Impact of acetochlor on ammonia-oxidizing bacteria in microcosm soils

jesc.a

c.cn

jesc.a

c.cn

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

AbstractAcetochlor 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 soilamended with three concentrations of acetochlor (50, 150, 250 mg/kg) and the control (0 mg acetochlor/kg soil) in a microcosmexperiment by PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) and the phylogenetic analysis ofexcised DGGE bands. DGGE profiles showed that acetochlor had a stimulating effect on AOB at the early stage after acetochloramended, 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 ofAOB. Cluster analysis of DGGE profiles showed that acetochlor had a greater effect on the community structure of AOB on day 60than on day 1. The phylogenetic analysis revealed that all the sequences of excised DGGE bands were closely related to members of thegenus Nitrosospira and formed two separate subclusters designated as subcluster 1 and subcluster 2 affiliated respectively with clusters3 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 paidmore attention in future.

Key words: ammonia-oxidizing bacteria (AOB); PCR-DGGE; acetochlor

Introduction

Heilongjiang Province in Northeast China is a maingrain production base of this country, and black soil is oneof its main agricultural soils. Herbicide acetochlor is usedwithin this area in an increasing amount to increase cropyield. Some studies were conducted regarding its impactson soil bacteria (Luo et al., 2004; Zhang et al., 2004a,2004b), but little is known about soil ammonia-oxidizingbacteria (AOB).

Nitrification is a key soil process affecting the globalnitrogen cycling, among which the AOB play an essentialrole. AOB are chemolithoautotrophs; they have in commonthe ability to utilize ammonia as the sole source of energyand carbon dioxide as the main source of C (Hooper etal., 1997). Furthermore, most strains belong to a single(monophyletic) evolutionary group within the β-subclassof Proteobacteria. Well-known genera in this group areNitrosomonas and Nitrosospira.

The AOB have been suggested as the model organismsin microbial ecology (Kowalchuk and Stephen, 2001) andare often used as indicators of soil perturbations (Stephenet 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) (Innereb-ner et al., 2006; Enwall et al., 2007; Ma et al., 2007),which is a powerful and convenient tool for analyzing thesequence diversity of complex natural microbial commu-nities (Muyzer et al., 1993).

In this study, the 16S rDNA genes were targeted aftersoil DNA extraction to investigate whether the applicationof acetochlor will have effects on the AOB communities byDGGE analysis.

1 Material and methods

1.1 Soil sampling and microcosm experiment

Surface soil samples (0–20 cm) without application ofagrochemicals for 8 years were collected from a field of theHailun Agro-ecological Experimental Station (47◦27′N,126◦55′E) in Hailun County of Heilongjiang Province,northeast China. The samples were well mixed, sievedthrough a 2-mm sieve, and stored at 4°C. The soil isclassified as a black soil or a Hapli-Udic Isohumosol ac-cording 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

jesc.a

c.cn

jesc.a

c.cn

No. 9 Impact of acetochlor on ammonia-oxidizing bacteria in microcosm soils 1127

adjusted to their field moisture content (18.9%, W/W)by adding distilled water, and then divided into 4 sub-portions to install four treatments (adding 0, 50, 150, and250 mg acetochlor/kg soil, respectively). Each treatmenthad three replicates, and 12 microcosms were prepared.The microcosm was a plastic cup filled with 300 g ofsoil, sealed with parafilm to minimize water loss, and wasincubated at 25°C in dark. No additional materials, exceptwater, were added, and sampling was done on day 1, 7, 30,and 60 of the 2 month incubation. Mixed triplicate sampleswere stored in a refrigerator for DNA extraction at –20°C.

1.2 DNA extraction and PCR amplification of 16S rD-NA

A direct lysis method described by Zhou et al. (1996)was adopted with a little modification. One gram soil wasmixed with 1,000 µl DNA extraction buffer (0.1 mol/LTris-HCl (pH 8.0), 0.1 mol/L sodium EDTA (pH 8.0), 0.1mol/L sodium phosphate (pH 8.0), 1.5 mol/L NaCl, 1%CTAB, and 1% SDS) in a microcentrifuge tube, vortexedfor 10 s, and incubated in a water bath at 65°C for30 min, with gentle end-over-end inversions every 15min. The sample was then frozen and thawed with threecycles from –70 to 65°C, and kept at 65°C for 1 h tomake all microbial cells be lysed completely. After 8,000r/min centrifugation for 10 min at room temperature, thesupernatant was collected, and transferred into anothermicrocentrifuge tube, mixing with an equal volume ofchloroformisoamyl alcohol (24:1, V/V). The aqueous phasewas recovered by centrifugation, and precipitated with0.6 volume of isopropanol at room temperature for 1 h.The pellet of crude nucleic acids was obtained by 12,000r/min centrifugation for 30 min at 4°C, washed with cold70% ethanol, and re-suspended in TE buffer to give afinal volume of 50 µl. The crude extract was purifiedwith Wizard PCR Preps (Promega, USA), according to themanufacturer’s instructions.

To amplify ammonia-oxidizer specific 16S rDNA fromsoils, we used a nested PCR approach. For the first-roundPCR, primer pair βAMOf/βAMOr (McCaig et al., 1994)was used, and for the second-round PCR, the AOB-specificprimer 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-mininitial denaturation at 94°C, 30 cycles at 94°C for 30 s, at55°C for 30 s, and at 72°C for 90 s, followed by a finalextension at 72°C for 10 min. PCR products were thendiluted 10−2–10−3 fold and used as template for PCR ofthe 16S rRNA genes using primers CTO189f /CTO654rwith a GC-clamp attached to the forward primer. The PCRprotocol included a 3-min initial denaturation at 94°C, 30cycles at 94°C for 30 s, at 55°C for 30 s, and at 72°C for45 s, followed by a final extension at 72°C for 5 min. Thefirst and second reaction mixtures (V = 50 µl) contained1× PCR reaction buffer (TaKaRa, Japan), 10 ng DNAtemplate, 20 pmol/L of forward and reverse primers, 200µmol/L dNTP mix, and 2.5 U of Ex Taq DNA Polymerase(TaKaRa, Japan).

1.3 DGGE analysis

DGGE analysis was performed with a DCode mutationdetection system (Bio-Rad, USA). Gels of 6% acrylamide(37.5:1 acrylamide-bisacrylamide) were formed between40% and 60% denaturant, with 100% denaturant defined as7 mol/L urea and 40% (V/V) formamide. Normally, 300 ngPCR products were loaded onto each lane of the gels. Thegels were run at voltage of 200 V for 5 h, and maintainedat 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-VBiological Technology Co., Ltd., China) and the imageswere analyzed with the software package Quantitiy one 2.1(Bio-Rad, USA). Cluster analysis and dendrograms werecalculated using UPGMA. The Shannon-Weaver diversityindex (H) (Shannon and Weaver, 1963) was calculated toexamine the structural diversity of the ammonia-oxidizercommunity.

1.4 Recovery and sequence analysis of bands fromDGGE gels

Dominant PCR-DGGE bands were manually excisedfrom the gel, suspended in 20 µl distilled water, andincubated overnight at room temperature. PCR-DGGEwas repeated using these samples as template until asingle band remained in each lane. A final PCR stepwas performed without the GC clamp attached to theforward primer. PCR products were then purified usinga Mini-DNA Fragment Rapid Purification Kit (BioDev,Beijing) and sequencing reactions were run on an ABI377 apparatus by Shanghai Sangon Biological EngineeringTechnology and Service Co. Ltd. The nucleotide sequencesobtained in this study have been deposited in the GenBankdatabase under accession numbers EF434160–EF434169.

1.5 Phylogenetic analysis of sequenced DGGE bands

Excised band sequences were subjected to a blastnsearch on the NCBI site to identify sequences with highestsimilarity. Highly similar sequences and some referencesequences of dominant ammonia-oxidizing groups wereadded to the data set for CLUSTAL W multiple sequencealignment and the phylogenetic tree was constructed inMEGA 3.1 by applying the neighbor-joining method with100 bootstrap resamplings.

2 Results

2.1 Effect of acetochlor on ammonia-oxidizing commu-nity

The PCR-DGGE banding profiles of soil ammonia-oxidizing communities under acetochlor stress (Fig.1)showed that there was a visible alternation in both theDGGE banding fingerprints and the amounts among differ-ent treatments. H was calculated to estimate the diversityof the AOB communities in acetochlor-amended and con-trol soils (Table 1). The diversity indices changed fromincreasing at the initial stage of incubation to decreasingat the end of the incubation for all acetochlor-amended

jesc.a

c.cn

jesc.a

c.cn

1128 LI Xinyu et al. Vol. 20

Table 1 Shannon-Weaver diversity indices (H) of AOB communitiesfrom DGGE profiles of control (CK) and acetochlor-amended samples

Sampling time (d) Acetochlor amended level (mg/kg)0 (CK) 50 150 250

1 2.039 2.106 2.284 2.0547 1.371 2.048 2.050 1.35730 1.772 1.684 1.936 1.77060 2.161 1.601 1.098 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 thatof the medium-acetochlor amended samples (AM) stillincreased on day 30 and started to decline at the endof the incubation. The diversity index of high-acetochloramended samples (AH) was slightly higher than that of thecontrol samples on day 7, and then had a similar value tothat of the control samples, and at last decreased on day60. Based on the DGGE profiles (Fig.1), the increasingindices in acetochlor-amended samples on day 1 may resultfrom the rich number of bands and high brightness insome bands, suggesting that acetochlor may stimulate theincrease of AOB. From day 1 to day 60, the changeswere obvious in band number and position, especially forthe AM and AH samples. On day 60, the lower diver-sity indices in acetochlor-amended samples were mainlyascribed to the decrease of the band number. It indicatedthat the acetochlor had a stimulating effect on AOB at theearly stage of incubation, and the order of intensity andduration was AM >AL >AH. When the concentration was

higher than 150 mg/kg, the stimulating effect weakened inintensity and shortened in duration of time. At the end of60 d incubation, acetochlor had a negative effect on thediversity of AOB.

Clustering analysis of acetochlor-amended and con-trol samples sampled on day 1 and 60 are shown inFig.2. The dendrogram was composed of three main

Fig. 2 UPGMA dendrogram constructed with AOB DGGE profiles fromcontrol and acetochlor-amended samples on day 1 and 60. Samples weremarked by number (1, 2, 3, and 4 stand for the four acetochlor-amendedlevels of 0, 50, 150, and 250 mg acetochlor/kg soil, respectively) andsampling 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 labeledbands 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: 250mg/kg.

jesc.a

c.cn

jesc.a

c.cn

No. 9 Impact of acetochlor on ammonia-oxidizing bacteria in microcosm soils 1129

clusters as follows: one cluster included three concentra-tion acetochlor-amended samples on day 1 and the controlon day 1 and 60; one cluster had AL and AM sampleson day 60; and the last cluster contained only AH sampleon day 60. The results showed that acetochlor had greatereffect on the community structure of AOB on day 60 thanon day 1. From the results of the diversity indices andclustering analysis, we can conclude that acetochlor hada toxic effect on AOB community.

2.2 Sequencing and phylogenetic analyses

Based on the BLASTN results, highly similar Gen-Bank sequences and AOB reference sequences were addedto the data set to analyze the phylogenetic relationshipof ammonia-oxidizing bacteria in this study. CLUSTAL

W multiple sequence alignment was performed and thephylogenetic distance tree was constructed as shown inFig.3. As seen in Fig.3, all the sequences of excisedDGGE bands were closely related to members of the genusNitrosospira forming two separate subclusters designatedas subcluster 1 and subcluster 2. A5–A7 and A17 belongedto subcluster 2 and sequence similarities within subcluster2 ranged from 96% to 99%. Bands A8–A13 belonged toNitrosospira subcluster 1 and sequence similarities withinthe subcluster 1 ranged from 96% to 99%. Subcluster 1 wasaffiliated with Nitrosospira cluster 3 comprising most ofthe soil 16S rDNA sequences from cultured Nitrosospirasp. as defined by Stephen et al. (1996). Subcluster 2 wasaffiliated 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-joiningmethod 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).

jesc.a

c.cn

jesc.a

c.cn

1130 LI Xinyu et al. Vol. 20

kinds of change patterns according to the DGGE profiles.(1) A5–A6 and A11–A13 all increased in acetochlor-amended samples; however, these bands appeared toincrease at different times of incubation. A5–A6 in sub-cluster 2 only increased in the early stage of incubation;A11–A12 in subcluster 1 was on day 30 and A13 wasboth on day 1 and 30. Dominant AOB in diversity changedfrom subcluster 2 to subcluster 1 over time. (2) A7 insubcluster 1 and A9 in subcluster 2 belong to the commonammonia-oxidizing populations given that these two bandswere found in most treatments during the entire incubationperiod. (3) A8–A10 in subcluster 1 and A17 in subcluster 2varied frequently in four treatments during 60 d incubation.The change was greater in AM and AH samples than inAL samples. The results from the phylogenetic analysisindicated that major temporal changes in composition ofthe AOB communities from subcluster 2 to subcluster 1occurred in acetochlor-amended soils during the period ofthe experiment. All the sequences of excised bands werehighly similar to the Nitrosospira sp. with the similaritylevels ranging from 98% to 100%, except A10, whichwas highly similar to Nitrosovibrio tenuis Nv1 (with thesimilarity level of 98.61%).

3 Discussion

In this study, the excised bands were closely related toNitrosospira clusters 3, SM, and 4 comprising most of theβAOB 16S rDNA sequences of cultured or yet unculturedNitrosospira 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). Analysisof different habitats revealed that Nitrosospira cluster 1is formed exclusively by 16S rDNA sequences retrievedfrom marine sediments (Stephen et al., 1996), sea water(Phillips et al., 1999), or coastal sand dunes (Kowalchuk etal., 1997), whereas clusters 2–4 are comprised of rDNA se-quences retrieved from cultured strains and clonal analysesfrom soil, sewage or rhizosphere environments, and coastalsand dunes (Utaker et al., 1996; Kowalchuk et al., 1997,1999; Speksnijder et al., 1998). In contrast, Nitrosomonassubclusters 5–7 represent 16S rDNA sequences fromvarious 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 tothe definition of Stephen et al. (1996). The close relation-ship between environmental habitats and the phylogeneticstructure of the respective βAOB populations was alsoobserved in this study. The above close relationship wasalso observed in other researches for coastal sand dunesand agricultural soil, and may reflect the physiologicaldifferences (Kowalchuk et al., 1997; Stephen et al., 1998;Haleem et al., 2000).

In this study, there are two dominant types of AOBdivided into subcluster 1 and subcluster 2. At the earlystage of the experiment, the diversity of AOB in subcluster

2 increased under acetochlor stress, while the diversity ofAOB in subcluster 1 was high on day 30 after incubation.The change of the dominant AOB in diversity over timemay result from the difference of utilizing degradatedproducts with degradation of acetochlor, suggesting thephysiological differences in subcluster 1 (in cluster 3 ) andsubcluster 2 ( in cluster 4).

At the end of 60 d incubation, the diversity indicesof AOB in AM and AH samples were extremely low,resulting in only two bands in the DGGE analysis. Incontrast, the ammonia oxidizers had a high diversity in thecontrol sample. The results showed that acetochlor had anegative effect on the AOB on a long-term basis and thechronic effect of acetochlor should be paid more attentionin 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 acetochlorhad a stimulating effect on AOB community at the earlytreatment, and the order in intensity and duration was AM(150 mg/kg) > AL (50 mg/kg) > AH (250 mg/kg). At theend of 60 d incubation, acetochlor had a negative effect onthe diversity of AOB community. The cluster analysis ofDGGE profiles indicated that acetochlor had more effecton the community structure of AOB on day 60 than on day1.

The phylogenetic analysis revealed that all sequences ofexcised DGGE bands were closely related to members ofthe genus Nitrosospira forming two separate subclusters,designated as subcluster 1 and subcluster 2, and affiliatedrespectively with Nitrosospira clusters 3 and 4 as definedby Stephen et al. (1996). There was a major temporalchange in the composition of the AOB community fromsubcluster 2 to subcluster 1 that occurred in acetochlor-treated soils during the period of study.

Based on the results described above, it is found thatacetochlor had a negative effect on the AOB on a long-term basis, and the chronic effect of acetochlor should bepaid more attention.

Acknowledgments

This work was supported by the Natural Science Foun-dation for Young Scientists of China (No. 40701088).

References

Chang Y J, Anwar Hussain A K M, Stephen J R, Mullens MD, White D C, Peacock A, 2001. Impact of herbicideson the abundance and structure of indigenous β-subgroupammonia-oxidizer communities in soil microcosms. Envi-ron Toxicol and Chem, 20: 2462–2468.

CRGCST (Cooperative Research Croup on Chinese Soil Taxono-my). 2001. Chinese Soil Taxonomy. Beijing and New York:Science Press. 123.

Enwall K, Nyberg K, Bertilsson S, Cederlund H, Stenstrom J,Hallin S, 2007. Long-term impact of fertilization on activityand composition of bacterial communities and metabolicguilds in agricultural soil. Soil Biol Biochem, 39: 106–115.

jesc.a

c.cn

jesc.a

c.cn

No. 9 Impact of acetochlor on ammonia-oxidizing bacteria in microcosm soils 1131

Haleem D A E, von Wintzingerode F, Moter A, Moawad H, GobelU B, 2000. Phylogenetic analysis of rhizosphere-associatedβ-subclass proteobacterial ammonia oxidizers in a munic-ipal wastewater treatment plant based on rhizoremediationtechnology. 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 bybacteria. Antonie Van Leeuwenhoek, 71: 59–67.

Innerebner G, Knapp B, Vasara T, Romantschuk M, InsamH, 2006. Traceability of ammonia-oxidizing bacteria incompost-treated soils. Soil Biol Biochem, 38: 1092–1100.

Kowalchuk G A, Stephen J R, 2001. Ammonia-oxidizing bac-teria, a model for molecular microbial ecology. Annu RevMicrobiol, 55: 485–529.

Kowalchuk G A, Stephen J R, de Boer W, Prosser J I, Embley TM, Woldendorp J W, 1997. Analysis of ammonia-oxidizingbacteria of the β-subdivision of the class Proteobacteria incoastal sand dunes by denaturing gradient gel electrophore-sis and sequencing of PCR-amplified 16S ribosomal DNAfragments. 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 analysisof ammonia-oxidizing bacteria of the β-subdivision of theclass of Proteobacteria in compost and composted materi-als. Appl Environ Microbiol, 65: 396–403.

Luo H F, Qi H Y, Zhang H X, 2004. The impact of acetochlor onthe 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 potentialof ammonia oxidizing bacteria to produce nitrous oxide insoils 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 analysisof enrichment cultures of marine ammonia oxidizers. FEMSMicrobiol Lett, 120: 363–368.

Muyzer G, De Waal E C, Uitterlinden A G, 1993. Profilingof complex microbial populations by denaturing gradientgel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S RNA. Appl EnvironMicrobiol, 3: 695–700.

Nyberg K, Schnurer A, Sundh I, Jarvis Å, Hallin S, 2006. Am-monia oxidising communities in agricultural soil incubatedwith organic waste residues. Biol Fert Soils, 42: 315–323.

Phillips C J, Smith Z, Embley T M, Prosser J I, 1999. Phyloge-netic difference between particle-associated and planktonic

ammonia-oxidizing bacteria of the β-subdivision of the classProteobacteria in the northwestern Mediterranean sea. ApplEnviron Microbiol, 65: 779–786.

Shannon C E, Weaver W, 1963. The Mathematical Theory ofCommunication. 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 sequencegroup 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 toxicmetals on indigenous soil β-subgroup proteobacterium am-monia oxidizer community structure and protection againsttoxicity by inoculated metal-resistant bacteria. Appl EnvironMicrobiol, 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 β-subgroup proteobacterial ammonia oxidizer populations insoil by denaturing gradient gel electrophoresis analysis andhierarchical phylogenetic probing. Appl Environ Microbiol,64: 2958–2965.

Stephen J R, McCaig A E, Smith Z, Prosser J I, EmbleyT M, 1996. Molecular diversity of soil and marine 16SrRNA gene sequences related to beta-subgroup ammonia-oxidizing bacteria. Appl Environ Microbiol, 62: 4147–4154.

Utaker J B, Bakken L R, Jiang Q Q, Nes I F, 1996. Phylogeneticanalysis of 7 new isolates of ammonia-oxidizing bacteriabased on 16S ribosomal RNA gene sequences. Syst andAppl Microbiol, 18: 549–559.

Yu J L, Zhao D Y, Liu B H, Lin X C, Jiang B G, 1998. Residueanalysis of acetochlor in soybean and soil. Pesticides, 37:28–30.

Zhang H W, Zhou Q X, Zhang Q R, Zhang C G, 2004a. Toxiceffects of acetochlor, methamidophos and their combinationon bacterial amount and population richness at molecularlevels in agricultural black soils. Environmental Science,25: 142–148.

Zhang Q R, Zhou Q X, Zhang H W, Cheng X S, 2004b. Jointeffects of acetochlor and Cu2+ on indigenous bacterialcommunities in phaeozem. Acta Scientiae Circumstantiae,24: 326–332.

Zhou J Z, Bruns M A, Tiedje J M, 1996. DNA recovery fromsoils of diverse composition. Appl Environ Microbiol, 62:316–322.