the activity as well as the community structure of the decomposing microflora could be influenced. We combined the analysis of straw decomposition and the study of the decomposing microflora to evaluate the impact of the multi-resistant Bt maize hybrid MON 89034 × MON 88017. Our approach includes the quantification of the metabolically active microbial groups by taxon-specific primers, and the T-RFLP analyses of the residue colonizing bacterial and fungal communities.

2. Material and methods 2.1. Maize cultivars Analyses were performed with the stacked Bt maize hybrid MON 89034 × MON 88017 (3-Bt), its near-isogenic control DKC 5143

Table 1 Selected physico-chemical soil propertiesa of the field release experiment in Braunschweig (Germany). For each row of the field release experiment mean values and standard errors of mean were presented. Field row

Soil texture

Sand (%) A B C D E Average a

64.2 57.3 50.6 46.7 47.1 53.2

± ± ± ± ± ±

2.7 1.4 1.8 2.1 2.0 7.0

Total organic C (%) Silt (%) 30.3 37.1 42.2 46.0 45.6 40.2

± ± ± ± ± ±

pH-Value

Clay (%) 2.6 1.3 2.2 1.5 2.1 6.2

5.4 5.6 7.2 7.2 7.3 6.5

± ± ± ± ± ±

0.6 0.5 1.7 0.7 0.3 1.2

2.4 2.6 2.5 2.4 2.4 2.5

± ± ± ± ± ±

1.0 0.9 0.1 0.1 0.2 0.6

5.9 5.9 6.0 6.0 6.0 6.0

± ± ± ± ± ±

0.06 0.02 0.05 0.03 0.04 0.06

Data of the soil texture were obtained from Niemeyer et al. (pers. comm).

(n-iso) which shares most of its genetic background with the transgenic hybrid, and the conventional varieties DKC 4250, Benicia, DKC 3420 and Agrano. MON 89034 × MON 88017 represents a F1 hybrid resulting from the hybridization of maize inbred MON 89034 with MON 88017. The transgenic hybrid produces the Cry1A.105 and Cry2Ab2 proteins, which are active against the European corn borer (Ostrinia nubilalis) and the Cry3Bb1 protein conferring resistance to corn rootworm (Diabrotica virgifera). Additionally, tolerance to glyphosate is provided by the expression of the cp4 epsps gene. Seeds of the maize varieties were obtained from Monsanto Agrar GmbH, Düsseldorf/Germany (MON 89034 × MON 88017, DKC 5143, DKC 4250), Pioneer Hi-Breed GmbH, Buxtehude/Germany (Benicia) and Saatbau Linz, Austria (DKC 3420, Agrano).

2.2. Field release experiment Bt maize MON 89034 × MON 88017, the nontransgenic control DKC 5143 and the conventional varieties DKC 4250 and Benicia were continuously grown over three years in a field experiment on a loamy sand in Braunschweig, Lower Saxony (Dohrmann et al., 2013). Physico-chemical soil properties are indicated in Table 1. The plots (30 m × 42 m) were evenly distributed over five rows in eight replications. With a 3 m distance between the rows and an 8 m wide strip planted with maize (DKC 4250), the experiment covered an area of 6.7 ha. The maize varieties were uniformly supplied with fertilizers and herbicides according to the integrated crop management. At grain maturity (October) the plots were harvested by threshing, the straw was chopped roughly and spread over the soil surface.

2.3. Sample collection and preparation Immediately after harvest, mixed straw samples (∼300 g) containing stems, leaves and tassels were taken from each plot (15 sampling points per plot) and dried (60 ◦ C) within 24 h to a constant weight. Then, the material was ground in an ultra-centrifugal mill with a vibratory feeder (ZM 200, Retsch Haan, Germany) to pass a 0.75-mm sieve and stored until used in the decomposition experiment. Mixed samples of rotting straw (∼300 g) were collected as described above four and eight weeks after harvest or during a frostfree period (January–March) and at the beginning of vegetation (April). Directly after sampling, the material was transported under cool conditions to the laboratory. Rotting straw without loosely adhering soil was taken with tweezers to get a subsample of 5 g which was ground in liquid nitrogen using mortar and pestle. Two aliquots of 150 mg each were stored at −80 ◦ C until DNA and RNA extraction.

R. Becker et al. / Applied Soil Ecology 73 (2014) 9–18

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Table 2 Primer systems for quantification of metabolically active microbial groups by real-time PCR. Phylogenetic target

Primers

Annealing temperature (◦ C)

PCR specificity (%)

Reference

Bacteria Fungi Actinobacteria ␣-Proteobacteria ␤-Proteobacteria Pseudomonas

8f + Eub518 SSU-0817 + SSU-1536 Eub338F + Act1159R ADF681F + 1392R Beta680F + 1392R 8f + PSMG

53 56 60 51 57 53

100 100 89 83 94 100

Blackwood et al. (2005); Fierer et al. (2005) Edel-Hermann et al. (2004) Blackwood et al. (2005) a Blackwood et al. (2005) a Blackwood et al. (2005) a Reiter et al. (2003)

a

Annealing temperatures were modified.

2.4. Decomposition experiments The decomposition of the straw from the four maize varieties collected from the field release experiment was evaluated with a substrate-induced respiration method (Anderson and Domsch, 1978), using an automated infra-red gas analysis system (Heinemeyer et al., 1989). One gram of the ground straw of each plot was mixed with 100 g finely sieved (1 mm) soil obtained from the field trial and incubated in Perspex tubes at 20 ◦ C over a period of 100 h in triplicate. The substrate-induced release of CO2 was continuously measured over 100 h, averaged over the three parallel measurements and displayed as cumulative CO2 –C release. In addition, the decomposition was analyzed in a 14 C pulse labelling experiment using maize plants of the four field grown variants and the varieties DKC 3420 and Agrano. The plants were grown in greenhouse in Mitscherlich pots filled with a fertilized loamy sand soil. After 7 weeks the pots were transferred in labelling chambers to obtain 14 C labelled plant matter. In total 18 plants of the same developmental stage (flowering, three per each genotype) were selected and labelled by a pulse of 30 MBq of 14 CO2 for 8 h. The temperature in the growth chamber during day and night was maintained at 22 ◦ C for 14 h (light intensity: 350 ␮E m−2 s−1 ) and 18 ◦ C for 10 h, respectively. The relative humidity was maintained at 65% throughout the experiment. The plants were harvested 31 days after labelling and dried at 104 ◦ C by separating the corncob from shoot. Only stalks, tassels and leaves of each genotype were ground twice with silica sand to obtain homogeneous 14 C sources which were used as substrates for the mineralization experiment. The carbon contents and 14 C activity of each source was determined by combustion at 1200 ◦ C in the presence of continuous O2 flow. The CO2 from the elemental analyzer CS 500 (ELTRA GmbH, Neuss, Germany) was absorbed in a CO2 -trap (Qureshi et al., 1985) by 7 ml Carbo-Sorb E (PerkinElmer). Aliquots of 3 ml trap solution were mixed with 12 ml of Permafluor-Scintillator (PerkinElmer, Rodgau, Germany) to measure the 14 C activity using a liquid scintillation counter (Tri-Carb 2900 TR, PerkinElmer, Rodgau, Germany). Afterwards, 1000 g finely sieved (2 mm) top soil of albic luvisol obtained from arable site near Müncheberg, Germany was mixed with 14 C labelled plant material (containing 0.7 g C) and filled in the glass bottles which comprises in- and outlet for gas exchange. For this, a total of 24 bottles (4 per genotype) was used to investigate the decomposition of labelled maize straw at 21 ◦ C for 56 days. A continuous air flow (30 ml per min) was pumped through the head space of each bottle, subsequently trapping the CO2 downstream (each CO2 -trap was divided into 3 glass vials containing 12 ml of 1 M NaOH). The NaOH solution was renewed every second day up to the end of experiment. The amount of 14 C in the respired CO2 was determined using Tri-Carb 2900 TR by dissolving 3 ml aliquots in 12 ml of UltimaGoldScintillator (PerkinElmer) as well. The 14 C activity was displayed as cumulative carbon release (percentage of 14 C turnover from the source).

2.5. Isolation of total DNA and RNA, and quantification of cDNA by real-time PCR Total RNA and DNA was extracted in parallel from the frozen samples (150 mg) by the FastRNA Pro Soil-Direct Kit and the FastDNA Spin Kit for Soil, respectively, according to the manufacturer’s instructions (MP Biomedicals, Germany) using the FastPrep-24 Instrument for cell lysis (40 s at 6.0 m s−1 ). RNA was additionally treated with DNaseI (Sigma-Aldrich, Germany), checked for contaminating DNA by PCR amplification with primers 8f and Eub518 and subsequently transcribed with the AMV Reverse Transcription System (Promega, Germany). Diluted cDNA samples were subjected to real-time PCR using the Power SYBR Green PCR Master Mix (Applied Biosystems, Germany) in a reaction mixture of 20 ␮l containing 0.5 ␮M of each primer. PCR conditions were 10 min at 95 ◦ C, followed by 40 cycles of 95 ◦ C for 30 s, 30 s at the annealing temperature, and 72 ◦ C for 60 s. The annealing temperature was adjusted according to the primer pair listened in Table 2. Each sample was run in duplicate on an ABI Prism 7500 fast thermal cycler (Applied Biosystems). The standard curves were obtained by serial dilutions of genomic DNA from Pseudomonas putida F1 (for Bacteria and Pseudomonas), Streptomyces avermitilis DSM 46492 (for Actinobacteria), Rhizobium radiobacter GV2260 (for ˛-Proteobacteria) and Burkholderia phymatum DSM 17167 (for ˇ-Proteobacteria) or plasmid DNA harbouring the 18S rRNA gene fragment from Fusarium graminearum M22 (for Fungi). 16S rRNA gene copy numbers were calculated based on the genome size and the number of 16S rRNA genes present in the respective strains. Serials dilutions of the samples were used to prove an equal PCR efficiency of standard and cDNA samples. Only single peaks were observed in the dissociation curves from both the standards and samples, indicating specific amplification with each set of primers. To test the group specificity of the assays, respective PCR products were cloned using the TOPO TA cloning kit (Invitrogen, Germany). Plasmid DNA from about 50 positive clones were purified by the Invisorb® Spin Plasmid Mini Two kit (Invitrogen, Germany) and sequenced with the M13 reverse primer. The sequences were evaluated using the programme Chimaera Check from the RDP to detect chimeric artefacts (Cole et al., 2005). The classification of the clones was based on the BLAST analysis and on the taxonomic assignment by the Classifier programme of the RDP.

2.6. T-RFLP analysis T-RFLP analysis was performed from DNA and cDNA by amplification of 16S rRNA and 18S rRNA gene fragments using the primers 8f labelled with 6-FAM and 926r (Ulrich and Becker, 2006) as well as nu-SSU-0817 labelled with 6-FAM and nuSSU-1536r (EdelHermann et al., 2004). The PCR products were digested with the restriction enzymes HhaI or Alu and Mbo, respectively, mixed with 0.2 ␮l ROX-labelled MapMarker 1000 (BioVentures, Murfreesboro,

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USA) and subsequently separated on an ABI 310 DNA sequencer (Applied Biosystems, Germany). 2.7. Data processing and statistical analysis

3.1. Straw decomposition In a first approach, the straw decomposition rate of the maize varieties grown in the field release experiment was measured by CO2 –C release from soil enriched with ground straw obtained at harvest in three consecutive years. Analysis of cumulative CO2 –C respired from soil over 100 h indicated in two of the three years a significant impact of the maize variety on the decomposition rate (Fig. 1). In the first year, the cumulative soil respiration of MON 89034 × MON 88017 and the near-isogenic control DKC 5143 were proven to be significantly higher than those of the varieties DKC 4250 and Benicia. This effect was found to be less pronounced in the second year, so that only the difference between the isogenic control and Benicia was statistically significant. In the third year, all variants displayed very similar levels of CO2 –C respiration. Generally in all three years under study, a significant difference between the transgenic hybrid and its nontransgenic counterpart could not be detected. For a more specific insight into straw degradation, the organic matter transformation was additionally analyzed in a mineralization experiment using 14 C-labelled plant residues of MON 89034 × MON 88017, the near-isogenic control and four

conventional varieties (DKC 4250, Benicia, DKC 3420, Agrano). Similar to the first approach, distinct variety effects became apparent after 118 h of incubation (Fig. 2). At that time, the turnover of Benicia was found to be significantly faster than those of the other conventional varieties, but similar to the Bt maize hybrid.

70

C labelled straw (%)

60

50

14

3. Results

Fig. 1. Cumulative respiration of soil enriched with straw of Bt maize MON 89034 × MON 88017 (3-Bt), the near-isogenic control DKC 5143 (n-iso), DKC 4250 and Benicia in three consecutive years. Different letters indicate significant differences between the variants (ANOVA or Kruskal–Wallis Analysis with post hoc Tukey test, p < 0.05). Error bars represent the standard error of mean.

40

Cumulative respiration of

Results of decomposition experiments and real-time PCR analysis of microbial groups are given as mean values with standard errors of mean. Data sets were tested for normal distribution (Shapiro–Wilk test) and homogeneity of variances (Levene test). Differences between the experimental variants were tested by ANOVA with the post hoc test Tukey HSD or Tamhane if equal variance test failed (p < 0.05) or Kruskal–Wallis Analysis (if normality test failed) followed by post hoc determination of mutual differences using SigmaStat (vers. 3.5) or SPSS (vers. 14.0.1) software. T-RFLP profiles were evaluated by the GeneScan Analytical Software version 3.1.2. The ABI files were directly converted into the GelCompar curve format and analyzed with the GelCompar II software package v. 2.5 (Applied Maths, Ghent, Belgium). The densitometric curves showing both the presence and the relative abundance of T-RFs were analyzed through Pearson correlation (Ulrich and Becker, 2006). This avoids the problem that even minor differences in separation, combined with the presence of peaks differing in less than one base pair, require a manual alignment of the profiles (Blackwood et al., 2003). Densitometric curves in the range of 30–940 bp were used for calculation of a similarity matrix based on Pearson product-moment correlation coefficients and clustering by the Ward algorithm (Ward, 1963) as well as for ordination analysis by means of non-metric multidimensional scaling (NMS). PC-ORD v.6.08 (McCune and Mefford, 2011) was used to perform the NMS. Stress values were in the range of 7.2 to 10.8 indicating a reliable test performance. The proportion of variance explained by each axis was determined by calculating the coefficient of determination between distances in ordination space and distances in the original sample space. Pearson product-moment correlations were calculated between the NMS scores and physico-chemical soil properties of samples. Soil characteristics showing significant correlation with at least one NMS axis (p < 0.05) were included as vectors on the ordination plot. Significant differences between the T-RFLP profiles were tested using a permutation test as described by Kropf et al. (2004).

30

Cumulative decomposition ( %) of 14 C labelled straw after 118 hours 1344 hours

20

Cultivars 3-Bt n-iso DKC 4250 Benicia Agrano DKC 3420

10

30.5 ± 0.9 bc 27.9 ± 0.5 ab 28.2 ± 0.6 ab 31.3 ± 0.5 c 28.3 ± 0.3 ab 26.1 ± 0.3 a

68.7 ± 1.9 abc 62.7 ± 1.4 abc 65.3 ± 0.7 b 71.4 ± 0.5 c 63.4 ± 0.4 b 59.2 ± 0.3 a

0 0

200

400

600

800

1000

1200

Time (hours) Fig. 2. Respiration rates from soil enriched with 14 C labelled residues from Bt maize MON 89034 × MON 88017 (3-Bt), DKC 5143 (n-iso), DKC 4250, DKC 3420, Benicia and Agrano. The table shows mean values and the standard error of mean after 118 and 1344 h of decomposition. Different letters indicate significant differences between the variants (ANOVA with post hoc Tukey or Tamhane test, p < 0.05).

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Fig. 3. Abundance of the metabolically active straw decomposing microbial groups as determined by the quantification of the small-subunit rRNA copy numbers. Different letters indicate significant differences between the variants at the respective sampling date (ANOVA or Kruskal–Wallis test with post hoc Tukey test, p < 0.05). Error bars represent the standard error of mean.

By comparing the Bt maize and its nontransgenic counterpart, a significant difference could not be detected. The distances in the turnover observed at 118 h increased continuously until the end of the experiment. After 1344 h (56 days) of incubation, Benicia again reached significantly higher respiration rates than the conventional varieties DKC 3420, Agrano and DKC 4250. In general, the transgenic Bt maize did not show significant differences to both the near-isogenic control and the other varieties. 3.2. Quantification of metabolically active microbial groups Metabolically active microbial groups involved in straw decomposition were quantified on the basis of primers targeting the small-subunit ribosomal RNA of Bacteria, Fungi, Actinobacteria, ˛Proteobacteria, ˇ-Proteobacteria and Pseudomonas. The specificity of the primers was tested by cloning and sequencing of the PCR products. The primer pairs for the Bacteria and Fungi as well as the genus Pseudomonas amplified exclusively sequences belonging to the respective group. Primers targeting the other microbial groups reached a specificity of 83–94% (Table 2). The clones from each primer system applied could be assigned to a broad spectrum of phylogenetically different Bacteria and Fungi.

Real-time PCR data obtained for the three sampling dates in three years under study showed an abundance of metabolically active Bacteria in a range of 3 × 1011 –2 × 1013 16S rRNA copies per g straw. Copy numbers of the analyzed bacterial taxa ranged from 2–9% of total active bacteria. Active Fungi were quantified in a range of 1 × 1010 –7 × 1011 18S rRNA copies per g straw. By comparing the maize variants within a sampling, the abundances of microbial groups varied only within one order of magnitude and rarely showed a dependence of the plant genotype, which was limited to a single sampling as well. In the first year under study, statistical differences were found between the variants Benicia and DKC 4250 four weeks after harvest for the group of Actinobacteria as well as for the ˛- and ˇ-Proteobacteria (Fig. 3). Moreover, a significant difference between the transgenic hybrid and Benicia was found at sampling in spring (April) for the group of Actinobacteria. No distinguishable effects of the transgenic hybrid and the near-isogenic control DKC 5143 were found besides these variety effects (Fig. 3). Data analyses of the second year generally revealed no significant effects of the variants tested (data not shown). In the third year, merely a variety influence became obvious at the first sampling date. Here, the variant DKC 4250 showed significantly less Bacteria in comparison to the transgenic hybrid

A6 D7 A5 D6 A2 B1 B6 B4 B8 A7 B5 B3 A4 D2 D1 C2 C8 C4 E7 C3 E5 C7 C6 D8 D3 E1 D4 E2 E8 C5 E4 A1

DNA-based

3-Bt 3-Bt n-iso Benicia Benicia n-iso n-iso 3-Bt DKC 4250 DKC 4250 Benicia n-iso DKC 4250 n-iso Benicia Benicia Benicia DKC 4250 DKC 4250 3-Bt 3-Bt DKC 4250 3-Bt n-iso DKC 4250 DKC 4250 Benicia 3-Bt Benicia n-iso n-iso 3-Bt

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R. Becker et al. / Applied Soil Ecology 73 (2014) 9–18

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D3 D1 C6 A5 C7 A2 C2 C3 C5 A1 E5 E2 D7 D8 E1 E8 E7 E4 B4 B6 D2 B7 B5 C4 D4 D6 C8 A4 A7 A6 B3 B1

RNA-based

DKC 4250 Benicia 3-Bt n-iso DKC 4250 Benicia Benicia 3-Bt n-iso 3-Bt 3-Bt 3-Bt 3-Bt n-iso DKC 4250 Benicia DKC 4250 n-iso 3-Bt n-iso n-iso DKC 4250 Benicia DKC 4250 Benicia Benicia Benicia DKC 4250 DKC 4250 3-Bt n-iso n-iso

Fig. 4. Dendrogams showing the similarity of T-RFLP profiles of the decomposing bacterial communities obtained from plant residues of Bt maize MON 89034 × MON 88017 (3-Bt), DKC 5143 (n-iso), DKC 4250 and Benicia collected 4 weeks after harvest (first sampling campaign). Left: total communities; Right: metabolically active communities.

and the near-isogenic control (2 × 1011 vs. 7–8 × 1011 16S rRNA copies per g straw). Across the three years, seasonal effects on the abundance of the straw decomposing microorganisms were obvious and found to be more pronounced than the influence of the plant genotype. All microbial taxa except the total Bacteria showed significant differences between the sampling dates (two Way ANOVA, p < 0.05, Fig. 3). All in all, an impact of the transgenic modification of maize could not be detected on the abundance of metabolically active bacteria and fungi residing on rotting maize straw.

T-RFLP analysis of the 16S rRNA gene was performed to analyze the metabolically active as well as the total bacterial communities residing on the rotting maize straw. Both the active and the total communities (RNA and DNA-based) displayed complex and reproducible T-RFLP profiles. The number of T-RFs in the individual profiles ranged from 35–60 in the total and from 27–52 in the active part of the bacterial communities. The profiles of both groups were found to be similar with regard to the number and the relative abundance of the dominant T-RFs. Therefore, most of the total bacterial

A1

3-Bt n-iso DKC 4250 Benicia NMS axis 2 (6%)

3.3. T-RFLP analysis of microbial communities

E8 A2 E2 E5 E4 A7 B4

B1

A5 B3 B5 C4

E7 E1 D8

Sand (0.58, 0.08) C6

A4 C2

B6

D6 A6

C3

pH (0.37, 0.09) C5

C8

D3

D7

Silt

(0.61, 0.08)

C7 D1

B8 D2

D4

NMS axis 1 (91%) Fig. 5. Nonmetric Multidimensional Scaling (NMS) ordinations of metabolically active bacterial communities (RNA-based) obtained from plant residues collected 5 months after harvest (first sampling campaign). NMS stress was 7.2 for the shown two-dimensional solution. Statistically significant correlations (p < 0.05) of soil characteristics were indicated by arrows. Values in brackets show r2 to Axis 1 and 2.

DNA-based

A6 B3 A1 B6 B8 A2 A5 B4 C2 C3 A7 C4 B5 A4 B1 D2 D8 E8 E7 E4 C5 C6 C7 E5 D4 D6 D3 C8 E2 D7 E1 D1

3-Bt n-iso 3-Bt n-iso DKC 4250 Benicia n-iso 3-Bt Benicia 3-Bt DKC 4250 DKC 4250 Benicia DKC 4250 n-iso n-iso n-iso Benicia DKC 4250 n-iso n-iso 3-Bt DKC 4250 3-Bt Benicia Benicia DKC 4250 Benicia 3-Bt 3-Bt DKC 4250 Benicia

RNA-based

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B4 B3 A2 A6 A4 C7 D1 A1 C5 C8 A7 B1 B5 D2 E8 C6 E1 E4 E2 E5 E7 C3 B8 C2 B6 A5 C4 D8 D7 D6 D3 D4

3-Bt n-iso Benicia 3-Bt DKC 4250 DKC 4250 Benicia 3-Bt n-iso Benicia DKC 4250 n-iso Benicia n-iso Benicia 3-Bt DKC 4250 n-iso 3-Bt 3-Bt DKC 4250 3-Bt DKC 4250 Benicia n-iso n-iso DKC 4250 n-iso 3-Bt Benicia DKC 4250 Benicia

Fig. 6. Dendrogams showing the similarity of T-RFLP profiles of the decomposing fungal communities obtained from plant residues of Bt maize MON 89034 × MON 88017 (3-Bt), DKC 5143 (n-iso), DKC 4250 and Benicia collected 4 weeks after harvest (first sampling campaign). Left: total communities; Right: metabolically active communities.

community detected on the rotting straw seems to be metabolically active. By comparing the profiles from the different maize varieties, no clear differences became visible. As exemplarily shown for the first sampling of the first year under study, cluster analysis revealed no impact of the maize varieties on the total and active residue colonizing communities (Fig. 4). The profiles of the transgenic Bt maize hybrid and the other variants were often evenly distributed over the clusters. Accordingly, the permutation tests did not show significant differences between the transgenic hybrid, the near-isogenic control and the conventional varieties. However, the branching of the profiles indicated in some cases a dependence of the communities from the soil heterogeneity within the release trial. As particularly visible on the third sampling of the first year (April), the profiles belonging to the field rows A and B were clearly separated from those of the rows D and E (Fig. 5). This separation was mainly based on Axis 1, which explains 91% of the variability. Differentiation between rows A and B vs. D and E as well as the nearly separate grouping of the rows D and E were proven as statistically significant (P < 0.05). The NMS was also used to reveal the main drivers within the soil properties. As a result, especially the silt and sand fraction of the topsoil showed a clear correlation to the NMS Axis 1 (r2 = 0.61 and 0.58). A weaker correlation was found for the pH (r2 = 0.37). Compared with bacteria, the T-RFLP profiles of the fungal community were found to be less complex, displaying only 10–15 T-RFs. The fingerprints of the total and active fungal communities again showed corresponding dominant peaks. Thus, the fungal microflora analyzed in the sampling period seems to be in large part metabolically active as well. Both the occurrence and the relative abundance of T-RFs varied independently from the experimental variants. Results of cluster analyses, as exemplarily shown for the

first sampling date in Fig. 6, indicated no distinguishable impact of the maize varieties on the total and active communities. Accordingly, significant differences between the transgenic hybrid and its near-isogenic control as well as the conventional varieties were not detected. In contrast, results of some samplings revealed again an effect of site characteristics on the community structure. For example, the NMS of the active fungal communities from the third sampling date of the first year revealed a separation of profiles from the rows A and B from those of the rows D and E (Fig. 7) which was proved as significant (p < 0.05). However, this effect was less pronounced compared to the bacteria analyzed at the same sampling date (Fig. 5). NMS analysis of the fungal T-RFLP profiles resulted in a strong correlation of the NMS axis 1 to the silt and sand fraction (r2 = 0.37 and 0.36, Fig. 7).

4. Discussion Our study was carried out to assess potential impacts of the multi-resistant Bt maize hybrid MON 89034 × MON 88017 on the structure and function of the microbial communities involved in straw decomposition. Both the presence of the insecticidal cry proteins in plant residues and the potential changes of macroscopic plant characteristics by unintended pleiotropic effects might affect the residue-colonizing bacterial and fungal communities and consequently the organic matter transformation. Based on two experimental approaches differing in incubation time and the growth conditions of the maize plants, we found significant differences in the straw decomposition rates only between the conventional varieties. These genotypic effects could be observed after a short-term incubation of 100 h and up to the mineralization of about 65% at 56 days as well. Such a genotypic variability

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R. Becker et al. / Applied Soil Ecology 73 (2014) 9–18

E8 B6 A1

E2

A5 B3

NMS axis 2 (18%)

A7 A4

A2

B5 C2 C8 C5

E7 E1

Sand (0.36, 0.04)

C3 C4 C6 B8 C7

D3

B1

Silt (0.37, 0.05)

B4

D7

D1

E5

E4

D8

D2 D6 D4

3-Bt n-iso DKC 4250 Benicia

A6

NMS axis 1 (77%) Fig. 7. Nonmetric Multidimensional Scaling (NMS) ordinations of metabolically active fungal communities (RNA-based) obtained from plant residues collected 5 months after harvest (first sampling campaign). NMS stress was 10.8 for the shown two-dimensional solution. Statistically significant correlations (p < 0.05) of soil characteristics were indicated by arrows. Values in brackets show r2 to Axis 1 and 2.

was also found in two other studies analysing residue decomposition of Bt maize in comparison to a range of conventionally bred varieties (Fließbach et al., 2012; Zurbrügg et al., 2010). Comparing the turnover of the Bt maize to the nontransgenic control, recent studies revealed no significant differences between Bt and non-Bt straw (Lehman et al., 2010; Tarkalson et al., 2008; Xue et al., 2011; Zurbrügg et al., 2010) or even a potential higher susceptibility to decomposition due to higher N concentrations in Bt stems (Yanni et al., 2011). Similar to the results obtained from transgenic plants with a single event, our results also indicated no differences in decomposition rates between the multi-resistant Bt hybrid and its nontransgenic counterpart. Correspondingly, Lehman et al. (2008) showed a comparable turnover rate of a multi-resistant Bt maize variety as measured by the litter bag technique. Taking all data together, environmental factors seemed to have an essential impact on the decomposition in both experiments. Thus, measuring of CO2 –C release over 100 h indicated a significant variety effect in only two of the three years. On the other hand, the comparison of the decomposition rates obtained in the two approaches displayed an altered ranking of the maize varieties tested. Environmental effects also became visible in studies analysing the organic matter transfer of Bt maize as well as wheat and rye straw under different agronomical conditions, such as incorporation of the plant material into the soil or nitrogen application. Here, the agricultural practice and the handling of the straw in the incubation experiment (mixed vs. unmixed with soil) decisively influenced the decomposition of the plant residues (Al-Kaisi ˜ and Guzman, 2013; Londono-R et al., 2013; Nicolardot et al., 2007). When considering these aspects, changes in growth conditions or other environmental factors may differently influence the decomposition rate of the single varieties. Apart from this, under all conditions tested in this study, the transgenic hybrid could not be distinguished from its near-isogenic control. To detect potential shifts in the straw-colonizing microflora, the abundance of different microbial taxa as well as the structure of the bacterial and fungal communities was analyzed. The study focused on the metabolically active microbial community (RNA-based) to reveal a closer link to organic matter transformation. Our data obtained for the abundance of the metabolically active microbial groups, analyzed at three sampling dates per year, did not reveal

a distinguishable effect of the transgenic hybrid, but in some cases a significant effect of the maize variety. However, this influence detected for one or the other microbial group was always temporally restricted and not consistent over the sampling period. As already described for several other microbial parameters analyzed on Bt maize residues (Tan et al., 2010; Xue et al., 2011), impacts of the transgenic maize were generally not detected but, e.g., seasonal factors (different sampling dates) were identified as an essential driver of the metabolically active microbes during decomposition. T-RFLP analyses based on the same samples revealed mostly corresponding dominant peaks for the metabolically active and total communities suggesting that the straw colonizing bacteria and fungi were in large part viable and active (Mengoni et al., 2005; Pennanen et al., 2004). Established microbial habitats as arable soil that harbour both active microbes and cells in starvation often display deviating profiles of active and total communities in response to changing environmental conditions (Girvan et al., 2005; Hagn et al., 2003; Lillis et al., 2009). However, freshly colonized habitats as newly introduced plant residues can be considered to be colonized only with microbial cells which are metabolically active. As expected, T-RFLP profiles of the fungal communities displayed a reduced number of T-RF’s as compared to the bacteria, since the profiles were generated using the fungal 18S rRNA gene as target sequence. The more variable ITS region of the rRNA genes was not suitable for the RNA-based analysis. In general, results of all T-RFLP analyses performed over three years indicated no apparent effect of the experimental variants on the decomposer communities residing on the rotting straw. Neither the transgenic Bt maize hybrid containing the three cry-proteins (Cry 1A.105, Cry2Ab2, Cry3Bb1) nor the different conventional maize varieties had distinguishable influences on the straw decomposing bacterial and fungal communities. This is consistent with several other studies on different Bt maize varieties, which revealed no changes in the structure of the soil or residue-colonizing microbial communities during organic mat˜ et al., 2013; Tan et al., 2010; Xue ter transformation (Londono-R et al., 2011). In contrast, environmental or agronomical conditions were proven as driving factors on the studied bacterial and fungal ˜ et al., 2013). This impact was also observed microflora (Londono-R in our study as the analyses of the T-RFLP profiles revealed a significant correlation with the small differences in soil texture and pH across the field trial. 5. Conclusions The results of this study are in line with numerous scientific data suggesting that the cultivation of Bt maize has no significant impact on soil organic matter transformation and the involved bacterial and fungal communities. Our study focussed on metabolically active bacteria and fungi which were likewise unaffected by the transgenic maize. In this context, the analysis of the multi-resistant Bt hybrid MON 89034 × MON 88017 showed that this stacked genetically modified maize provided similar results as obtained for transgenic hybrids expressing just a single insecticidal cry protein. Acknowledgements We thank Florian Hackelsperger and the colleagues at the Experimental Research Station (FLI, Braunschweig) for conducting the field trial and Jürgen Niemeyer for providing the detailed data of the soil properties of the field plots. We are grateful to Sigune Weinert and Martina Wiemer for the excellent technical assistance and we express appreciation to the members of the joint research project “Biosafety field studies on maize with multiple genes providing protection against the European corn borer and the Western

R. Becker et al. / Applied Soil Ecology 73 (2014) 9–18

rootworm”, especially Stefan Rauschen for collaboration and support. The study was financed by the Federal Ministry of Education and Research (Grant 0315215H).

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