- Email: [email protected]
Effect of fungicide iprodione on soil bacterial community
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 59 (2004) 127–132
Effect of fungicide iprodione on soil bacterial community Yei-Shung Wang, Chih-Yuan Wen, Tzu-Chuan Chiu, and Jui-Hung Yen* Department of Agricultural Chemistry, National Taiwan University, Roosevelt Road 1, Sec. 4, Taipei 10617, Taiwan Received 3 March 2003; received in revised form 8 October 2003; accepted 28 January 2004
Abstract The effect of the fungicide iprodione on soil bacterial communities was studied by treating two kinds of soils with different concentrations of iprodione. Degradation rates of iprodione in sterile and unsterile soils were also investigated. Residues of iprodione were measured by using high-performance liquid chromatography (HPLC) and a change of bacterial communities was performed with denaturing gradient gel electrophoresis (DGGE) by counting the 16S rDNA band on DGGE patterns. The degradation rate of iprodione was slower in sterile soil than in unsterile soil in both Da-Hu sandy loam and Kuan-Shi loam. After treatment with fungicide, soil bacterial communities were changed and recovered rapidly to the original status when incubated at a lower temperature (15 C) and a lower iprodione concentration (5 mg/g). At the same temperature but with more iprodione (50 mg/g) added, the soil bacterial community increases slowly and regains the original status slowly. However, when incubated at the higher temperature (30 C), the soil bacterial community is more complex than that at the lower temperature. The response of the soil bacterial community to the iprodione is faster at the higher than at the lower temperature. At 30 C and with 50 mg/g iprodione, the amounts of soil bacterial communities increased quickly but cannot be reduced to the original status after incubation for 23 days. r 2004 Elsevier Inc. All rights reserved. Keywords: Fungicide iprodione; Soil bacterial community; 16S rDNA,DGGE
1. Introduction The diversity of the microbial community in soil is an important issue in modern soil microbiology. The diversity of microorganisms in our environment is the main indicator of risk assessment in the use of pesticides in agriculture, but traditional enrichment techniques always underestimate the diversity of soil microorganisms so that we cannot correctly understand the real impact of pesticides on microorganisms. Generally, the effect of a pesticide on soil microbial communities was studied by traditional methods based on cultivation (Wardle and Parkinson, 1990). But it is well known that more than 90% of the microorganisms existing in soil are unculturable on artificial media (Amann et al., 1995; Ranjard et al., 2000). Many molecular biological techniques were introduced to solve such problem (Schloter et al., 2000). A new method, PCR amplification of 16S rDNA followed by denaturing gradient gel electrophoresis (PCR-DGGE), was recently proposed for studying complex microbial populations (Gelsomino *Corresponding author. Fax: +886-2-23620155. E-mail address: [email protected] (J.-H. Yen). 0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.01.008
et al., 1999; Kozdroj and Elsas, 2001). The 16S rDNA are present in all known bacteria and their sequence difference can reflect the phylogenetic distance. It is suitable for diversity analysis at a community level. And the DGGE is based on the electrophoresis of PCRamplified 16S rDNA fragments in polyacrylamide gels containing a linearly increasing gradient of denaturants. From the DGGE gels, DNA fragments of the same length but with different base pairs (G+C or A+T) can be separated (Muyzer et al., 1993). Fungicide iprodione, 3-(3,5-dichlorophenyl)-N-isopropyl-2,4-dioxoimidazoli dine-1-carboximide, has been introduced to control a variety of crop diseases. It is recommended for controlling fungal diseases in strawberry, tobacco, and pear in Taiwan. There are many reports that iprodione may inhibit the organisms in the environment (Wauchope et al., 1992). Based on the findings, we thought it would be interesting to know the iprodione degradation in soil, as well as its impact on the diversity of soil microbial communities. In these studies, the effect of the fungicide iprodione on the soil bacterial communities under different concentrations was described. In order to comprehend the relationship between pesticide usage and bacterial
ARTICLE IN PRESS Y.-S. Wang et al. / Ecotoxicology and Environmental Safety 59 (2004) 127–132
128
community changes in soil at the molecular level of biological techniques, not only the degradation rates of pesticides at different temperatures and concentrations were investigated, but also the PCR-amplified 16S ribosomal DNA (16S rDNA) fragments were analyzed by using the denaturing gradient gel electrophoresis.
with 1 mL acetonitrile at regular intervals. Solvent extracts were passed through 0.2-mm filters and analyzed by using HPLC (Hitachi) with a C-18 reverse-phase column (LiChroCART 250-4 HPLC-cartridge LiChrospher 100 RP-18 endcapped, 5 mm). The mobile phase consisted of acetonitrile and water (75:25; v/v), with a flow rate of 1.0 mL/min, and iprodione was detected with a UV–Vis detector at 216 nm.
2. Materials and methods 2.4. DNA extraction and purification 2.1. Materials Soil samples were collected from a strawberry orchard located at Da-Hu and Kuan-Shi of Mia Lei County which is the most important strawberry production area in northern Taiwan. Soil was sampled at a depth of 0–10 cm on July 2001 and stored at 0 C during the experimental period. The properties of the soils are listed in Table 1. Fungicide iprodione, 3-(3,5-dichlorophenyl)-N-isopropyl-2,4-dioxoimi dazolidine-1-carboximide, with a purity of 99.0% was purchased from Dr. Ehrenstorfer (Augsburg, Germany). 2.2. Methods Cultural medium consisted of (in g/L) NH4Cl (0.535), Na2HPO4 (4.26), KH2PO4 (2.74), Na2SO4 (0.1136), MgSO4 7H2O (0.049), CaCl2 (6.23 103), FeSO4 7H2O (3.8 103), ZnCl2 (1.36 105), CuCl2 (2.7 105), NaBr (1.03105), Na2MoO4 2H2O (6.95 106), MnCl2 4H2O (1.97 105), KI (1.67 105), H3BO3 (1.24 105), CoCl2 6H2O (2.77 105), and NiCl2 6H2O (2.38 105). Soil culture was prepared by adding 300 g soil with 500 mL cultural medium in a 1-L serum bottle. A batch experiment was carried out by shaking with 120 rpm in a 125-mL serum bottle containing 10 mL of soil culture and 40 mL of culture medium. Sterile control was performed by autoclaving the soil culture at 121 C for 30 min. Fungicide iprodione was added to every bottle in 5 or 50 mg/g soil. Two different incubation temperatures were set at 15 C and 30 C. 2.3. Iprodione analysis In order to determine the remaining fungicide iprodione, 1 mL of soil culture was taken and extracted
Genomic DNA was extracted using the method described by Liu et al. (1997). In brief, 1 mL of batch culture was centrifuged in 6000 rpm and the supernatant was removed. The cells and soil pellets were resuspended in 1 mL of lysis buffer containing lysozyme (0.1 mg/mL) and achloropeptidase (0.1 mg/mL) and incubated in 37 C for 30 min to lyse cells. Proteinase K (0.2 mg/mL) and 20% SDS 75 mL were added to the samples and then incubated at 37 C in a water bath for 2 h. After being extracted by using chloroform and isoamylalcohol (24:1), DNA was precipitated with isopropanol and ammonium acetate. Extracted DNA was dissolved in 50 mL sterile deionized water, and further removal of humic acids was completed by electrophoresis in a 1% agarose gel. The DNA band was excised from the gel and recovered with a QIAquick gel extraction kit. 2.5. PCR-DGGE PCR was performed with ABgene DNA polymerase and a buffer kit obtained from ABsystem. The PCR mixture (50 mL) used contained 0.2 mM of each dNTP, 2.0 mM MgCl2, 1X buffer solution, 3 units of DNA polymerase, 0.4 mM of each primer, and 2 mL of template DNA. Primers 968F and 1401R were used for the amplification of bacterial 16S rDNA (Duarte et al., 1998). A GC-clamp was attached to the 50 -end of a 968F (Muyzer et al., 1993). GeneAMP PCR system 9700 (Applied Biosystems, USA) was used in PCR amplification. The PCR condition was 35 cycles of 92 C for 1 min, 55 C for 1 min, and 72 C for 1 min, followed by a final extension at 72 C for 10 min. The PCR products were confirmed by using 1% (w/v) agarose gel electrophoresis and ethidium bromide staining. DGGE analysis was performed by using a D-Code universal mutation detection system (Bio-Rad, USA). Samples
Table 1 Selected properties of soils Soil
Da-Hu Kuan-Shi
Texture
Sandy loam Loam
Sand
Loam
Clay
(%)
(%)
(%)
67.3 48.6
18.8 29.8
14.0 21.6
pH
Organic carbon fraction (%)
6.45 6.58
0.9 0.9
ARTICLE IN PRESS Y.-S. Wang et al. / Ecotoxicology and Environmental Safety 59 (2004) 127–132
of 20 mL of PCR products were loaded onto 8% (w/v) polyacrylamide gel that contained 30–70% denaturing gradient of formamide and urea. The electrophoresis was run at 60 C in 1X TAE for 8 h at a constant voltage of 120 V. After the electrophoresis, the polyacrylamide gel was stained with SYBR Green I nucleic acid gel stain and visualized on a UV transilluminator. The gel was photographed with a CCD camera.
2.6. Statistical comparison of DGGE pattern The DGGE profiles were analyzed by UPGMA (unweighted pair-group method using arithmetic averages) and the similarity was calculated by coefficient of DICE, by using the Phoretix 1D Professional software (Nonlinear Dynamics, UK). The UPGMA method was used to reveal the change of bacterial community among incubation days.
129
3. Results 3.1. Degradation of iprodione in soils The degradation of the fungicide iprodione in Da-Hu sandy loam and Kuan-Shi loam soils is shown in Figs. 1 and 2. From these results, iprodione has a slower degradation rate in sterile than in nonsterile soil culture. This means that biological degradation of iprodione is of more importance than chemical degradation in both soils at 15 C. Joan and Maria (1998) found that the iprodione degradation rate is influenced by the number of application times. The degradation rate is faster on the second application and even more after the third. Moreover, when incubated at 30 C, the degradation rate of iprodione is faster than that at 15 C, in either sterile or nonsterile soil culture. It seems that iprodione is unstable at higher temperatures, causing a fast degradation even under sterile conditions. The degradation of iprodione at 30 C and nonsterile soil conditions was combined with biological and chemical effects. 80
70
70
60 Residues (ug/g)
Residues (ug/g)
60
50 40 30
50 40 30
20
20
10
10
0
0
0
20
40
60
80
Time (days)
Fig. 1. Degradation of iprodione (50 mg/g) in Da-Hu sandy loam under sterile (J) and nonsterile conditions (K) at 15 C and sterile (&) and nonsterile conditions (’) at 30 C.
0
20
40
60
80
Time (days)
Fig. 2. Degradation of iprodione (50 mg/g) in Kuan-Shi loam under sterile (J) and nonsterile conditions (K) at 15 C and sterile (&) and nonsterile conditions (’) at 30 C.
Fig. 3. DGGE patterns of 16S rDNA fragments from Da-Hu sandy loam incubation with iprodione for 0, 1,3,5,7,16, and 23 days at 15 C. (A) No iprodione added; (B) 5 mg/g iprodione added; (C) 50 mg/g iprodione added.
ARTICLE IN PRESS Y.-S. Wang et al. / Ecotoxicology and Environmental Safety 59 (2004) 127–132
130
3.2. Soil bacterial community affected by iprodione and temperature In parallel with the residual iprodione analysis, DGGE analysis of 16S rDNA fragments was used to investigate the effect of the iprodione on the soil bacterial community. Fig. 3 shows the DGGE patterns of the 16S rDNA fragments which were extracted from the soil at 15 C incubation. Figs. 3A–C depict the soils spiked at 0,5 and 50 mg/g of iprodione, respectively. The preponderant bands on the gels can be considered the most abundant bacterial species in each treatment. Fig. 3A shows no change in the bacterial community at 23 days of incubation. However, the number of bands increased at the early stage (ca. 3–7 days) as observed from Fig. 3B. The number of bands decreased after 16
Coefficient: Dice
Algorithm: UPGMA Day 16
days and returned to the same bands as in the beginning. This result implies that the bacterial community will recover after 23 days incubation of adding 5 mg/g iprodione. Fig. 4 demonstrates the similarity among the band patterns in Fig. 3B by using DICE coefficients. The result shows that the difference in the microbial community will increase after 5 mg/g iprodione added and then slowly decrease to approximately the original situation. The similarity of 0 and 5 days is 71%, but it returns to 76% after 23 days (data not shown). For understanding the effect of the higher dosage iprodione on the soil bacterial community, 50 mg/g iprodione was added to the soil and incubation was at 15 C (Fig. 3C) and 30 C (Fig. 5C). Fig. 3C shows that the band number increase has a little delay of up to 5–7 days. And then, till 23 days, there still is little difference in band numbers from that in the beginning. To confirm this phenomenon, another soil sample (Kuan-Shi loam, Table 1) underwent the same procedure. The same result could Coefficient: Dice
Algorithm: UPGMA
Day 23
Day 23
Day 0
Day 0
Day 1
Day 7 Day 1
Day 7
Day 5
Day 3
Day 3
Day 5
0.72 0.8
0.9
Fig. 4. Similarity of DGGE pattern of 16S rDNA fragments from DaHu sandy loam incubation with iprodione for different days at 15 C with 5 mg/g iprodione added.
Day 16
0.65 0.7
0.8
0.9
Fig. 6. Similarity of DGGE pattern of 16S rDNA fragments from DaHu sandy loam incubation with iprodione for different days at 30 C with 5 mg/g iprodione added.
Fig. 5. DGGE patterns of 16S rDNA fragments from Da-Hu sandy loam incubation with iprodione for 0, 1,3,5,7,16, and 23 days at 30 C. (A) No iprodione added; (B) 5 mg/g iprodione added; (C) 50 mg/g iprodione added.
ARTICLE IN PRESS Y.-S. Wang et al. / Ecotoxicology and Environmental Safety 59 (2004) 127–132
131
Fig. 7. DGGE patterns of 16S rDNA fragments from Kuan-Shi loam incubation with iprodione for 0, 1,3,5,7,16, and 23 days at 15 C. (A) No iprodione added; (B) 5 mg/g iprodione added; (C) 50 mg/g iprodione added.
Coefficient: Dice
Algorithm: UPGMA Day 0 Day 7 Day 23 Day 16 Day 1 Day 3 Day 5
0.78 0.8
0.9
Fig. 8. Similarity of DGGE pattern of 16S rDNA fragments from Kuan-Shi loam incubation with iprodione for different days at 15 C with 5 mg/g iprodione added.
be observed as shown in Figs. 7 and 9. A similar analysis of Figs. 7B and 9B is shown in Figs. 8 and 10, respectively (Figs. 5–10).
4. Discussion At a 15 C incubation temperature, microorganisms adapted quickly to the iprodione-inoculated soil, the bacterial community number increasing very fast. Since the iprodione dissipated gradually from the soil, the bacterial community decreased to its original situation. From this result we observed a transformation of the soil bacterial community during the soil self-purification procession, and the impact of iprodione on the soil bacterial community was impermanent and recoverable. Even a high dosage of iprodione will delay the bacterial community increase but it is recoverable after iprodione dissipation.
Figs. 5 and 9 show the same iprodione concentration in different soil incubations at 30 C. According to Figs. 3 and 5 (at 15 C) and Figs. 7 and 9 (at 30 C), the bacterial community is not always more complex at 30 C than at 15 C incubation. From Figs. 5B and 6, the bacterial community increases from the first day of iprodione treatment and kept the same band numbers for 7 days. As time elapses, the band numbers decrease and return to the original status (0 day) after 23 days. When more iprodione was introduced, Figs. 5C and 9C, the bacterial community number did not return to the status before treatment. The reason is attributed to the special induced bacteria at 30 C incubation, or more time is needed after iprodione treatment. Overall, the effect of iprodione on the soil bacterial community under lower incubation temperatures and lower chemical concentrations was that the soil bacterial community soon returned to its original status. Similarly, with lower temperatures but more iprodione added, the soil bacterial community diversity increased slowly and then slowly decreased to its original status, too. However, at higher incubation temperatures the diversity of the soil bacterial community is initially more complex than that at the lower temperature condition. The response of soil bacterial community to the iprodione at higher temperatures is faster than that at the lower temperature condition. At higher temperatures and higher iprodione concentrations, the soil bacterial community also increased very fast, but could not return to the original status during a 23-day incubation periods.
5. Conclusion Iprodione concentration and soil temperature impact the soil bacterial community in soil suspensions. High temperatures and high iprodione concentrations extend the effect on the diversity of soil bacterial community.
ARTICLE IN PRESS Y.-S. Wang et al. / Ecotoxicology and Environmental Safety 59 (2004) 127–132
132
Fig. 9. DGGE patterns of 16S rDNA fragments from Kuan-Shi loam incubation with iprodione for 0, 1,3,5,7,16, and 23 days at 30 C. (A) No iprodione added; (B) 5 mg/g iprodione added; (C) 50 mg/g iprodione added.
Coefficient: Dice
Algorithm: UPGMA
Day 0
Day 1 Day 3
Day 16
Day 7 Day 5 Day 23
0.71 0.8
0.9
Fig. 10. Similarity of DGGE pattern of 16S rDNA fragments from Kuan-Shi loam incubation with iprodione for different days at 30 C with 5 mg/g iprodione added.
Acknowledgments This investigation was supported by funds provided by the National Science Council, Republic of China, under Agreement No. NSC 91-2313-B-002-350.
References Amann, R.I., Ludwig, W., Schleifer, K.-H., 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143–169.
Duarte, G.F., Rosado, A.S., Seldin, L., Keijzer, A.C., 1998. Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacterial community. J. Microbiol. Methods 32, 21–29. Gelsomino, A., Wolters, A.C.K., Cacco, G., van Elsas, J.D., 1999. Assessment of bacterial community structure in soil by polymerase chain reaction and denaturing gradient gel electrophoresis. J. Microbiol. Methods 38, 1–15. Joan, G.C., Maria, X.V., 1998. Monitoring degradation of dicarbocimidic fungicide residues in soils. J. Agric. Food Chem. 46, 2845–2850 (DOI: 10.1021/jf971025t). Kozdroj, J., Elsas, D.V., 2001. Structure diversity of microbial communities in arable soils of a heavily industrialized area determined by PCR-DGGE fingerprinting and FAME profiling. Appl. Soil. Ecol. 17, 31–42. Liu, W.T., Marsh, T.L., Cheng, H., Forney, L.J., 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63, 4516–4522. Muyzer, G., de Waal, E.C., Uitterlinden, A., 1993. Profiling of complex microbial populations using denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59, 695–700. Ranjard, L., Poly, F., Nazaret, S., 2000. Monitoring complex bacterial communities using culture-independent molecular techniques: application to soil environment. Res. Microbiol. 151, 167–177. Schloter, M., Lebuhn, M., Heulin, T., Hartmann, A., 2000. Ecology and evolution of bacterial microdiversity. FEMS. Microbiol. Rev. 24, 647–660. Wardle, D.A., Parkinson, D., 1990. Effects of three herbicides on soil microbial biomass and activity. Plant Soil 122, 21–28. Wauchope, R.D., Buttler, T.M., Hornsby, A.G., Augustijn Beckers, P.W.M., Burt, J.P., 1992. Pesticide properties database for environmental decisionmaking. Rev. Environ. Contam. Toxicol. 123, 1–157.
Ecotoxicology and Environmental Safety 59 (2004) 127–132
Effect of fungicide iprodione on soil bacterial community Yei-Shung Wang, Chih-Yuan Wen, Tzu-Chuan Chiu, and Jui-Hung Yen* Department of Agricultural Chemistry, National Taiwan University, Roosevelt Road 1, Sec. 4, Taipei 10617, Taiwan Received 3 March 2003; received in revised form 8 October 2003; accepted 28 January 2004
Abstract The effect of the fungicide iprodione on soil bacterial communities was studied by treating two kinds of soils with different concentrations of iprodione. Degradation rates of iprodione in sterile and unsterile soils were also investigated. Residues of iprodione were measured by using high-performance liquid chromatography (HPLC) and a change of bacterial communities was performed with denaturing gradient gel electrophoresis (DGGE) by counting the 16S rDNA band on DGGE patterns. The degradation rate of iprodione was slower in sterile soil than in unsterile soil in both Da-Hu sandy loam and Kuan-Shi loam. After treatment with fungicide, soil bacterial communities were changed and recovered rapidly to the original status when incubated at a lower temperature (15 C) and a lower iprodione concentration (5 mg/g). At the same temperature but with more iprodione (50 mg/g) added, the soil bacterial community increases slowly and regains the original status slowly. However, when incubated at the higher temperature (30 C), the soil bacterial community is more complex than that at the lower temperature. The response of the soil bacterial community to the iprodione is faster at the higher than at the lower temperature. At 30 C and with 50 mg/g iprodione, the amounts of soil bacterial communities increased quickly but cannot be reduced to the original status after incubation for 23 days. r 2004 Elsevier Inc. All rights reserved. Keywords: Fungicide iprodione; Soil bacterial community; 16S rDNA,DGGE
1. Introduction The diversity of the microbial community in soil is an important issue in modern soil microbiology. The diversity of microorganisms in our environment is the main indicator of risk assessment in the use of pesticides in agriculture, but traditional enrichment techniques always underestimate the diversity of soil microorganisms so that we cannot correctly understand the real impact of pesticides on microorganisms. Generally, the effect of a pesticide on soil microbial communities was studied by traditional methods based on cultivation (Wardle and Parkinson, 1990). But it is well known that more than 90% of the microorganisms existing in soil are unculturable on artificial media (Amann et al., 1995; Ranjard et al., 2000). Many molecular biological techniques were introduced to solve such problem (Schloter et al., 2000). A new method, PCR amplification of 16S rDNA followed by denaturing gradient gel electrophoresis (PCR-DGGE), was recently proposed for studying complex microbial populations (Gelsomino *Corresponding author. Fax: +886-2-23620155. E-mail address: [email protected] (J.-H. Yen). 0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.01.008
et al., 1999; Kozdroj and Elsas, 2001). The 16S rDNA are present in all known bacteria and their sequence difference can reflect the phylogenetic distance. It is suitable for diversity analysis at a community level. And the DGGE is based on the electrophoresis of PCRamplified 16S rDNA fragments in polyacrylamide gels containing a linearly increasing gradient of denaturants. From the DGGE gels, DNA fragments of the same length but with different base pairs (G+C or A+T) can be separated (Muyzer et al., 1993). Fungicide iprodione, 3-(3,5-dichlorophenyl)-N-isopropyl-2,4-dioxoimidazoli dine-1-carboximide, has been introduced to control a variety of crop diseases. It is recommended for controlling fungal diseases in strawberry, tobacco, and pear in Taiwan. There are many reports that iprodione may inhibit the organisms in the environment (Wauchope et al., 1992). Based on the findings, we thought it would be interesting to know the iprodione degradation in soil, as well as its impact on the diversity of soil microbial communities. In these studies, the effect of the fungicide iprodione on the soil bacterial communities under different concentrations was described. In order to comprehend the relationship between pesticide usage and bacterial
ARTICLE IN PRESS Y.-S. Wang et al. / Ecotoxicology and Environmental Safety 59 (2004) 127–132
128
community changes in soil at the molecular level of biological techniques, not only the degradation rates of pesticides at different temperatures and concentrations were investigated, but also the PCR-amplified 16S ribosomal DNA (16S rDNA) fragments were analyzed by using the denaturing gradient gel electrophoresis.
with 1 mL acetonitrile at regular intervals. Solvent extracts were passed through 0.2-mm filters and analyzed by using HPLC (Hitachi) with a C-18 reverse-phase column (LiChroCART 250-4 HPLC-cartridge LiChrospher 100 RP-18 endcapped, 5 mm). The mobile phase consisted of acetonitrile and water (75:25; v/v), with a flow rate of 1.0 mL/min, and iprodione was detected with a UV–Vis detector at 216 nm.
2. Materials and methods 2.4. DNA extraction and purification 2.1. Materials Soil samples were collected from a strawberry orchard located at Da-Hu and Kuan-Shi of Mia Lei County which is the most important strawberry production area in northern Taiwan. Soil was sampled at a depth of 0–10 cm on July 2001 and stored at 0 C during the experimental period. The properties of the soils are listed in Table 1. Fungicide iprodione, 3-(3,5-dichlorophenyl)-N-isopropyl-2,4-dioxoimi dazolidine-1-carboximide, with a purity of 99.0% was purchased from Dr. Ehrenstorfer (Augsburg, Germany). 2.2. Methods Cultural medium consisted of (in g/L) NH4Cl (0.535), Na2HPO4 (4.26), KH2PO4 (2.74), Na2SO4 (0.1136), MgSO4 7H2O (0.049), CaCl2 (6.23 103), FeSO4 7H2O (3.8 103), ZnCl2 (1.36 105), CuCl2 (2.7 105), NaBr (1.03105), Na2MoO4 2H2O (6.95 106), MnCl2 4H2O (1.97 105), KI (1.67 105), H3BO3 (1.24 105), CoCl2 6H2O (2.77 105), and NiCl2 6H2O (2.38 105). Soil culture was prepared by adding 300 g soil with 500 mL cultural medium in a 1-L serum bottle. A batch experiment was carried out by shaking with 120 rpm in a 125-mL serum bottle containing 10 mL of soil culture and 40 mL of culture medium. Sterile control was performed by autoclaving the soil culture at 121 C for 30 min. Fungicide iprodione was added to every bottle in 5 or 50 mg/g soil. Two different incubation temperatures were set at 15 C and 30 C. 2.3. Iprodione analysis In order to determine the remaining fungicide iprodione, 1 mL of soil culture was taken and extracted
Genomic DNA was extracted using the method described by Liu et al. (1997). In brief, 1 mL of batch culture was centrifuged in 6000 rpm and the supernatant was removed. The cells and soil pellets were resuspended in 1 mL of lysis buffer containing lysozyme (0.1 mg/mL) and achloropeptidase (0.1 mg/mL) and incubated in 37 C for 30 min to lyse cells. Proteinase K (0.2 mg/mL) and 20% SDS 75 mL were added to the samples and then incubated at 37 C in a water bath for 2 h. After being extracted by using chloroform and isoamylalcohol (24:1), DNA was precipitated with isopropanol and ammonium acetate. Extracted DNA was dissolved in 50 mL sterile deionized water, and further removal of humic acids was completed by electrophoresis in a 1% agarose gel. The DNA band was excised from the gel and recovered with a QIAquick gel extraction kit. 2.5. PCR-DGGE PCR was performed with ABgene DNA polymerase and a buffer kit obtained from ABsystem. The PCR mixture (50 mL) used contained 0.2 mM of each dNTP, 2.0 mM MgCl2, 1X buffer solution, 3 units of DNA polymerase, 0.4 mM of each primer, and 2 mL of template DNA. Primers 968F and 1401R were used for the amplification of bacterial 16S rDNA (Duarte et al., 1998). A GC-clamp was attached to the 50 -end of a 968F (Muyzer et al., 1993). GeneAMP PCR system 9700 (Applied Biosystems, USA) was used in PCR amplification. The PCR condition was 35 cycles of 92 C for 1 min, 55 C for 1 min, and 72 C for 1 min, followed by a final extension at 72 C for 10 min. The PCR products were confirmed by using 1% (w/v) agarose gel electrophoresis and ethidium bromide staining. DGGE analysis was performed by using a D-Code universal mutation detection system (Bio-Rad, USA). Samples
Table 1 Selected properties of soils Soil
Da-Hu Kuan-Shi
Texture
Sandy loam Loam
Sand
Loam
Clay
(%)
(%)
(%)
67.3 48.6
18.8 29.8
14.0 21.6
pH
Organic carbon fraction (%)
6.45 6.58
0.9 0.9
ARTICLE IN PRESS Y.-S. Wang et al. / Ecotoxicology and Environmental Safety 59 (2004) 127–132
of 20 mL of PCR products were loaded onto 8% (w/v) polyacrylamide gel that contained 30–70% denaturing gradient of formamide and urea. The electrophoresis was run at 60 C in 1X TAE for 8 h at a constant voltage of 120 V. After the electrophoresis, the polyacrylamide gel was stained with SYBR Green I nucleic acid gel stain and visualized on a UV transilluminator. The gel was photographed with a CCD camera.
2.6. Statistical comparison of DGGE pattern The DGGE profiles were analyzed by UPGMA (unweighted pair-group method using arithmetic averages) and the similarity was calculated by coefficient of DICE, by using the Phoretix 1D Professional software (Nonlinear Dynamics, UK). The UPGMA method was used to reveal the change of bacterial community among incubation days.
129
3. Results 3.1. Degradation of iprodione in soils The degradation of the fungicide iprodione in Da-Hu sandy loam and Kuan-Shi loam soils is shown in Figs. 1 and 2. From these results, iprodione has a slower degradation rate in sterile than in nonsterile soil culture. This means that biological degradation of iprodione is of more importance than chemical degradation in both soils at 15 C. Joan and Maria (1998) found that the iprodione degradation rate is influenced by the number of application times. The degradation rate is faster on the second application and even more after the third. Moreover, when incubated at 30 C, the degradation rate of iprodione is faster than that at 15 C, in either sterile or nonsterile soil culture. It seems that iprodione is unstable at higher temperatures, causing a fast degradation even under sterile conditions. The degradation of iprodione at 30 C and nonsterile soil conditions was combined with biological and chemical effects. 80
70
70
60 Residues (ug/g)
Residues (ug/g)
60
50 40 30
50 40 30
20
20
10
10
0
0
0
20
40
60
80
Time (days)
Fig. 1. Degradation of iprodione (50 mg/g) in Da-Hu sandy loam under sterile (J) and nonsterile conditions (K) at 15 C and sterile (&) and nonsterile conditions (’) at 30 C.
0
20
40
60
80
Time (days)
Fig. 2. Degradation of iprodione (50 mg/g) in Kuan-Shi loam under sterile (J) and nonsterile conditions (K) at 15 C and sterile (&) and nonsterile conditions (’) at 30 C.
Fig. 3. DGGE patterns of 16S rDNA fragments from Da-Hu sandy loam incubation with iprodione for 0, 1,3,5,7,16, and 23 days at 15 C. (A) No iprodione added; (B) 5 mg/g iprodione added; (C) 50 mg/g iprodione added.
ARTICLE IN PRESS Y.-S. Wang et al. / Ecotoxicology and Environmental Safety 59 (2004) 127–132
130
3.2. Soil bacterial community affected by iprodione and temperature In parallel with the residual iprodione analysis, DGGE analysis of 16S rDNA fragments was used to investigate the effect of the iprodione on the soil bacterial community. Fig. 3 shows the DGGE patterns of the 16S rDNA fragments which were extracted from the soil at 15 C incubation. Figs. 3A–C depict the soils spiked at 0,5 and 50 mg/g of iprodione, respectively. The preponderant bands on the gels can be considered the most abundant bacterial species in each treatment. Fig. 3A shows no change in the bacterial community at 23 days of incubation. However, the number of bands increased at the early stage (ca. 3–7 days) as observed from Fig. 3B. The number of bands decreased after 16
Coefficient: Dice
Algorithm: UPGMA Day 16
days and returned to the same bands as in the beginning. This result implies that the bacterial community will recover after 23 days incubation of adding 5 mg/g iprodione. Fig. 4 demonstrates the similarity among the band patterns in Fig. 3B by using DICE coefficients. The result shows that the difference in the microbial community will increase after 5 mg/g iprodione added and then slowly decrease to approximately the original situation. The similarity of 0 and 5 days is 71%, but it returns to 76% after 23 days (data not shown). For understanding the effect of the higher dosage iprodione on the soil bacterial community, 50 mg/g iprodione was added to the soil and incubation was at 15 C (Fig. 3C) and 30 C (Fig. 5C). Fig. 3C shows that the band number increase has a little delay of up to 5–7 days. And then, till 23 days, there still is little difference in band numbers from that in the beginning. To confirm this phenomenon, another soil sample (Kuan-Shi loam, Table 1) underwent the same procedure. The same result could Coefficient: Dice
Algorithm: UPGMA
Day 23
Day 23
Day 0
Day 0
Day 1
Day 7 Day 1
Day 7
Day 5
Day 3
Day 3
Day 5
0.72 0.8
0.9
Fig. 4. Similarity of DGGE pattern of 16S rDNA fragments from DaHu sandy loam incubation with iprodione for different days at 15 C with 5 mg/g iprodione added.
Day 16
0.65 0.7
0.8
0.9
Fig. 6. Similarity of DGGE pattern of 16S rDNA fragments from DaHu sandy loam incubation with iprodione for different days at 30 C with 5 mg/g iprodione added.
Fig. 5. DGGE patterns of 16S rDNA fragments from Da-Hu sandy loam incubation with iprodione for 0, 1,3,5,7,16, and 23 days at 30 C. (A) No iprodione added; (B) 5 mg/g iprodione added; (C) 50 mg/g iprodione added.
ARTICLE IN PRESS Y.-S. Wang et al. / Ecotoxicology and Environmental Safety 59 (2004) 127–132
131
Fig. 7. DGGE patterns of 16S rDNA fragments from Kuan-Shi loam incubation with iprodione for 0, 1,3,5,7,16, and 23 days at 15 C. (A) No iprodione added; (B) 5 mg/g iprodione added; (C) 50 mg/g iprodione added.
Coefficient: Dice
Algorithm: UPGMA Day 0 Day 7 Day 23 Day 16 Day 1 Day 3 Day 5
0.78 0.8
0.9
Fig. 8. Similarity of DGGE pattern of 16S rDNA fragments from Kuan-Shi loam incubation with iprodione for different days at 15 C with 5 mg/g iprodione added.
be observed as shown in Figs. 7 and 9. A similar analysis of Figs. 7B and 9B is shown in Figs. 8 and 10, respectively (Figs. 5–10).
4. Discussion At a 15 C incubation temperature, microorganisms adapted quickly to the iprodione-inoculated soil, the bacterial community number increasing very fast. Since the iprodione dissipated gradually from the soil, the bacterial community decreased to its original situation. From this result we observed a transformation of the soil bacterial community during the soil self-purification procession, and the impact of iprodione on the soil bacterial community was impermanent and recoverable. Even a high dosage of iprodione will delay the bacterial community increase but it is recoverable after iprodione dissipation.
Figs. 5 and 9 show the same iprodione concentration in different soil incubations at 30 C. According to Figs. 3 and 5 (at 15 C) and Figs. 7 and 9 (at 30 C), the bacterial community is not always more complex at 30 C than at 15 C incubation. From Figs. 5B and 6, the bacterial community increases from the first day of iprodione treatment and kept the same band numbers for 7 days. As time elapses, the band numbers decrease and return to the original status (0 day) after 23 days. When more iprodione was introduced, Figs. 5C and 9C, the bacterial community number did not return to the status before treatment. The reason is attributed to the special induced bacteria at 30 C incubation, or more time is needed after iprodione treatment. Overall, the effect of iprodione on the soil bacterial community under lower incubation temperatures and lower chemical concentrations was that the soil bacterial community soon returned to its original status. Similarly, with lower temperatures but more iprodione added, the soil bacterial community diversity increased slowly and then slowly decreased to its original status, too. However, at higher incubation temperatures the diversity of the soil bacterial community is initially more complex than that at the lower temperature condition. The response of soil bacterial community to the iprodione at higher temperatures is faster than that at the lower temperature condition. At higher temperatures and higher iprodione concentrations, the soil bacterial community also increased very fast, but could not return to the original status during a 23-day incubation periods.
5. Conclusion Iprodione concentration and soil temperature impact the soil bacterial community in soil suspensions. High temperatures and high iprodione concentrations extend the effect on the diversity of soil bacterial community.
ARTICLE IN PRESS Y.-S. Wang et al. / Ecotoxicology and Environmental Safety 59 (2004) 127–132
132
Fig. 9. DGGE patterns of 16S rDNA fragments from Kuan-Shi loam incubation with iprodione for 0, 1,3,5,7,16, and 23 days at 30 C. (A) No iprodione added; (B) 5 mg/g iprodione added; (C) 50 mg/g iprodione added.
Coefficient: Dice
Algorithm: UPGMA
Day 0
Day 1 Day 3
Day 16
Day 7 Day 5 Day 23
0.71 0.8
0.9
Fig. 10. Similarity of DGGE pattern of 16S rDNA fragments from Kuan-Shi loam incubation with iprodione for different days at 30 C with 5 mg/g iprodione added.
Acknowledgments This investigation was supported by funds provided by the National Science Council, Republic of China, under Agreement No. NSC 91-2313-B-002-350.
References Amann, R.I., Ludwig, W., Schleifer, K.-H., 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143–169.
Duarte, G.F., Rosado, A.S., Seldin, L., Keijzer, A.C., 1998. Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacterial community. J. Microbiol. Methods 32, 21–29. Gelsomino, A., Wolters, A.C.K., Cacco, G., van Elsas, J.D., 1999. Assessment of bacterial community structure in soil by polymerase chain reaction and denaturing gradient gel electrophoresis. J. Microbiol. Methods 38, 1–15. Joan, G.C., Maria, X.V., 1998. Monitoring degradation of dicarbocimidic fungicide residues in soils. J. Agric. Food Chem. 46, 2845–2850 (DOI: 10.1021/jf971025t). Kozdroj, J., Elsas, D.V., 2001. Structure diversity of microbial communities in arable soils of a heavily industrialized area determined by PCR-DGGE fingerprinting and FAME profiling. Appl. Soil. Ecol. 17, 31–42. Liu, W.T., Marsh, T.L., Cheng, H., Forney, L.J., 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63, 4516–4522. Muyzer, G., de Waal, E.C., Uitterlinden, A., 1993. Profiling of complex microbial populations using denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59, 695–700. Ranjard, L., Poly, F., Nazaret, S., 2000. Monitoring complex bacterial communities using culture-independent molecular techniques: application to soil environment. Res. Microbiol. 151, 167–177. Schloter, M., Lebuhn, M., Heulin, T., Hartmann, A., 2000. Ecology and evolution of bacterial microdiversity. FEMS. Microbiol. Rev. 24, 647–660. Wardle, D.A., Parkinson, D., 1990. Effects of three herbicides on soil microbial biomass and activity. Plant Soil 122, 21–28. Wauchope, R.D., Buttler, T.M., Hornsby, A.G., Augustijn Beckers, P.W.M., Burt, J.P., 1992. Pesticide properties database for environmental decisionmaking. Rev. Environ. Contam. Toxicol. 123, 1–157.