assessing the effects of genetically modified cmv-r...

1

Assessing the effects of genetically modified CMV-resistant 1

tomato plant on soil microbial communities by PCR-DGGE 2

analysis 3

Chih-Hui Lin and Tzu-Ming Pan*

4

5

Author address: Institute of Microbiology and Biochemistry, College of Life 6

Science, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617 7

Taiwan. 8

9

Title running head: Effects of GM tomato on soil microbial communities 10

11

*Corresponding author: Institute of Microbiology and Biochemistry, College of 12

Life Science, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 13

10617 Taiwan. 14

Tel: +886-2-33664519 ext. 10. Fax: +886-2-33663838. E-mail: [email protected] 15

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00018-10 AEM Accepts, published online ahead of print on 26 March 2010

on June 29, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

http://aem.asm.org/

2

ABSTRACT 16

The effects of genetically modified Cucumber Mosaic Virus (CMV)-resistant 17

tomato on soil microbial communities were evaluated in this study. In comparison of 18

to the effect of tomato genotype, soil position and environmental factors played a 19

more dominant role in the variation of soil microbial communities. 20

21

KEYWORDS: genetically modified crop, soil microbial communities, denaturing 22

gradient gel electrophoresis (DGGE). 23

24


.asm.org/

Dow

nloaded from

http://aem.asm.org/

3

Microbial communities are integral parts of soil processes such as organic 25

matter decomposition and nutrient cycling (3). Interaction between plant 26

species/genotypes and rhizosphere microorganisms has been revealed in several 27

reports (9, 10, 15, 16, 19, 20). A genetically modified Cucumber Mosaic Virus 28

(CMV)-resistant tomato (Lycopersicon esculentum) was developed by the Asian 29

Vegetable Research and Development Center - The World Vegetable Center 30

(AVRDC, Tainan, Taiwan). The Taiwan Department of Health has conducted a safety 31

assessment (project code: DOH95-FS031) of this CMV-resistant tomato and 32

concluded it as safe (18). Although no significant impact on soil processes caused by 33

GM crop plants has been reported, case by case detailed study of accessible and 34

relevant indicators of soil ecosystem is the most feasible strategy until we more fully 35

understand soil ecosystems (5). Plant-microbe-soil nitrogen cycling is an essential 36

part of ecological functions and processes in ecosystems. The influence of plants on 37

nitrogen transformation comes from the interaction between plant roots and microbial 38

communities, as microbes are key players in soil nitrogen processes (4). To evaluate 39

the impact of GM tomato plant on soil microbial communities, general DGGE 40

profiles of bacteria (22), fungi (24), and actinomycetes (11), as well as three 41

functional bacteria communities involved in nitrogen cycling: free-living 42


.asm.org/

Dow

nloaded from

http://aem.asm.org/

4

nitrogen-fixing bacteria (6), ammonium-oxidizing bacteria (13, 17) and 43

nitrate-reducing bacteria (8, 14, 21, 25), were investigated in this study. 44

45

The transgenic CMV-resistant tomato (line R8) and its original plant line L4783 46

were provided and planted by the AVRDC. Each GMO test field (5 m × 5 m) was an 47

independent net house separated by bush, wire fence and fosse barriers with 9 m gap 48

between houses. Tomatoes were ridge-furrow cultivated with 120 cm gaps between 49

plants. Ridges were covered with silver-black plastic sheets for weed prevention and 50

control of soil temperature. Test fields were routinely managed through watering, pest 51

control and nipping. 52

53

Soil samples (sandy clay loam) were collected in April, 2007 from two test fields. 54

The tomato plants were approximately 100 days old at the time of sampling. Samples 55

were collected 10-15 cm below the soil surface of furrows and ridges; the ridge 56

sample was obtained from directly below the tomato plant and contained some root 57

material. Each test field was sampled at five positions, each of which included one 58

furrow and one ridge sample. A total of 20 samples were analyzed. The total nitrogen 59

content of soil was determined by the Kjeldahl method (2). The total organic carbon 60


.asm.org/

Dow

nloaded from

http://aem.asm.org/

5

(TOC) was determined by the Walkey-Black method (27). The moisture content of 61

soil samples was determined by weight loss. Agar plate enumeration of total microbes, 62

fungi and actinomycetes was carried out using nutrient agar (BD Biosciences, 63

Franklin Lakes, NJ, USA), rose bengal agar (BD Biosciences) and glycerol-yeast 64

extract agar, respectively. Univariate analysis was performed using SPSS software 65

(SPSS ver. 12.0, SPSS Inc.) on collected data. Duncan’s multiple range test or 66

Dunnett’s T3 test was used in Post Hoc tests according to the homogeneity of 67

variances (29). 68

69

Soil DNA was extracted from 1 g samples using an UltraCleanTM

soil DNA kit 70

(MO BIO Laboratories Inc., Carlsbad, CA, USA). DGGE was performed using the 71

DcodeTM

system (Bio-Rad Laboratories, Hercules, CA, USA). DGGE images were 72

processed and converted into an unweighted binary pattern (ImageJ software (1) 73

absent=0, present=1). Similarity metrics of microbial community profiles were 74

generated using SPSS software with the Dice's measure. Principal component analysis 75

(PCA) of the similarity metrics was performed using GenStat Discovery Edition 3 76

software (VSN International Ltd., Hemel Hempstead, UK). Cluster analysis of 77

microbial community profiles was performed using Phylip 3.68 with the UPGMA 78


.asm.org/

Dow

nloaded from

http://aem.asm.org/

6

method (7). Box-whisker plots were generated using SigmaPlot 8.0 (SPSS Inc., 79

Chicago, IL, USA). 80

81

The total microbes, fungus and actinomycetes in soil samples were approximately 82

106-10

7, 10

5-10

6 and 10

4 CFU/dried soil (g), respectively. Although the average 83

number of soil microbes measured from the GM tomato test field was higher, there 84

was no significant difference in the number of soil microbes between the GM and 85

wild-type (WT) tomato test fields. In addition, no significant difference in soil 86

properties between the GM and corresponding WT tomato test field was observed 87

(data not shown). DGGE profiles of soil samples have shown diverse patterns with 88

few common bands regardless of tomato plant type (data not shown). PCA and cluster 89

analysis of DGGE patterns have revealed that the effect of soil position was stronger 90

than the effect of GM tomato plant on the soil microbial communities (Fig. 1). Minor 91

correlations between the tomato plant and the variations of 16S rRNA gene and 92

ammonium-oxidizer communities in the furrow soils were present in the cluster 93

analysis results. However, the correlations were weakened by the low similarity 94

among individual soil samples from the same treatment. A box plot of similarity 95

showed that the similarities between ridge soils in the bacteria and 96


.asm.org/

Dow

nloaded from

http://aem.asm.org/

7

ammonium-oxidizing bacteria DGGE profiles were relatively low, with an average of 97

approximately 0.5 (Fig. 2). 98

99

Tomato L4783 (WT-plant) belongs to a unique popular group of Taiwan cultivar 100

whose fruit is harvested and consumed at the breaker phase. The ridge - furrow plot 101

with support sticks is a popular tomato cultivation method in Taiwan. The ridges are 102

typically covered with silver-black plastic sheets that create a physical barrier for 103

weed prevention and control of soil temperature. In contrast of ridges, furrows incur 104

frequent human activities including watering, fertilizing, nipping and walking during 105

the husbandry of tomatoes. The isolation created by the plastic covering of the ridges, 106

and the frequent human activity occurring at the furrows, poteintially contribute to the 107

difference between ridge and furrow soils in the test fields. Because furrow soil is less 108

relevant to the plant and directly exposed to the environment, we think that the minor 109

correlations between plant type and furrow soil microbial communities in this study 110

(Fig. 1) resulted from environmental factors such as relative position of field to the 111

drain, wind direction and human activities. The effect of tomato genotype on the 112

variations of DGGE profiles was considered minor because the effects of soil position 113

or environment factors were stronger than the effect of plant genotype. The results of 114

this study show that CMV-resistant GM tomato plant only had minor effect of on soil 115


.asm.org/

Dow

nloaded from

http://aem.asm.org/

8

microbial communities (12, 23, 26, 28).116


.asm.org/

Dow

nloaded from

http://aem.asm.org/

9

Literature Cited 117

(1) Abramoff, M. D., P. J. Magelhaes, and S. J. Ram. Image processing with 118

ImageJ. 2004. Biophotonics Int. 11:36-42. 119

(2) AOAC Official Methods of Analysis. 1990. Protein (Crude) in animal feed, semi 120

automated method No. 976.06. 121

(3) Arias, M. E., J. A. Gonzalez-Perez, F. J. Gonzalez-Vila, and A. S. Ball. 2005. 122

Soil health-a new challenge for microbiologists and chemists. Int. Microbiol. 123

8:13-21. 124

(4) Bloem, J., P. de Ruiter, and L. A. Bouwman. 1997. Soil food webs and nutrient 125

cycling in agro-ecosystem, p. 245-278. In J. D. Van Elsas, J. T. Trevors, and H. 126

M. E. Wellington (ed.) Modern Soil Microbiology, Marcel Dekker, New York, 127

NY. 128

(5) Bruinsma, M., G. A. Kowalchuk, and J. A. van Veen. 2003. Effects of 129

genetically modified plants on microbial communities and processes in soil. Biol. 130

Fertil. Soils. 37:329-337. 131

(6) Diallo, M. D., A. Willems, N. Vloemans, S. Cousin, T. T. Vandekerckhove, P. 132

de Lajude, M. Neyra, W. Vyverman, M. Gillis, and K. van der Gucht. 2004. 133

Polymerase chain reaction denaturing gradient gel electrophoresis analysis of the 134

N2-fixing bacterial diversity in soil under Acacia tortilis ssp. raddiana and 135


.asm.org/

Dow

nloaded from

http://aem.asm.org/

10

Balanites aegyptiaca in the dryland part of Senegal. Environ. Microbiol. 136

6:400-415. 137

(7) Felsenstein, J. 1989. PHYLIP - Phylogeny Inference Package (Version 3.2). 138

Cladistics 5:164-166. 139

(8) Flanagan, D. A., L. G. Gregory, J. P.Carter, A. K. Sen, D. J.Richardson, and 140

S. Sprio. 1999. Detection of genes for periplasmic nitrate reductase in nitrate 141

respiring bacteria and in community DNA. FEMS Microbiol. Lett. 177:263-270. 142

(9) Grayston, S. J., S. Wang, C. D. Campbell, and A. C. Edwards. 1998. Selective 143

influence of plant species on microbial diversity in the rhizosphere. Soil Biol. 144

Biochem. 30:369-378. 145

(10) Haichar, F. E., C. Marol, O. Berge, J. I. Rangel-Castro, J. I. Prosser, J. 146

Balesdent, T. Heulin, and W. Achouak. 2008. Plant host habitat and root 147

exudates shape soil bacterial community structure. ISME J. 2:1221-1230. 148

(11) Heuer, H., M. Krsek, P. Baker, K. Smmalla, and E. M. H. Wellington. 1997. 149

Analysis of actinomycete communities by specific amplification of gene 150

encoding 16S rRNA gene and gel-electrophoretic separation in denaturing 151

gradients. Appl. Environ. Microbiol. 63:3233-3241. 152

(12) Heuer, H., R. M. Kroppenstedt, J. Lottmann, G. Berg, and K. Smalla. 2002. 153

Effects of T4 lysozyme release from transgenic potato roots on bacterial 154


.asm.org/

Dow

nloaded from

http://aem.asm.org/

11

rhizosphere communities are negligible relative to natural factors. Appl. Environ. 155

Microbiol. 68:1325-1335. 156

(13) Innerebner, G., B. Knapp, T. Vasara, M. Romantschuk, and H. Insam. 2006. 157

Traceability of ammonium-oxidizing bacteria in compost-treated soils. Soil Biol. 158

Biochem. 38:1092-1100. 159

(14) Jackson, L. E., M. Buger and, T. R. Cavagnaro. 2008. Roots, nitrogen 160

transformations, and ecosystem services. Annu. Rev. Plant Biol. 59:341-363. 161

(15) Kowalchuk, G. A., D. S. Buma, W. De Boer, P. G. L. Klinkhammer, and H. 162

van Veen. 2002. Effects of above-ground plant species composition and 163

diversity on the diversity of soil-borne microorganism. Antonie van 164

Leeuwenhoek J. Microbiol. 81:509-520. 165

(16) Kowalchuk, G. A., M. Bruinsma, and J. A. Van Veen. 2003. Assessing 166

responses of soil microorganisms to GM plants. Trends. Ecol. Evol. 18:403-410. 167

(17) Kowalchuk, G. A., J. R. Stephen, W. De Boer, J. I. Prosser, T. M. Embley 168

and J. W. Woldendorp. 1997. Analysis of ammonia oxidizing bacteria of β 169

subdivision of the class proteobacteria in coastal sand dunes by denaturing 170

gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal 171

DNA fragments. Appl. Environ. Microbiol. 67:4742-4751. 172


.asm.org/

Dow

nloaded from

http://aem.asm.org/

12

(18) Lin, C. H., and T. M. Pan. 2008. Safety assessment of the genetically modified 173

tomato against Cucumber Mosaic Virus. National Science and Technology 174

Program for Agricultural Biotechnology in Taiwan, Taipei, Taiwan. 175

(19) Marschner, P., C. H. Yang, R. Lieberei, and D. E. Crowley. 2001. Soil and 176

plant specific effects on bacterial community composition in the rhizosphere. 177

Soil Biol. Biochem. 33:1437-1445. 178

(20) Marschner, P., Z. Solaiman, and Z. Rengel. 2006. Rhizosphere properties of 179

Poaceae genotypes under P-limiting condition. Plant Soil 283:11-24. 180

(21) McDevitt, C., P. Burrell, L. L. Blackall, and A. G. McEwan. 2000. Aerobic 181

nitrate respiration in a nitrite-oxidising bioreactor. FEMS Microbiol. Lett. 182

184:113-118. 183

(22) Muyzer, G., E. C. de Waal, and A. G. Uitterlinden. 1993. Profiling of 184

complex microbial populations by denaturing gradient gel electrophoresis 185

analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. 186

Appl. Environ. Microbiol. 59:695-700. 187

(23) Milling, A., K. Smalla, F. X. Maidi, M. Schloter, and J. C. Munch. 2004. 188

Effects of transgenic potatoes with an altered starch composition on the diversity 189

of soil and rhizosphere bacteria and fungi. Plant Soil 266:23-39. 190


.asm.org/

Dow

nloaded from

http://aem.asm.org/

13

(24) Oros-Sichler, M., N. C. M. Gomes, G. Neuber, and K. Smalla. 2006. A new 191

semi-nested PCR protocol to amplify large 18S rRNA gene fragments for 192

PCR-DGGE analysis of soil fungal communities. J. Microbiol. Methods 193

65:63-75. 194

(25) Patra, A. K., A. Luc, C. J. Annie, D. Valerie, J. G. Susan, G. Nadine, L. 195

Pierre, L. Frederique, M. Shahid, N. Sylvie, P. Laurent, P. Franck, I. P. 196

James, and L. R. Xavier. 2006. Effects of management regime and plant 197

species on the enzyme activity and genetic structure of N2-fixing, denitrifying 198

and nitrifying bacterial communities in grassland soils. Environ. Microbiol. 199

8:1005-1016. 200

(26) van Overbeek, L., and J. D. van Elsas. 2008. Effects of plant genotype and 201

growth stage on the structure of bacterial communities associated with potato 202

(Solanum tuberosum L.). FEMS Microbiol. Ecol. 64:283-296. 203

(27) Walkey, A., and I. A. Black. 1934. An examination of the Degtjareff method for 204

determining soil organic matter and a proposed modification of the chromic acid 205

titration method. Soil Sci. 37:29-38. 206

(28) Weinert, N., R. Meincke, C. Gottwald, H. Heuer, N. C. M. Gomes, M. G. 207

Schloter, H. Heur, R. M. Kroppenstedt, J. Lottmann, G. Berg, and K. 208

Smalla. 2002. Effects of T4 lysozyme release from transgenic potato roots on 209


.asm.org/

Dow

nloaded from

http://aem.asm.org/

14

bacterial rhizosphere communities are negligible relative to natural factors. Appl. 210

Environ. Microbiol. 68:1325-1335. 211

(29) Wu, M. L. 2007. SPSS operation and application-practice analysis of variance. 212

Wu-Nan Book Inc. Taipei, Taiwan. 213


.asm.org/

Dow

nloaded from

http://aem.asm.org/

15

Figure Legends 214

Fig. 1. PCA and clustering analysis of DGGE profiles of 16S rRNA gene and 215

ammonium-oxidizing bacteria specific 16S rRNA. (A) PCA anlysis of 16S rRNA 216

gene. (B) Clustering analysis of 16S rRNA gene. (C) PCA analysis of 217

ammonium-oxidizing bacteria specific 16S rRNA gene. (D) Clustering analysis of 218

ammonium-oxidizing bacteria specific 16S rRNA gene. Solid circle: ridge soil 219

samples with GM tomato. Open circle: ridge soil samples with WT tomato. Solid 220

triangle: furrow soil samples with GM tomato. Open triangle: furrow soil samples 221

with WT tomato. 222

223

Fig. 2. Box plot of the similarity of selected DGGE profiles among soil samples. 224

(A) Bacteria community (16S rRNA gene) of ridge soil. (B) Ammonium-oxidizing 225

bacteria community of ridge soil. (C) Bacteria community (16S rRNA gene) of 226

furrow soil. (D) Ammonium-oxidizing bacteria community of furrow soil. GM-GM: 227

similarity between soil samples of GM test field. WT-WT: similarity between soil 228

samples of WT test field. GM-WT: similarity between soil samples of GM and WT 229

test field. Dotted line: means. Solid line: medians. Gray box: quartiles. End of 230

whiskers: 90th and 10th percentiles. Open circle: outliers. Dashed lines: 95% 231

confidence intervals for GM-WT.232


.asm.org/

Dow

nloaded from

http://aem.asm.org/

16

233

234

235

236

237

238

239

240

241

Figure 1 242


.asm.org/

Dow

nloaded from

http://aem.asm.org/

17

243

244

245

246

247

248

249

250

251

252

253

Figure 2 254

255


.asm.org/

Dow

nloaded from

http://aem.asm.org/

assessing the effects of genetically modified cmv-r...

Documents