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Impact of acetochlor on ammonia-oxidizing bacteria in microcosm soils
Journal of Environmental Sciences 20(2008) 1126–1131
Impact of acetochlor on ammonia-oxidizing bacteria in microcosm soils LI Xinyu1 , ZHANG Huiwen1,∗, WU Minna1,2 , SU Zhencheng1 , ZHANG Chenggang1 1. Microbiolog Resource and Ecology Group, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China. E-mail: [email protected] 2. Graduate School of Chinese Academy of Sciences, Beijing 100039, China Received 25 October 2007; revised 13 December 2007; accepted 16 January 2008
Abstract Acetochlor is an increasingly used herbicide on corn in North China. Currently, the effect of acetochlor on soil ammonia-oxidizing bacteria (AOB) communities is not well documented. Here, we studied the diversity and community composition of AOB in soil amended with three concentrations of acetochlor (50, 150, 250 mg/kg) and the control (0 mg acetochlor/kg soil) in a microcosm experiment by PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) and the phylogenetic analysis of excised DGGE bands. DGGE profiles showed that acetochlor had a stimulating effect on AOB at the early stage after acetochlor amended, and the order of intensity and duration is medium-acetochlor amended samples (AM) > low-acetochlor amended samples (AL) > high-acetochlor amended samples (AH). At the end of 60 d microcosm, acetochlor had a negative effect on the diversity of AOB. Cluster analysis of DGGE profiles showed that acetochlor had a greater effect on the community structure of AOB on day 60 than on day 1. The phylogenetic analysis revealed that all the sequences of excised DGGE bands were closely related to members of the genus Nitrosospira and formed two separate subclusters designated as subcluster 1 and subcluster 2 affiliated respectively with clusters 3 and 4 in Nitrosospira as defined by Stephen. Some dominant AOB had a change from subcluster 2 to subcluster 1 with the incubation. The results showed that acetochlor had an effect on the AOB on a long-term basis and the chronic effect of acetochlor should be paid more attention in future. Key words: ammonia-oxidizing bacteria (AOB); PCR-DGGE; acetochlor
Introduction Heilongjiang Province in Northeast China is a main grain production base of this country, and black soil is one of its main agricultural soils. Herbicide acetochlor is used within this area in an increasing amount to increase crop yield. Some studies were conducted regarding its impacts on soil bacteria (Luo et al., 2004; Zhang et al., 2004a, 2004b), but little is known about soil ammonia-oxidizing bacteria (AOB). Nitrification is a key soil process affecting the global nitrogen cycling, among which the AOB play an essential role. AOB are chemolithoautotrophs; they have in common the ability to utilize ammonia as the sole source of energy and carbon dioxide as the main source of C (Hooper et al., 1997). Furthermore, most strains belong to a single (monophyletic) evolutionary group within the β-subclass of Proteobacteria. Well-known genera in this group are Nitrosomonas and Nitrosospira. The AOB have been suggested as the model organisms in microbial ecology (Kowalchuk and Stephen, 2001) and are often used as indicators of soil perturbations (Stephen et al., 1999; Chang et al., 2001; Nyberg et al., 2006). Fingerprints of AOB communities are often analyzed by * Corresponding author. E-mail: [email protected].
denaturing gradient gel electrophoresis (DGGE) (Innerebner et al., 2006; Enwall et al., 2007; Ma et al., 2007), which is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial communities (Muyzer et al., 1993). In this study, the 16S rDNA genes were targeted after soil DNA extraction to investigate whether the application of acetochlor will have effects on the AOB communities by DGGE analysis.
1 Material and methods 1.1 Soil sampling and microcosm experiment Surface soil samples (0–20 cm) without application of agrochemicals for 8 years were collected from a field of the Hailun Agro-ecological Experimental Station (47◦ 27 N, 126◦ 55 E) in Hailun County of Heilongjiang Province, northeast China. The samples were well mixed, sieved through a 2-mm sieve, and stored at 4°C. The soil is classified as a black soil or a Hapli-Udic Isohumosol according to the Chinese Soil Taxonomy (CRGCST, 2001). The properties of the soil were as follows: pH (water), 6.2; moisture, 11.2% (W/W); organic matter, 15.9 g/kg. For microcosm experiment, the samples were first
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Impact of acetochlor on ammonia-oxidizing bacteria in microcosm soils
adjusted to their field moisture content (18.9%, W/W) by adding distilled water, and then divided into 4 subportions to install four treatments (adding 0, 50, 150, and 250 mg acetochlor/kg soil, respectively). Each treatment had three replicates, and 12 microcosms were prepared. The microcosm was a plastic cup filled with 300 g of soil, sealed with parafilm to minimize water loss, and was incubated at 25°C in dark. No additional materials, except water, were added, and sampling was done on day 1, 7, 30, and 60 of the 2 month incubation. Mixed triplicate samples were stored in a refrigerator for DNA extraction at –20°C. 1.2 DNA extraction and PCR amplification of 16S rDNA A direct lysis method described by Zhou et al. (1996) was adopted with a little modification. One gram soil was mixed with 1,000 µl DNA extraction buffer (0.1 mol/L Tris-HCl (pH 8.0), 0.1 mol/L sodium EDTA (pH 8.0), 0.1 mol/L sodium phosphate (pH 8.0), 1.5 mol/L NaCl, 1% CTAB, and 1% SDS) in a microcentrifuge tube, vortexed for 10 s, and incubated in a water bath at 65°C for 30 min, with gentle end-over-end inversions every 15 min. The sample was then frozen and thawed with three cycles from –70 to 65°C, and kept at 65°C for 1 h to make all microbial cells be lysed completely. After 8,000 r/min centrifugation for 10 min at room temperature, the supernatant was collected, and transferred into another microcentrifuge tube, mixing with an equal volume of chloroformisoamyl alcohol (24:1, V/V). The aqueous phase was recovered by centrifugation, and precipitated with 0.6 volume of isopropanol at room temperature for 1 h. The pellet of crude nucleic acids was obtained by 12,000 r/min centrifugation for 30 min at 4°C, washed with cold 70% ethanol, and re-suspended in TE buffer to give a final volume of 50 µl. The crude extract was purified with Wizard PCR Preps (Promega, USA), according to the manufacturer’s instructions. To amplify ammonia-oxidizer specific 16S rDNA from soils, we used a nested PCR approach. For the first-round PCR, primer pair βAMOf/βAMOr (McCaig et al., 1994) was used, and for the second-round PCR, the AOB-specific primer pair CTO189f+GC/CTO654r (Kowalchuk et al., 1997) was used to obtain DNA fragments 465 bp in length. All PCRs were performed with a PTC-200 thermal cycler (MJ Research, USA). The PCR protocol included a 5-min initial denaturation at 94°C, 30 cycles at 94°C for 30 s, at 55°C for 30 s, and at 72°C for 90 s, followed by a final extension at 72°C for 10 min. PCR products were then diluted 10−2 –10−3 fold and used as template for PCR of the 16S rRNA genes using primers CTO189f /CTO654r with a GC-clamp attached to the forward primer. The PCR protocol included a 3-min initial denaturation at 94°C, 30 cycles at 94°C for 30 s, at 55°C for 30 s, and at 72°C for 45 s, followed by a final extension at 72°C for 5 min. The first and second reaction mixtures (V = 50 µl) contained 1× PCR reaction buffer (TaKaRa, Japan), 10 ng DNA template, 20 pmol/L of forward and reverse primers, 200 µmol/L dNTP mix, and 2.5 U of Ex Taq DNA Polymerase (TaKaRa, Japan).
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1.3 DGGE analysis DGGE analysis was performed with a DCode mutation detection system (Bio-Rad, USA). Gels of 6% acrylamide (37.5:1 acrylamide-bisacrylamide) were formed between 40% and 60% denaturant, with 100% denaturant defined as 7 mol/L urea and 40% (V/V) formamide. Normally, 300 ng PCR products were loaded onto each lane of the gels. The gels were run at voltage of 200 V for 5 h, and maintained at a constant temperature of 60°C in 7 L of 1× TAE buffer (40 mmol/L Tris-acetate, 1 mmol/L EDTA, and pH 8.0). The DGGE gels were stained with GeneFinder (Bio-V Biological Technology Co., Ltd., China) and the images were analyzed with the software package Quantitiy one 2.1 (Bio-Rad, USA). Cluster analysis and dendrograms were calculated using UPGMA. The Shannon-Weaver diversity index (H) (Shannon and Weaver, 1963) was calculated to examine the structural diversity of the ammonia-oxidizer community. 1.4 Recovery and sequence analysis of bands from DGGE gels Dominant PCR-DGGE bands were manually excised from the gel, suspended in 20 µl distilled water, and incubated overnight at room temperature. PCR-DGGE was repeated using these samples as template until a single band remained in each lane. A final PCR step was performed without the GC clamp attached to the forward primer. PCR products were then purified using a Mini-DNA Fragment Rapid Purification Kit (BioDev, Beijing) and sequencing reactions were run on an ABI 377 apparatus by Shanghai Sangon Biological Engineering Technology and Service Co. Ltd. The nucleotide sequences obtained in this study have been deposited in the GenBank database under accession numbers EF434160–EF434169. 1.5 Phylogenetic analysis of sequenced DGGE bands Excised band sequences were subjected to a blastn search on the NCBI site to identify sequences with highest similarity. Highly similar sequences and some reference sequences of dominant ammonia-oxidizing groups were added to the data set for CLUSTAL W multiple sequence alignment and the phylogenetic tree was constructed in MEGA 3.1 by applying the neighbor-joining method with 100 bootstrap resamplings.
2 Results 2.1 Effect of acetochlor on ammonia-oxidizing community The PCR-DGGE banding profiles of soil ammoniaoxidizing communities under acetochlor stress (Fig.1) showed that there was a visible alternation in both the DGGE banding fingerprints and the amounts among different treatments. H was calculated to estimate the diversity of the AOB communities in acetochlor-amended and control soils (Table 1). The diversity indices changed from increasing at the initial stage of incubation to decreasing at the end of the incubation for all acetochlor-amended
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Table 1 Shannon-Weaver diversity indices (H) of AOB communities from DGGE profiles of control (CK) and acetochlor-amended samples Sampling time (d)
Acetochlor amended level (mg/kg) 0 (CK) 50 150 250
1 7 30 60
2.039 1.371 1.772 2.161
2.106 2.048 1.684 1.601
2.284 2.050 1.936 1.098
2.054 1.357 1.770 1.087
samples compared with that for the control sample. First, the diversity index of the low-acetochlor amended samples (AL) started to decline after 30 d incubation, while that of the medium-acetochlor amended samples (AM) still increased on day 30 and started to decline at the end of the incubation. The diversity index of high-acetochlor amended samples (AH) was slightly higher than that of the control samples on day 7, and then had a similar value to that of the control samples, and at last decreased on day 60. Based on the DGGE profiles (Fig.1), the increasing indices in acetochlor-amended samples on day 1 may result from the rich number of bands and high brightness in some bands, suggesting that acetochlor may stimulate the increase of AOB. From day 1 to day 60, the changes were obvious in band number and position, especially for the AM and AH samples. On day 60, the lower diversity indices in acetochlor-amended samples were mainly ascribed to the decrease of the band number. It indicated that the acetochlor had a stimulating effect on AOB at the early stage of incubation, and the order of intensity and duration was AM > AL > AH. When the concentration was
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higher than 150 mg/kg, the stimulating effect weakened in intensity and shortened in duration of time. At the end of 60 d incubation, acetochlor had a negative effect on the diversity of AOB. Clustering analysis of acetochlor-amended and control samples sampled on day 1 and 60 are shown in Fig.2. The dendrogram was composed of three main
Fig. 2 UPGMA dendrogram constructed with AOB DGGE profiles from control and acetochlor-amended samples on day 1 and 60. Samples were marked by number (1, 2, 3, and 4 stand for the four acetochlor-amended levels of 0, 50, 150, and 250 mg acetochlor/kg soil, respectively) and sampling time (1 and 60 d, respectively).
Fig. 1 DGGE community fingerprints of AOB in soil samples amended with different amounts of acetochlor sampled on day 1, 7, 30, 60. The labeled bands were excised from the gel, reamplified, and subjected to sequence analysis. lane 1: control soil; lane 2: 50 mg/kg; lane 3: 150 mg/kg; lane 4: 250 mg/kg.
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clusters as follows: one cluster included three concentration acetochlor-amended samples on day 1 and the control on day 1 and 60; one cluster had AL and AM samples on day 60; and the last cluster contained only AH sample on day 60. The results showed that acetochlor had greater effect on the community structure of AOB on day 60 than on day 1. From the results of the diversity indices and clustering analysis, we can conclude that acetochlor had a toxic effect on AOB community. 2.2 Sequencing and phylogenetic analyses Based on the BLASTN results, highly similar GenBank sequences and AOB reference sequences were added to the data set to analyze the phylogenetic relationship of ammonia-oxidizing bacteria in this study. CLUSTAL
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W multiple sequence alignment was performed and the phylogenetic distance tree was constructed as shown in Fig.3. As seen in Fig.3, all the sequences of excised DGGE bands were closely related to members of the genus Nitrosospira forming two separate subclusters designated as subcluster 1 and subcluster 2. A5–A7 and A17 belonged to subcluster 2 and sequence similarities within subcluster 2 ranged from 96% to 99%. Bands A8–A13 belonged to Nitrosospira subcluster 1 and sequence similarities within the subcluster 1 ranged from 96% to 99%. Subcluster 1 was affiliated with Nitrosospira cluster 3 comprising most of the soil 16S rDNA sequences from cultured Nitrosospira sp. as defined by Stephen et al. (1996). Subcluster 2 was affiliated with Nitrosospira cluster 4. Nine excised DGGE bands can be divided into three
Fig. 3 Phylogenetic relationship of excised DGGE bands and reference AOB sequences. The dendrogram was generated by the neighbor-joining method with 100 bootstrap resamplings. Branching points supported by bootstrap values > 50% are indicated in the text. The scale bar represents 10% sequence divergence. The designation of clusters 1–7 and Nitrosospira subcluster SM refers to Stephen et al. (1996) and Haleem et al. (2000).
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kinds of change patterns according to the DGGE profiles. (1) A5–A6 and A11–A13 all increased in acetochloramended samples; however, these bands appeared to increase at different times of incubation. A5–A6 in subcluster 2 only increased in the early stage of incubation; A11–A12 in subcluster 1 was on day 30 and A13 was both on day 1 and 30. Dominant AOB in diversity changed from subcluster 2 to subcluster 1 over time. (2) A7 in subcluster 1 and A9 in subcluster 2 belong to the common ammonia-oxidizing populations given that these two bands were found in most treatments during the entire incubation period. (3) A8–A10 in subcluster 1 and A17 in subcluster 2 varied frequently in four treatments during 60 d incubation. The change was greater in AM and AH samples than in AL samples. The results from the phylogenetic analysis indicated that major temporal changes in composition of the AOB communities from subcluster 2 to subcluster 1 occurred in acetochlor-amended soils during the period of the experiment. All the sequences of excised bands were highly similar to the Nitrosospira sp. with the similarity levels ranging from 98% to 100%, except A10, which was highly similar to Nitrosovibrio tenuis Nv1 (with the similarity level of 98.61%).
3 Discussion In this study, the excised bands were closely related to Nitrosospira clusters 3, SM, and 4 comprising most of the βAOB 16S rDNA sequences of cultured or yet uncultured Nitrosospira sp. from soil and rhizosphere. Stephen et al. (1996) showed that Nitrosospira sp. and Nitrosomonas sp. could be subdivided into at least seven rDNA clusters, designated as 1–4 and 5–7, respectively (Fig.3). Analysis of different habitats revealed that Nitrosospira cluster 1 is formed exclusively by 16S rDNA sequences retrieved from marine sediments (Stephen et al., 1996), sea water (Phillips et al., 1999), or coastal sand dunes (Kowalchuk et al., 1997), whereas clusters 2–4 are comprised of rDNA sequences retrieved from cultured strains and clonal analyses from soil, sewage or rhizosphere environments, and coastal sand dunes (Utaker et al., 1996; Kowalchuk et al., 1997, 1999; Speksnijder et al., 1998). In contrast, Nitrosomonas subclusters 5–7 represent 16S rDNA sequences from various environmental sources such as marine sediments (Stephen et al., 1996), seawater (Phillips et al., 1999), freshwater (Speksnijder et al., 1998), or coastal sand dunes (Kowalchuk et al., 1997). Recently, Haleem et al. (2000) found another novel subcluster (SM cluster) in addition to the definition of Stephen et al. (1996). The close relationship between environmental habitats and the phylogenetic structure of the respective βAOB populations was also observed in this study. The above close relationship was also observed in other researches for coastal sand dunes and agricultural soil, and may reflect the physiological differences (Kowalchuk et al., 1997; Stephen et al., 1998; Haleem et al., 2000). In this study, there are two dominant types of AOB divided into subcluster 1 and subcluster 2. At the early stage of the experiment, the diversity of AOB in subcluster
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2 increased under acetochlor stress, while the diversity of AOB in subcluster 1 was high on day 30 after incubation. The change of the dominant AOB in diversity over time may result from the difference of utilizing degradated products with degradation of acetochlor, suggesting the physiological differences in subcluster 1 (in cluster 3 ) and subcluster 2 ( in cluster 4). At the end of 60 d incubation, the diversity indices of AOB in AM and AH samples were extremely low, resulting in only two bands in the DGGE analysis. In contrast, the ammonia oxidizers had a high diversity in the control sample. The results showed that acetochlor had a negative effect on the AOB on a long-term basis and the chronic effect of acetochlor should be paid more attention in future even if acetochlor is low toxic and easy to degrade (Yu et al., 1998).
4 Conclusions The results described in this article show that acetochlor had a stimulating effect on AOB community at the early treatment, and the order in intensity and duration was AM (150 mg/kg) > AL (50 mg/kg) > AH (250 mg/kg). At the end of 60 d incubation, acetochlor had a negative effect on the diversity of AOB community. The cluster analysis of DGGE profiles indicated that acetochlor had more effect on the community structure of AOB on day 60 than on day 1. The phylogenetic analysis revealed that all sequences of excised DGGE bands were closely related to members of the genus Nitrosospira forming two separate subclusters, designated as subcluster 1 and subcluster 2, and affiliated respectively with Nitrosospira clusters 3 and 4 as defined by Stephen et al. (1996). There was a major temporal change in the composition of the AOB community from subcluster 2 to subcluster 1 that occurred in acetochlortreated soils during the period of study. Based on the results described above, it is found that acetochlor had a negative effect on the AOB on a longterm basis, and the chronic effect of acetochlor should be paid more attention. Acknowledgments This work was supported by the Natural Science Foundation for Young Scientists of China (No. 40701088).
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Haleem D A E, von Wintzingerode F, Moter A, Moawad H, Gobel U B, 2000. Phylogenetic analysis of rhizosphere-associated β-subclass proteobacterial ammonia oxidizers in a municipal wastewater treatment plant based on rhizoremediation technology. Lett Appl Microbiol, 31: 34–38. Hooper A B, Vannelli T, Bergmann D J, Arciero D, 1997. Enzymology of the oxidation of ammonia to nitrite by bacteria. Antonie Van Leeuwenhoek, 71: 59–67. Innerebner G, Knapp B, Vasara T, Romantschuk M, Insam H, 2006. Traceability of ammonia-oxidizing bacteria in compost-treated soils. Soil Biol Biochem, 38: 1092–1100. Kowalchuk G A, Stephen J R, 2001. Ammonia-oxidizing bacteria, a model for molecular microbial ecology. Annu Rev Microbiol, 55: 485–529. Kowalchuk G A, Stephen J R, de Boer W, Prosser J I, Embley T M, Woldendorp J W, 1997. Analysis of ammonia-oxidizing bacteria of the β-subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments. Appl Environ Microbiol, 63(4): 1489–1497. Kowalchuk G A, Naoumenko Z S, Derikx P J L, Felske A, Stephen J R, Arkhipchenko I A, 1999. Molecular analysis of ammonia-oxidizing bacteria of the β-subdivision of the class of Proteobacteria in compost and composted materials. Appl Environ Microbiol, 65: 396–403. Luo H F, Qi H Y, Zhang H X, 2004. The impact of acetochlor on the bacterial diversity in soil. Acta Microbiologica Sinica, 44: 519–522. Ma W K, Schautz A, Fishback L A E, Bedard-Haughn A, Farrell R E, Siciliano S D, 2007. Assessing the potential of ammonia oxidizing bacteria to produce nitrous oxide in soils of a high arctic lowland ecosystem on Devon Island, Canada. Soil Biol Biochem, 39: 2001–2013. McCaig A E, Embley T M, Prosser J I, 1994. Molecular analysis of enrichment cultures of marine ammonia oxidizers. FEMS Microbiol Lett, 120: 363–368. Muyzer G, De Waal E C, Uitterlinden A G, 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reactionamplified genes coding for 16S RNA. Appl Environ Microbiol, 3: 695–700. Nyberg K, Schn¨urer A, Sundh I, Jarvis Å, Hallin S, 2006. Ammonia oxidising communities in agricultural soil incubated with organic waste residues. Biol Fert Soils, 42: 315–323. Phillips C J, Smith Z, Embley T M, Prosser J I, 1999. Phylogenetic difference between particle-associated and planktonic
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ammonia-oxidizing bacteria of the β-subdivision of the class Proteobacteria in the northwestern Mediterranean sea. Appl Environ Microbiol, 65: 779–786. Shannon C E, Weaver W, 1963. The Mathematical Theory of Communication. Urbana: University of Illinois Press. 1– 125. Speksnijder A G, Kowalchuk G A, Roest K, Laanbroek H J, 1998. Recovery of a Nitrosomonas-like 16S rDNA sequence group from freshwater habitats. Syst and Appl Microbiol, 21: 321–330. Stephen J R, Chang Y J, Macnaughton S J, Kowalchuk G A, Leung K, Flemming C A, White D C, 1999. Effect of toxic metals on indigenous soil β-subgroup proteobacterium ammonia oxidizer community structure and protection against toxicity by inoculated metal-resistant bacteria. Appl Environ Microbiol, 65: 95–101. Stephen J R, Kowalchuk G A, Bruns M A V, McCaig A E, Phillips C J, Embley T M, Prosser J I, 1998. Analysis of βsubgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing. Appl Environ Microbiol, 64: 2958–2965. Stephen J R, McCaig A E, Smith Z, Prosser J I, Embley T M, 1996. Molecular diversity of soil and marine 16S rRNA gene sequences related to beta-subgroup ammoniaoxidizing bacteria. Appl Environ Microbiol, 62: 4147–4154. Utaker J B, Bakken L R, Jiang Q Q, Nes I F, 1996. Phylogenetic analysis of 7 new isolates of ammonia-oxidizing bacteria based on 16S ribosomal RNA gene sequences. Syst and Appl Microbiol, 18: 549–559. Yu J L, Zhao D Y, Liu B H, Lin X C, Jiang B G, 1998. Residue analysis of acetochlor in soybean and soil. Pesticides, 37: 28–30. Zhang H W, Zhou Q X, Zhang Q R, Zhang C G, 2004a. Toxic effects of acetochlor, methamidophos and their combination on bacterial amount and population richness at molecular levels in agricultural black soils. Environmental Science, 25: 142–148. Zhang Q R, Zhou Q X, Zhang H W, Cheng X S, 2004b. Joint effects of acetochlor and Cu2+ on indigenous bacterial communities in phaeozem. Acta Scientiae Circumstantiae, 24: 326–332. Zhou J Z, Bruns M A, Tiedje J M, 1996. DNA recovery from soils of diverse composition. Appl Environ Microbiol, 62: 316–322.
Impact of acetochlor on ammonia-oxidizing bacteria in microcosm soils LI Xinyu1 , ZHANG Huiwen1,∗, WU Minna1,2 , SU Zhencheng1 , ZHANG Chenggang1 1. Microbiolog Resource and Ecology Group, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China. E-mail: [email protected] 2. Graduate School of Chinese Academy of Sciences, Beijing 100039, China Received 25 October 2007; revised 13 December 2007; accepted 16 January 2008
Abstract Acetochlor is an increasingly used herbicide on corn in North China. Currently, the effect of acetochlor on soil ammonia-oxidizing bacteria (AOB) communities is not well documented. Here, we studied the diversity and community composition of AOB in soil amended with three concentrations of acetochlor (50, 150, 250 mg/kg) and the control (0 mg acetochlor/kg soil) in a microcosm experiment by PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) and the phylogenetic analysis of excised DGGE bands. DGGE profiles showed that acetochlor had a stimulating effect on AOB at the early stage after acetochlor amended, and the order of intensity and duration is medium-acetochlor amended samples (AM) > low-acetochlor amended samples (AL) > high-acetochlor amended samples (AH). At the end of 60 d microcosm, acetochlor had a negative effect on the diversity of AOB. Cluster analysis of DGGE profiles showed that acetochlor had a greater effect on the community structure of AOB on day 60 than on day 1. The phylogenetic analysis revealed that all the sequences of excised DGGE bands were closely related to members of the genus Nitrosospira and formed two separate subclusters designated as subcluster 1 and subcluster 2 affiliated respectively with clusters 3 and 4 in Nitrosospira as defined by Stephen. Some dominant AOB had a change from subcluster 2 to subcluster 1 with the incubation. The results showed that acetochlor had an effect on the AOB on a long-term basis and the chronic effect of acetochlor should be paid more attention in future. Key words: ammonia-oxidizing bacteria (AOB); PCR-DGGE; acetochlor
Introduction Heilongjiang Province in Northeast China is a main grain production base of this country, and black soil is one of its main agricultural soils. Herbicide acetochlor is used within this area in an increasing amount to increase crop yield. Some studies were conducted regarding its impacts on soil bacteria (Luo et al., 2004; Zhang et al., 2004a, 2004b), but little is known about soil ammonia-oxidizing bacteria (AOB). Nitrification is a key soil process affecting the global nitrogen cycling, among which the AOB play an essential role. AOB are chemolithoautotrophs; they have in common the ability to utilize ammonia as the sole source of energy and carbon dioxide as the main source of C (Hooper et al., 1997). Furthermore, most strains belong to a single (monophyletic) evolutionary group within the β-subclass of Proteobacteria. Well-known genera in this group are Nitrosomonas and Nitrosospira. The AOB have been suggested as the model organisms in microbial ecology (Kowalchuk and Stephen, 2001) and are often used as indicators of soil perturbations (Stephen et al., 1999; Chang et al., 2001; Nyberg et al., 2006). Fingerprints of AOB communities are often analyzed by * Corresponding author. E-mail: [email protected].
denaturing gradient gel electrophoresis (DGGE) (Innerebner et al., 2006; Enwall et al., 2007; Ma et al., 2007), which is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial communities (Muyzer et al., 1993). In this study, the 16S rDNA genes were targeted after soil DNA extraction to investigate whether the application of acetochlor will have effects on the AOB communities by DGGE analysis.
1 Material and methods 1.1 Soil sampling and microcosm experiment Surface soil samples (0–20 cm) without application of agrochemicals for 8 years were collected from a field of the Hailun Agro-ecological Experimental Station (47◦ 27 N, 126◦ 55 E) in Hailun County of Heilongjiang Province, northeast China. The samples were well mixed, sieved through a 2-mm sieve, and stored at 4°C. The soil is classified as a black soil or a Hapli-Udic Isohumosol according to the Chinese Soil Taxonomy (CRGCST, 2001). The properties of the soil were as follows: pH (water), 6.2; moisture, 11.2% (W/W); organic matter, 15.9 g/kg. For microcosm experiment, the samples were first
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adjusted to their field moisture content (18.9%, W/W) by adding distilled water, and then divided into 4 subportions to install four treatments (adding 0, 50, 150, and 250 mg acetochlor/kg soil, respectively). Each treatment had three replicates, and 12 microcosms were prepared. The microcosm was a plastic cup filled with 300 g of soil, sealed with parafilm to minimize water loss, and was incubated at 25°C in dark. No additional materials, except water, were added, and sampling was done on day 1, 7, 30, and 60 of the 2 month incubation. Mixed triplicate samples were stored in a refrigerator for DNA extraction at –20°C. 1.2 DNA extraction and PCR amplification of 16S rDNA A direct lysis method described by Zhou et al. (1996) was adopted with a little modification. One gram soil was mixed with 1,000 µl DNA extraction buffer (0.1 mol/L Tris-HCl (pH 8.0), 0.1 mol/L sodium EDTA (pH 8.0), 0.1 mol/L sodium phosphate (pH 8.0), 1.5 mol/L NaCl, 1% CTAB, and 1% SDS) in a microcentrifuge tube, vortexed for 10 s, and incubated in a water bath at 65°C for 30 min, with gentle end-over-end inversions every 15 min. The sample was then frozen and thawed with three cycles from –70 to 65°C, and kept at 65°C for 1 h to make all microbial cells be lysed completely. After 8,000 r/min centrifugation for 10 min at room temperature, the supernatant was collected, and transferred into another microcentrifuge tube, mixing with an equal volume of chloroformisoamyl alcohol (24:1, V/V). The aqueous phase was recovered by centrifugation, and precipitated with 0.6 volume of isopropanol at room temperature for 1 h. The pellet of crude nucleic acids was obtained by 12,000 r/min centrifugation for 30 min at 4°C, washed with cold 70% ethanol, and re-suspended in TE buffer to give a final volume of 50 µl. The crude extract was purified with Wizard PCR Preps (Promega, USA), according to the manufacturer’s instructions. To amplify ammonia-oxidizer specific 16S rDNA from soils, we used a nested PCR approach. For the first-round PCR, primer pair βAMOf/βAMOr (McCaig et al., 1994) was used, and for the second-round PCR, the AOB-specific primer pair CTO189f+GC/CTO654r (Kowalchuk et al., 1997) was used to obtain DNA fragments 465 bp in length. All PCRs were performed with a PTC-200 thermal cycler (MJ Research, USA). The PCR protocol included a 5-min initial denaturation at 94°C, 30 cycles at 94°C for 30 s, at 55°C for 30 s, and at 72°C for 90 s, followed by a final extension at 72°C for 10 min. PCR products were then diluted 10−2 –10−3 fold and used as template for PCR of the 16S rRNA genes using primers CTO189f /CTO654r with a GC-clamp attached to the forward primer. The PCR protocol included a 3-min initial denaturation at 94°C, 30 cycles at 94°C for 30 s, at 55°C for 30 s, and at 72°C for 45 s, followed by a final extension at 72°C for 5 min. The first and second reaction mixtures (V = 50 µl) contained 1× PCR reaction buffer (TaKaRa, Japan), 10 ng DNA template, 20 pmol/L of forward and reverse primers, 200 µmol/L dNTP mix, and 2.5 U of Ex Taq DNA Polymerase (TaKaRa, Japan).
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1.3 DGGE analysis DGGE analysis was performed with a DCode mutation detection system (Bio-Rad, USA). Gels of 6% acrylamide (37.5:1 acrylamide-bisacrylamide) were formed between 40% and 60% denaturant, with 100% denaturant defined as 7 mol/L urea and 40% (V/V) formamide. Normally, 300 ng PCR products were loaded onto each lane of the gels. The gels were run at voltage of 200 V for 5 h, and maintained at a constant temperature of 60°C in 7 L of 1× TAE buffer (40 mmol/L Tris-acetate, 1 mmol/L EDTA, and pH 8.0). The DGGE gels were stained with GeneFinder (Bio-V Biological Technology Co., Ltd., China) and the images were analyzed with the software package Quantitiy one 2.1 (Bio-Rad, USA). Cluster analysis and dendrograms were calculated using UPGMA. The Shannon-Weaver diversity index (H) (Shannon and Weaver, 1963) was calculated to examine the structural diversity of the ammonia-oxidizer community. 1.4 Recovery and sequence analysis of bands from DGGE gels Dominant PCR-DGGE bands were manually excised from the gel, suspended in 20 µl distilled water, and incubated overnight at room temperature. PCR-DGGE was repeated using these samples as template until a single band remained in each lane. A final PCR step was performed without the GC clamp attached to the forward primer. PCR products were then purified using a Mini-DNA Fragment Rapid Purification Kit (BioDev, Beijing) and sequencing reactions were run on an ABI 377 apparatus by Shanghai Sangon Biological Engineering Technology and Service Co. Ltd. The nucleotide sequences obtained in this study have been deposited in the GenBank database under accession numbers EF434160–EF434169. 1.5 Phylogenetic analysis of sequenced DGGE bands Excised band sequences were subjected to a blastn search on the NCBI site to identify sequences with highest similarity. Highly similar sequences and some reference sequences of dominant ammonia-oxidizing groups were added to the data set for CLUSTAL W multiple sequence alignment and the phylogenetic tree was constructed in MEGA 3.1 by applying the neighbor-joining method with 100 bootstrap resamplings.
2 Results 2.1 Effect of acetochlor on ammonia-oxidizing community The PCR-DGGE banding profiles of soil ammoniaoxidizing communities under acetochlor stress (Fig.1) showed that there was a visible alternation in both the DGGE banding fingerprints and the amounts among different treatments. H was calculated to estimate the diversity of the AOB communities in acetochlor-amended and control soils (Table 1). The diversity indices changed from increasing at the initial stage of incubation to decreasing at the end of the incubation for all acetochlor-amended
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Table 1 Shannon-Weaver diversity indices (H) of AOB communities from DGGE profiles of control (CK) and acetochlor-amended samples Sampling time (d)
Acetochlor amended level (mg/kg) 0 (CK) 50 150 250
1 7 30 60
2.039 1.371 1.772 2.161
2.106 2.048 1.684 1.601
2.284 2.050 1.936 1.098
2.054 1.357 1.770 1.087
samples compared with that for the control sample. First, the diversity index of the low-acetochlor amended samples (AL) started to decline after 30 d incubation, while that of the medium-acetochlor amended samples (AM) still increased on day 30 and started to decline at the end of the incubation. The diversity index of high-acetochlor amended samples (AH) was slightly higher than that of the control samples on day 7, and then had a similar value to that of the control samples, and at last decreased on day 60. Based on the DGGE profiles (Fig.1), the increasing indices in acetochlor-amended samples on day 1 may result from the rich number of bands and high brightness in some bands, suggesting that acetochlor may stimulate the increase of AOB. From day 1 to day 60, the changes were obvious in band number and position, especially for the AM and AH samples. On day 60, the lower diversity indices in acetochlor-amended samples were mainly ascribed to the decrease of the band number. It indicated that the acetochlor had a stimulating effect on AOB at the early stage of incubation, and the order of intensity and duration was AM > AL > AH. When the concentration was
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higher than 150 mg/kg, the stimulating effect weakened in intensity and shortened in duration of time. At the end of 60 d incubation, acetochlor had a negative effect on the diversity of AOB. Clustering analysis of acetochlor-amended and control samples sampled on day 1 and 60 are shown in Fig.2. The dendrogram was composed of three main
Fig. 2 UPGMA dendrogram constructed with AOB DGGE profiles from control and acetochlor-amended samples on day 1 and 60. Samples were marked by number (1, 2, 3, and 4 stand for the four acetochlor-amended levels of 0, 50, 150, and 250 mg acetochlor/kg soil, respectively) and sampling time (1 and 60 d, respectively).
Fig. 1 DGGE community fingerprints of AOB in soil samples amended with different amounts of acetochlor sampled on day 1, 7, 30, 60. The labeled bands were excised from the gel, reamplified, and subjected to sequence analysis. lane 1: control soil; lane 2: 50 mg/kg; lane 3: 150 mg/kg; lane 4: 250 mg/kg.
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clusters as follows: one cluster included three concentration acetochlor-amended samples on day 1 and the control on day 1 and 60; one cluster had AL and AM samples on day 60; and the last cluster contained only AH sample on day 60. The results showed that acetochlor had greater effect on the community structure of AOB on day 60 than on day 1. From the results of the diversity indices and clustering analysis, we can conclude that acetochlor had a toxic effect on AOB community. 2.2 Sequencing and phylogenetic analyses Based on the BLASTN results, highly similar GenBank sequences and AOB reference sequences were added to the data set to analyze the phylogenetic relationship of ammonia-oxidizing bacteria in this study. CLUSTAL
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W multiple sequence alignment was performed and the phylogenetic distance tree was constructed as shown in Fig.3. As seen in Fig.3, all the sequences of excised DGGE bands were closely related to members of the genus Nitrosospira forming two separate subclusters designated as subcluster 1 and subcluster 2. A5–A7 and A17 belonged to subcluster 2 and sequence similarities within subcluster 2 ranged from 96% to 99%. Bands A8–A13 belonged to Nitrosospira subcluster 1 and sequence similarities within the subcluster 1 ranged from 96% to 99%. Subcluster 1 was affiliated with Nitrosospira cluster 3 comprising most of the soil 16S rDNA sequences from cultured Nitrosospira sp. as defined by Stephen et al. (1996). Subcluster 2 was affiliated with Nitrosospira cluster 4. Nine excised DGGE bands can be divided into three
Fig. 3 Phylogenetic relationship of excised DGGE bands and reference AOB sequences. The dendrogram was generated by the neighbor-joining method with 100 bootstrap resamplings. Branching points supported by bootstrap values > 50% are indicated in the text. The scale bar represents 10% sequence divergence. The designation of clusters 1–7 and Nitrosospira subcluster SM refers to Stephen et al. (1996) and Haleem et al. (2000).
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kinds of change patterns according to the DGGE profiles. (1) A5–A6 and A11–A13 all increased in acetochloramended samples; however, these bands appeared to increase at different times of incubation. A5–A6 in subcluster 2 only increased in the early stage of incubation; A11–A12 in subcluster 1 was on day 30 and A13 was both on day 1 and 30. Dominant AOB in diversity changed from subcluster 2 to subcluster 1 over time. (2) A7 in subcluster 1 and A9 in subcluster 2 belong to the common ammonia-oxidizing populations given that these two bands were found in most treatments during the entire incubation period. (3) A8–A10 in subcluster 1 and A17 in subcluster 2 varied frequently in four treatments during 60 d incubation. The change was greater in AM and AH samples than in AL samples. The results from the phylogenetic analysis indicated that major temporal changes in composition of the AOB communities from subcluster 2 to subcluster 1 occurred in acetochlor-amended soils during the period of the experiment. All the sequences of excised bands were highly similar to the Nitrosospira sp. with the similarity levels ranging from 98% to 100%, except A10, which was highly similar to Nitrosovibrio tenuis Nv1 (with the similarity level of 98.61%).
3 Discussion In this study, the excised bands were closely related to Nitrosospira clusters 3, SM, and 4 comprising most of the βAOB 16S rDNA sequences of cultured or yet uncultured Nitrosospira sp. from soil and rhizosphere. Stephen et al. (1996) showed that Nitrosospira sp. and Nitrosomonas sp. could be subdivided into at least seven rDNA clusters, designated as 1–4 and 5–7, respectively (Fig.3). Analysis of different habitats revealed that Nitrosospira cluster 1 is formed exclusively by 16S rDNA sequences retrieved from marine sediments (Stephen et al., 1996), sea water (Phillips et al., 1999), or coastal sand dunes (Kowalchuk et al., 1997), whereas clusters 2–4 are comprised of rDNA sequences retrieved from cultured strains and clonal analyses from soil, sewage or rhizosphere environments, and coastal sand dunes (Utaker et al., 1996; Kowalchuk et al., 1997, 1999; Speksnijder et al., 1998). In contrast, Nitrosomonas subclusters 5–7 represent 16S rDNA sequences from various environmental sources such as marine sediments (Stephen et al., 1996), seawater (Phillips et al., 1999), freshwater (Speksnijder et al., 1998), or coastal sand dunes (Kowalchuk et al., 1997). Recently, Haleem et al. (2000) found another novel subcluster (SM cluster) in addition to the definition of Stephen et al. (1996). The close relationship between environmental habitats and the phylogenetic structure of the respective βAOB populations was also observed in this study. The above close relationship was also observed in other researches for coastal sand dunes and agricultural soil, and may reflect the physiological differences (Kowalchuk et al., 1997; Stephen et al., 1998; Haleem et al., 2000). In this study, there are two dominant types of AOB divided into subcluster 1 and subcluster 2. At the early stage of the experiment, the diversity of AOB in subcluster
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2 increased under acetochlor stress, while the diversity of AOB in subcluster 1 was high on day 30 after incubation. The change of the dominant AOB in diversity over time may result from the difference of utilizing degradated products with degradation of acetochlor, suggesting the physiological differences in subcluster 1 (in cluster 3 ) and subcluster 2 ( in cluster 4). At the end of 60 d incubation, the diversity indices of AOB in AM and AH samples were extremely low, resulting in only two bands in the DGGE analysis. In contrast, the ammonia oxidizers had a high diversity in the control sample. The results showed that acetochlor had a negative effect on the AOB on a long-term basis and the chronic effect of acetochlor should be paid more attention in future even if acetochlor is low toxic and easy to degrade (Yu et al., 1998).
4 Conclusions The results described in this article show that acetochlor had a stimulating effect on AOB community at the early treatment, and the order in intensity and duration was AM (150 mg/kg) > AL (50 mg/kg) > AH (250 mg/kg). At the end of 60 d incubation, acetochlor had a negative effect on the diversity of AOB community. The cluster analysis of DGGE profiles indicated that acetochlor had more effect on the community structure of AOB on day 60 than on day 1. The phylogenetic analysis revealed that all sequences of excised DGGE bands were closely related to members of the genus Nitrosospira forming two separate subclusters, designated as subcluster 1 and subcluster 2, and affiliated respectively with Nitrosospira clusters 3 and 4 as defined by Stephen et al. (1996). There was a major temporal change in the composition of the AOB community from subcluster 2 to subcluster 1 that occurred in acetochlortreated soils during the period of study. Based on the results described above, it is found that acetochlor had a negative effect on the AOB on a longterm basis, and the chronic effect of acetochlor should be paid more attention. Acknowledgments This work was supported by the Natural Science Foundation for Young Scientists of China (No. 40701088).
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