Environ Monit Assess DOI 10.1007/s10661-013-3581-9
Effects of fomesafen on soil enzyme activity, microbial population, and bacterial community composition Qingming Zhang & Lusheng Zhu & Jun Wang & Hui Xie & Jinhua Wang & Fenghua Wang & Fengxia Sun
Received: 16 September 2013 / Accepted: 3 December 2013 # Springer Science+Business Media Dordrecht 2013
Abstract Fomesafen is a diphenyl ether herbicide that has an important role in the removal of broadleaf weeds in bean and fruit tree fields. However, very little information is known about the effects of this herbicide on soil microbial community structure and activities. In the present study, laboratory experiments were conducted to examine the effects of different concentrations of fomesafen (0, 10, 100, and 500 μg/kg) on microbial community structure and activities during an exposure period of 60 days, using soil enzyme assays, plate counting, and denaturing gradient gel electrophoresis (DGGE). The results of enzymatic activity experiments showed that fomesafen had different stimulating effects on the activities of acid phosphatase, alkaline
Q. Zhang College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, China Q. Zhang : L. Zhu : J. Wang : H. Xie : J. Wang : F. Wang : F. Sun Key Laboratory of Agricultural Environment in the University of Shandong, National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, College of Resources and Environment, Shandong Agricultural University, Taian 271018, China L. Zhu (*) College of Resources and Environment, Shandong Agricultural University, 61 Daizong Road, Taian 271018, People’s Republic of China e-mail: [email protected] L. Zhu e-mail: [email protected]
phosphatase, and dehydrogenase, with dehydrogenase being most sensitive to fomesafen. On the tenth day, urease activity was inhibited significantly after treatment of different concentrations of fomesafen; this inhibiting effect then gradually disappeared and returned to the control level after 30 days. Plate counting experiments indicated that the number of bacteria and actinomycetes increased in fomesafen-spiked soil relative to the control after 30 days of incubation, while fungal number decreased significantly after only 10 days. The DGGE results revealed that the bacterial community varied in response to the addition of fomesafen, and the intensity of these six bands was greater on day 10. Sequencing and phylogenetic analyses indicated that the six excised DGGE bands were closely related to Emticicia, Bacillus, and uncultured bacteria. After 10 days, the bacterial community exhibited no obvious change compared with the control. Throughout the experiment, we concluded that 0–500 μg/kg of fomesafen could not produce significant toxic effects on soil microbial community structure and activities. Keywords Fomesafen . Soil enzyme . PCR-DGGE . Bacterial community structure
Introduction In modern agricultural production, pesticide application is a regular practice. However, the increased application of pesticide may cause soil contamination. In particular,
Environ Monit Assess
high toxicity and long residual herbicide could have hazardous impacts on the health of soil ecosystems and an adverse influence on soil biological processes by altering soil enzymatic activities, soil microbial population, and soil microbial community diversity (Ratcliff et al. 2006; Niemi et al. 2009; Sofo et al. 2012). Fomesafen (5-[2-chloro-4-(trifluoromethyl)phenoxy]N-(methylsulfonyl)2-nitrobenzamide) is a diphenyl ether herbicide that is usually applied as a pre- or postemergent foliar to remove broadleaf weeds in soybean plants, fruit trees, and rubber estate fields (Santos et al. 2006; Sikkema et al. 2009). In China, fomesafen has been used extensively with the succession and multiplication of weeds on soybean farmland in recent years; subsequently, the number of fomesafen production companies has increased annually (Liu 2010). However, due to the prolonged use of fomesafen over the years, this herbicide has lead to some adverse effects, such as injury to sensitive aftercrops and hazards to agricultural adjustment and security (Wang et al. 2003; Rauch et al. 2007). These adverse effects may be related to increased fomesafen residue concentration in the soil, and studies have shown that this pesticide has a relatively long halflife (approximately 37.5–56 d) in field soil (Mills and Simmons 1998; Cobucci et al. 1998). The soil environment can certainly be affected by fomesafen residue; unfortunately, information on such effects has been limited to date. Soil is a complex and dynamic living ecosystem, and certain factors influence its quality and health. In the assessment of soil environment quality, the living and dynamic nature of soil, indicated by such factors as soil enzymes and microorganisms, is usually used as a biomarker (Karlen et al. 1997; Sukul 2006). Soil enzymes are involved in catalyzing various reactions and metabolic processes occurring in organic matter metabolism, maintaining soil structure, cycling nutrients, detoxifying pollutants, and producing energy for both microorganisms and plants (Kızılkaya et al. 2004; Khan et al. 2010). Changes in soil enzymatic activities usually imply that natural and anthropogenic disturbances occurred in the soil ecosystem (Gianfreda and Rao 2008; Zhu et al. 2010). Similarly, soil microorganisms also have a crucial role in the cycling of nitrogen, sulfur, and phosphorus and the decomposition of organic residues (Nielsen and Winding 2002). Previous studies showed that microorganisms are very sensitive to changes in their surroundings and appear to be very suitable early warning indicators and predictors in soil health monitoring
(Nielsen and Winding 2002; Epelde et al. 2010). To date, some studies have used soil enzymatic activities and microbial community structure as indicators to determine toxicological influences of various pollutants on soil quality and health. With the rapid development of molecular biology methods, some techniques based on cultureindependent microbiology have been useful in research of soil microbial communities. Denaturing gradient gel electrophoresis (DGGE) is a new molecular biology method that has been widely applied in investigation of the effects of many pollutants on soil or sediment microbial communities, including pesticides (Sigler and Turco 2002; Ferreira et al. 2009), petroleum (Evans et al. 2004), polycyclic aromatic hydrocarbons (PAHs) (Andreoni et al. 2004; Tian et al. 2008), and heavy metals (Khan et al. 2010). Though DGGE is a highthroughput technique compared with other methods, the results of such analyses may be biased due to influencing factors such as the DNA extraction method, polymerase chain reaction (PCR) amplification, and DGGE procedure. Thus, the combination of both culture-independent and culture-dependent techniques might provide more useful and complementary information on the structural diversity of microbial communities (Aislabie et al. 2006; Li et al. 2008). In this study, laboratory simulation tests over a period of 60 days were conducted to evaluate the effects of herbicide fomesafen on soil microbial community structure and activities, using soil enzyme activity determination, plate counting, and 16S rDNA-PCR-DGGE analysis.
Materials and methods Chemicals and soils Fomesafen (98.2 % purity) was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Soil samples were collected from an experimental field (36.11′ N and 117.08 ′ E, 611 m above sea level) located in Shandong Agricultural University, Taian, China. In this region, the mean annual precipitation is 697 mm, and the mean annual temperature is 13 °C. This field remained under conventional farming practices and no history of fomesafen application in the last 5 years. Soil samples were taken from 0–20 cm of the bulk soil on the field prepared for the cultivation of corn after ploughing
Environ Monit Assess
in spring. The soil was classified as a typical Kandiudult (clay and loamy soil) in the USDA Soil Taxonomy System (Soil Survey Staff 1999) and some of its physical and chemical characteristics as follows: pH 7.6, organic matter 17.6 g/kg, available N 132.3 mg/kg, available P 18.4 mg/kg, available K 125.7 mg/kg, and maximal water holding capacity 18.5 %. To consider the spatial heterogeneity, soil samples were taken in triplicates, and each replicate consisted of five10-cm auger cores. Then, the soil samples were manually and gently crumbled, mixed thoroughly, air dried at room temperature, and sieved through a 2-mm mesh to remove stones and plant roots before being used. Soil treatment Concentrations were established according to the actual residual content levels of fomesafen in field soil; the fomesafen concentrations in the soil approximately range from 50 to 420 μg/kg when application rates were 180–375 g ai/ha (Rauch et al. 2007; Guo et al. 2000). Thus, the experiments were conducted with the following four treatments: 0 (solvent control), 10, 100, and 500 μg fomesafen/kg dry soil, labeled ck, F10, F100, and F500, respectively. Each treatment was replicated three times at each sampling stage. For each treatment, 1,200 g soil was thoroughly mixed with fomesafen stock acetone solution to achieve the setting concentration, and 80 g soil (three replicates in each treatment, five sampling intervals) was placed in a brown glass bottle (120 mL) as a microcosm system and sealed with a cotton plug to minimize water loss. All treatments were incubated in the dark at 25 °C for 60 days, and the soil moisture content levels were tested by weighing and adjusted to 60 % water holding capacity by adding deionized water every 2 days. Samples from each bottle were collected after 10, 20, 30, 40, and 60 d for enzyme assays, DNA extraction, and numeration of the microbial community. Enzymatic activity assay The acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) activities were determined as described by Tabatabai (1994). A 0.1 M p-nitrophenol phosphatase solution (pH 6.5 for acid phosphatase and pH 11 for alkaline phosphatase) was used as the substrate, and yellow-band absorbance of the filtrate due to p-nitrophenol was measured at 410 nm. The
p-nitrophenol concentration was calculated by reference to a calibration curve constructed using p-nitrophenol standards, and the results were expressed as micrograms of p-nitrophenol per gram of dry soil (oven-dried soil basis) per hour. The dehydrogenase (EC 1.1.1.1) activity was determined using the method described by Tabatabai (1994). The soluble 2, 3, 5-triphenyl tetrazolium chloride (TTC) can be reduced to the red compound triphenylformazan (TPF). Briefly, 1 g of sieved soil was placed in test tubes (15×150 mm) and mixed fully with 1 mL of 3 % TTC and 1 mL of deionized water. After 24 h of incubation at 37 °C, 10 mL of ethanol was added to each test tube, and then the suspension was mixed for 60 s by vortex. The resulting suspension was filtered through filter paper, and the concentration of TPF was colorimetrically determined at 485 nm by a spectrophotometer (Shimadzu UV-2550, Japan) using a calibration curve. The result was expressed as microgram of TPF per gram of dry soil every 24 h. The urease (EC 3.5.1.5) activity was measured according to the procedure of May and Douglas (1976). The result was estimated by determination of the formation of ammonium released during soil sample incubation with urea at 37 °C for 3 h by a spectrophotometer with wavelength adjusted to 578 nm, expressed as microgram of NH4+-N per gram of dry soil every 3 h.
Determination of microbial population The total number of culturable heterotrophic bacteria, actinomycetes, and fungi were obtained by the plate counting technique. Soil (10 g fresh weight) from each replicate of every treatment was put into 250 mL Erlenmeyer flasks containing 90 mL of sterile water, mechanically shaken for 20 min at 250 rpm, and stationed for 5 min. Each soil suspension was diluted serially in test tubes containing 9 mL of sterile water. Beef-extract–peptone-agar, Gauze's medium No. 1, and Martin agar medium (Xu and Zheng 1986) were used for the enumeration of fast-growing heterotrophic bacteria, actinomycetes, and fungi, respectively. The plates were inoculated with 100 μL of soil suspension and cultured in an incubator at 30 °C for 36 h, 5 d, and 2 d for the beef-extract–peptone-agar, Gauze's medium No. 1, and Martin agar medium, respectively. Data from triplicates were expressed as colony-forming units (CFU) per gram of dry soil.
Environ Monit Assess
DNA extraction and PCR-DGGE analysis Soil community DNA was extracted from 0.5 g of fresh soil using the fast soil DNA Kit (Omega), as described by the manufacturer. The yield and quality of extracted DNA were verified by 1.0 % agarose gel electrophoresis. A nested PCR method was applied to 16S rDNA fragment amplification of bacteria. The universal bacterial primers 27 F and 1492 R were used in the first PCR round, and 338 F-GC and 518 R were used in the second PCR round for amplification of the variable V3 region of 16S rDNA (Muyzer et al. 1993). PCR amplification was performed on a Bio-Rad iCycler Thermal Cycler using 50 μL reaction volumes. The reaction mixture contained 1 μL of template DNA (10 ng), 0.5 μmol of each primer, 0.4 mmol of each deoxyribonucleoside triphosphate (dNTP), 5 μL of 10× PCR buffer (free of Mg2+), 5 μL of MgCl2 (25 mmol/L), 5 U of Taq polymerase, and sterile filtered milli-Q water to a final volume of 50 μL. Amplification was performed using an initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, and a final extension of 10 min at 72 °C. Amplified products were examined with 1 % agarose gel. DGGE was performed with a Dcode system (Bio-Rad, USA). The PCR product for each sample (35 μL) was loaded into a 1-mm thick denaturing gradient polyacrylamide linear porosity gradient gel (8 % w/v; acrylamide/bisacrylamide, 37.5:1) with a denaturing gradient of 30–60 % urea and formamide (100 % denaturant contains 7 mol/L urea and 40 % formamide). The gel was run at 150 V for 5 h at 60 °C in 1× TAE running buffer. After electrophoresis, the gel was stained in 1× TAE containing 0.5 μg/mL SYBR Green I for 30 min, visualized under UV and photographed with Gel Doc XR (Bio-Rad, USA). The prominent and special DGGE bands were excised and eluted in 60 μL of sterile water overnight at 4 °C. The eluent (1 μL) was used for reamplification with primers 338-17 and 518 R. The DNA sequence of reamplification was analyzed by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd, China. Closest relatives were searched using BLAST algorithms of the National Center for Biotechnology (NCBI). Sequence alignment was performed by using Clustal W, and a phylogenetic tree (Neighbor-Joining) was constructed using MEGA 5.0. Sequences obtained in this study were deposited in
GenBank and are available under accession numbers JX853563 through JX853568. Data analysis n DGGE profiles, cluster analyses and dendrograms were calculated using an unweighted pair–group method with arithmetic averages (UPGMA) by Quantity One software (Bio-Rad, USA). For the enzymatic activity and microbial population, all data were analyzed by the SPSS 16.0 computer package. All of the values were presented as mean± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Duncan's Multiple Range test was carried out to test the differences among the mean values of different treatment at each exposure time. Significant difference between treatments is indicated as p
Effects of fomesafen on soil enzyme activity, microbial population, and bacterial community composition Qingming Zhang & Lusheng Zhu & Jun Wang & Hui Xie & Jinhua Wang & Fenghua Wang & Fengxia Sun
Received: 16 September 2013 / Accepted: 3 December 2013 # Springer Science+Business Media Dordrecht 2013
Abstract Fomesafen is a diphenyl ether herbicide that has an important role in the removal of broadleaf weeds in bean and fruit tree fields. However, very little information is known about the effects of this herbicide on soil microbial community structure and activities. In the present study, laboratory experiments were conducted to examine the effects of different concentrations of fomesafen (0, 10, 100, and 500 μg/kg) on microbial community structure and activities during an exposure period of 60 days, using soil enzyme assays, plate counting, and denaturing gradient gel electrophoresis (DGGE). The results of enzymatic activity experiments showed that fomesafen had different stimulating effects on the activities of acid phosphatase, alkaline
Q. Zhang College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, China Q. Zhang : L. Zhu : J. Wang : H. Xie : J. Wang : F. Wang : F. Sun Key Laboratory of Agricultural Environment in the University of Shandong, National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, College of Resources and Environment, Shandong Agricultural University, Taian 271018, China L. Zhu (*) College of Resources and Environment, Shandong Agricultural University, 61 Daizong Road, Taian 271018, People’s Republic of China e-mail: [email protected] L. Zhu e-mail: [email protected]
phosphatase, and dehydrogenase, with dehydrogenase being most sensitive to fomesafen. On the tenth day, urease activity was inhibited significantly after treatment of different concentrations of fomesafen; this inhibiting effect then gradually disappeared and returned to the control level after 30 days. Plate counting experiments indicated that the number of bacteria and actinomycetes increased in fomesafen-spiked soil relative to the control after 30 days of incubation, while fungal number decreased significantly after only 10 days. The DGGE results revealed that the bacterial community varied in response to the addition of fomesafen, and the intensity of these six bands was greater on day 10. Sequencing and phylogenetic analyses indicated that the six excised DGGE bands were closely related to Emticicia, Bacillus, and uncultured bacteria. After 10 days, the bacterial community exhibited no obvious change compared with the control. Throughout the experiment, we concluded that 0–500 μg/kg of fomesafen could not produce significant toxic effects on soil microbial community structure and activities. Keywords Fomesafen . Soil enzyme . PCR-DGGE . Bacterial community structure
Introduction In modern agricultural production, pesticide application is a regular practice. However, the increased application of pesticide may cause soil contamination. In particular,
Environ Monit Assess
high toxicity and long residual herbicide could have hazardous impacts on the health of soil ecosystems and an adverse influence on soil biological processes by altering soil enzymatic activities, soil microbial population, and soil microbial community diversity (Ratcliff et al. 2006; Niemi et al. 2009; Sofo et al. 2012). Fomesafen (5-[2-chloro-4-(trifluoromethyl)phenoxy]N-(methylsulfonyl)2-nitrobenzamide) is a diphenyl ether herbicide that is usually applied as a pre- or postemergent foliar to remove broadleaf weeds in soybean plants, fruit trees, and rubber estate fields (Santos et al. 2006; Sikkema et al. 2009). In China, fomesafen has been used extensively with the succession and multiplication of weeds on soybean farmland in recent years; subsequently, the number of fomesafen production companies has increased annually (Liu 2010). However, due to the prolonged use of fomesafen over the years, this herbicide has lead to some adverse effects, such as injury to sensitive aftercrops and hazards to agricultural adjustment and security (Wang et al. 2003; Rauch et al. 2007). These adverse effects may be related to increased fomesafen residue concentration in the soil, and studies have shown that this pesticide has a relatively long halflife (approximately 37.5–56 d) in field soil (Mills and Simmons 1998; Cobucci et al. 1998). The soil environment can certainly be affected by fomesafen residue; unfortunately, information on such effects has been limited to date. Soil is a complex and dynamic living ecosystem, and certain factors influence its quality and health. In the assessment of soil environment quality, the living and dynamic nature of soil, indicated by such factors as soil enzymes and microorganisms, is usually used as a biomarker (Karlen et al. 1997; Sukul 2006). Soil enzymes are involved in catalyzing various reactions and metabolic processes occurring in organic matter metabolism, maintaining soil structure, cycling nutrients, detoxifying pollutants, and producing energy for both microorganisms and plants (Kızılkaya et al. 2004; Khan et al. 2010). Changes in soil enzymatic activities usually imply that natural and anthropogenic disturbances occurred in the soil ecosystem (Gianfreda and Rao 2008; Zhu et al. 2010). Similarly, soil microorganisms also have a crucial role in the cycling of nitrogen, sulfur, and phosphorus and the decomposition of organic residues (Nielsen and Winding 2002). Previous studies showed that microorganisms are very sensitive to changes in their surroundings and appear to be very suitable early warning indicators and predictors in soil health monitoring
(Nielsen and Winding 2002; Epelde et al. 2010). To date, some studies have used soil enzymatic activities and microbial community structure as indicators to determine toxicological influences of various pollutants on soil quality and health. With the rapid development of molecular biology methods, some techniques based on cultureindependent microbiology have been useful in research of soil microbial communities. Denaturing gradient gel electrophoresis (DGGE) is a new molecular biology method that has been widely applied in investigation of the effects of many pollutants on soil or sediment microbial communities, including pesticides (Sigler and Turco 2002; Ferreira et al. 2009), petroleum (Evans et al. 2004), polycyclic aromatic hydrocarbons (PAHs) (Andreoni et al. 2004; Tian et al. 2008), and heavy metals (Khan et al. 2010). Though DGGE is a highthroughput technique compared with other methods, the results of such analyses may be biased due to influencing factors such as the DNA extraction method, polymerase chain reaction (PCR) amplification, and DGGE procedure. Thus, the combination of both culture-independent and culture-dependent techniques might provide more useful and complementary information on the structural diversity of microbial communities (Aislabie et al. 2006; Li et al. 2008). In this study, laboratory simulation tests over a period of 60 days were conducted to evaluate the effects of herbicide fomesafen on soil microbial community structure and activities, using soil enzyme activity determination, plate counting, and 16S rDNA-PCR-DGGE analysis.
Materials and methods Chemicals and soils Fomesafen (98.2 % purity) was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Soil samples were collected from an experimental field (36.11′ N and 117.08 ′ E, 611 m above sea level) located in Shandong Agricultural University, Taian, China. In this region, the mean annual precipitation is 697 mm, and the mean annual temperature is 13 °C. This field remained under conventional farming practices and no history of fomesafen application in the last 5 years. Soil samples were taken from 0–20 cm of the bulk soil on the field prepared for the cultivation of corn after ploughing
Environ Monit Assess
in spring. The soil was classified as a typical Kandiudult (clay and loamy soil) in the USDA Soil Taxonomy System (Soil Survey Staff 1999) and some of its physical and chemical characteristics as follows: pH 7.6, organic matter 17.6 g/kg, available N 132.3 mg/kg, available P 18.4 mg/kg, available K 125.7 mg/kg, and maximal water holding capacity 18.5 %. To consider the spatial heterogeneity, soil samples were taken in triplicates, and each replicate consisted of five10-cm auger cores. Then, the soil samples were manually and gently crumbled, mixed thoroughly, air dried at room temperature, and sieved through a 2-mm mesh to remove stones and plant roots before being used. Soil treatment Concentrations were established according to the actual residual content levels of fomesafen in field soil; the fomesafen concentrations in the soil approximately range from 50 to 420 μg/kg when application rates were 180–375 g ai/ha (Rauch et al. 2007; Guo et al. 2000). Thus, the experiments were conducted with the following four treatments: 0 (solvent control), 10, 100, and 500 μg fomesafen/kg dry soil, labeled ck, F10, F100, and F500, respectively. Each treatment was replicated three times at each sampling stage. For each treatment, 1,200 g soil was thoroughly mixed with fomesafen stock acetone solution to achieve the setting concentration, and 80 g soil (three replicates in each treatment, five sampling intervals) was placed in a brown glass bottle (120 mL) as a microcosm system and sealed with a cotton plug to minimize water loss. All treatments were incubated in the dark at 25 °C for 60 days, and the soil moisture content levels were tested by weighing and adjusted to 60 % water holding capacity by adding deionized water every 2 days. Samples from each bottle were collected after 10, 20, 30, 40, and 60 d for enzyme assays, DNA extraction, and numeration of the microbial community. Enzymatic activity assay The acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) activities were determined as described by Tabatabai (1994). A 0.1 M p-nitrophenol phosphatase solution (pH 6.5 for acid phosphatase and pH 11 for alkaline phosphatase) was used as the substrate, and yellow-band absorbance of the filtrate due to p-nitrophenol was measured at 410 nm. The
p-nitrophenol concentration was calculated by reference to a calibration curve constructed using p-nitrophenol standards, and the results were expressed as micrograms of p-nitrophenol per gram of dry soil (oven-dried soil basis) per hour. The dehydrogenase (EC 1.1.1.1) activity was determined using the method described by Tabatabai (1994). The soluble 2, 3, 5-triphenyl tetrazolium chloride (TTC) can be reduced to the red compound triphenylformazan (TPF). Briefly, 1 g of sieved soil was placed in test tubes (15×150 mm) and mixed fully with 1 mL of 3 % TTC and 1 mL of deionized water. After 24 h of incubation at 37 °C, 10 mL of ethanol was added to each test tube, and then the suspension was mixed for 60 s by vortex. The resulting suspension was filtered through filter paper, and the concentration of TPF was colorimetrically determined at 485 nm by a spectrophotometer (Shimadzu UV-2550, Japan) using a calibration curve. The result was expressed as microgram of TPF per gram of dry soil every 24 h. The urease (EC 3.5.1.5) activity was measured according to the procedure of May and Douglas (1976). The result was estimated by determination of the formation of ammonium released during soil sample incubation with urea at 37 °C for 3 h by a spectrophotometer with wavelength adjusted to 578 nm, expressed as microgram of NH4+-N per gram of dry soil every 3 h.
Determination of microbial population The total number of culturable heterotrophic bacteria, actinomycetes, and fungi were obtained by the plate counting technique. Soil (10 g fresh weight) from each replicate of every treatment was put into 250 mL Erlenmeyer flasks containing 90 mL of sterile water, mechanically shaken for 20 min at 250 rpm, and stationed for 5 min. Each soil suspension was diluted serially in test tubes containing 9 mL of sterile water. Beef-extract–peptone-agar, Gauze's medium No. 1, and Martin agar medium (Xu and Zheng 1986) were used for the enumeration of fast-growing heterotrophic bacteria, actinomycetes, and fungi, respectively. The plates were inoculated with 100 μL of soil suspension and cultured in an incubator at 30 °C for 36 h, 5 d, and 2 d for the beef-extract–peptone-agar, Gauze's medium No. 1, and Martin agar medium, respectively. Data from triplicates were expressed as colony-forming units (CFU) per gram of dry soil.
Environ Monit Assess
DNA extraction and PCR-DGGE analysis Soil community DNA was extracted from 0.5 g of fresh soil using the fast soil DNA Kit (Omega), as described by the manufacturer. The yield and quality of extracted DNA were verified by 1.0 % agarose gel electrophoresis. A nested PCR method was applied to 16S rDNA fragment amplification of bacteria. The universal bacterial primers 27 F and 1492 R were used in the first PCR round, and 338 F-GC and 518 R were used in the second PCR round for amplification of the variable V3 region of 16S rDNA (Muyzer et al. 1993). PCR amplification was performed on a Bio-Rad iCycler Thermal Cycler using 50 μL reaction volumes. The reaction mixture contained 1 μL of template DNA (10 ng), 0.5 μmol of each primer, 0.4 mmol of each deoxyribonucleoside triphosphate (dNTP), 5 μL of 10× PCR buffer (free of Mg2+), 5 μL of MgCl2 (25 mmol/L), 5 U of Taq polymerase, and sterile filtered milli-Q water to a final volume of 50 μL. Amplification was performed using an initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, and a final extension of 10 min at 72 °C. Amplified products were examined with 1 % agarose gel. DGGE was performed with a Dcode system (Bio-Rad, USA). The PCR product for each sample (35 μL) was loaded into a 1-mm thick denaturing gradient polyacrylamide linear porosity gradient gel (8 % w/v; acrylamide/bisacrylamide, 37.5:1) with a denaturing gradient of 30–60 % urea and formamide (100 % denaturant contains 7 mol/L urea and 40 % formamide). The gel was run at 150 V for 5 h at 60 °C in 1× TAE running buffer. After electrophoresis, the gel was stained in 1× TAE containing 0.5 μg/mL SYBR Green I for 30 min, visualized under UV and photographed with Gel Doc XR (Bio-Rad, USA). The prominent and special DGGE bands were excised and eluted in 60 μL of sterile water overnight at 4 °C. The eluent (1 μL) was used for reamplification with primers 338-17 and 518 R. The DNA sequence of reamplification was analyzed by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd, China. Closest relatives were searched using BLAST algorithms of the National Center for Biotechnology (NCBI). Sequence alignment was performed by using Clustal W, and a phylogenetic tree (Neighbor-Joining) was constructed using MEGA 5.0. Sequences obtained in this study were deposited in
GenBank and are available under accession numbers JX853563 through JX853568. Data analysis n DGGE profiles, cluster analyses and dendrograms were calculated using an unweighted pair–group method with arithmetic averages (UPGMA) by Quantity One software (Bio-Rad, USA). For the enzymatic activity and microbial population, all data were analyzed by the SPSS 16.0 computer package. All of the values were presented as mean± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Duncan's Multiple Range test was carried out to test the differences among the mean values of different treatment at each exposure time. Significant difference between treatments is indicated as p
