Isolation, Colonization, and Chlorpyrifos Degradation Mediation of the

Subscriber access provided by HACETTEPE UNIVERSITESI KUTUPHANESI

Article

Isolation, colonization and chlorpyrifos degradation mediation of the endophytic bacterium Sphingomonas strain HJY in Chinese chive (Allium tuberosum) Fayun Feng, Jing Ge, Yisong Li, Jinjin Cheng, Jianfeng Zhong, and Xiangyang Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05283 • Publication Date (Web): 19 Jan 2017 Downloaded from http://pubs.acs.org on January 24, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35

Journal of Agricultural and Food Chemistry

1

Isolation, colonization and chlorpyrifos degradation mediation of the

2

endophytic bacterium Sphingomonas strain HJY in Chinese chive (Allium

3

tuberosum)

4 5

Fayun Fenga,b#, Jing Gea,b#, Yisong Lic, Jinjin Chenga, Jianfeng Zhong b, Xiangyang

6

Yu a,b,*

7 8

a Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base,

9

50 Zhongling Street, Nanjing 210014, China

10

b Institute of Food Quality and Safety, Jiangsu Academy of Agricultural Sciences, 50 Zhongling

11

Street, Nanjing 210014, China

12

c Department of Plant Protection, Agricultural College, Shihezi University, Xinjiang, 832000

13 14 15 16 17 18 19 20 21 *Correspondence to: Xiangyang Yu, Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, 50 Zhongling Street, Nanjing 210014, China. E-mail: [email protected] # These authors contributed equally to this work

1

ACS Paragon Plus Environment


Page 2 of 35

22 23

ABSTRACT: The interaction of endophyte-plant can benefit the host in many

24

different ways. An endophytic bacterium strain (HJY) capable of degrading

25

chlorpyrifos (CP) was isolated from Chinese chives (Allium tuberosum Rottl. ex

26

Spreng). The isolated bacterium HJY classified as Sphingomonas sp. Strain HJY

27

could use chlorpyrifos as the sole carbon source. After being marked with the gfp

28

gene, the colonization and distribution of strain HJY-gfp was directly observed in

29

different tissues of Chinese chives with confocal laser scanning microscope. The

30

inoculation of strain HJY-gfp in Chinese chives resulted in a higher degradation of CP

31

inside the plants than in noninoculated plants. With drench application, up to 70% and

32

66% of CP was removed from shoots and roots of inoculated Chinese chives,

33

respectively. Moreover, up to 75% of CP was removed from the soil containing plants

34

inoculated with HJY-gfp. With foliage application, the applied concentration of

35

chlorpyrifos affected the degradation performance of strain HJY in Chinese chives.

36

Significant differences were only observed between inoculated and noninoculated

37

Chinese chives with the low applied concentration of CP. Together, other than natural

38

endophyte-assisted plant protection for food safety, interaction of HJY and plant may

39

be also a promising strategy for in situ bioremediation of soil contaminated with CP.

40 41

KEYWORDS:

42

Degradation, Food safety, Bioremediation

Sphigomonas

sp.,

Chlorpyrifos,

Endophyte-host

43 2


interaction,

Page 3 of 35


44

INTRODUCTION

45

The utilization of synthetic pesticides is the most effective measure to control

46

pests among all of the alternatives.1 However, there are increasing concerns about

47

excessive use of synthetic pesticides leading to residues in foodstuffs, pesticide

48

resistance, environmental contamination, etc. Availability of adequate food supplies

49

to sustain expanding food consumption becomes a serious issue, especially in China

50

which has the world’s largest population and limited arable land. Therefore,

51

agrichemical use is crucial to ensure food supply. While applying properly, the

52

residues should be lower than the statutory Maximum Residue Levels (MRLs).2

53

Understanding the factors responsible for the metabolism and detoxification of

54

pesticide residues in plants can aid the development of new technology to efficiently

55

control pests while resulting in minimal residues in food.

56

Endophytes are nonpathogenic microorganisms that colonize healthy plant tissues

57

intracellularly and/or intercellularly.3 Endophytic bacteria have been found in virtually

58

every plant studied. They colonize internal tissues of host plants and can form a range

59

of different relationships, including symbiosis, mutualism, commensalism and

60

trophobiosis.4 Endophytes play an important role in the adaptation of plants to the

61

polluted environments and in the improvement of phytoremediation. Endophytes can

62

alleviate the phytotoxicity of pollutants5, 6, reduce the bioaccumulation of pollutants in

63

plants6, engage in the transportation and accumulation of metal ions in plants, increase

64

plants’ tolerances to heavy metals (and even be utilized in heavy metal

65

bioremediation)7, 8, promote plant growth at contaminated sites9, 3


10

, and produce


Page 4 of 35

66

chemical defense compounds to protect the host.11 Recently, Dimitroula et al reported

67

that the endophytic bacteria Pseudomonas sp. strain R16 and Ochrobactrum sp. strain

68

R24 were chosen for chromium (VI) reduction assays.12 To date, several persistent

69

organic pollutants (POP)-degrading endophytic bacteria have been isolated from

70

plants grown in POP-contaminated soils.10, 12, 13 Some of the endophytic bacteria that

71

have been successfully colonized in plants improved in planta degradation of

72

pollutants and reduced evapotranspiration.3, 14 However, no report was found to use

73

pesticide-degrading endophytes and plants interactions for the consideration of food

74

safety. Pesticide residue is a critical issue on agricultural products nowadays,

75

plant-endophyte partnership can be used to accelerate degradation of pesticides in

76

crops, thereby decreasing the accumulation of pesticides and reducing the risks to

77

human health.

78

Chlorpyrifos

(CP),

O,

O-diethyl-O-(3,

5,

79

6-trichloro-2-pyridinyl)-phosphorothioate), is a broad-spectrum, moderately toxic

80

chlorinated organophosphate insecticide that is used extensively in agricultural

81

industries.15 In recent years, it has also been widely employed worldwide for the

82

control of household and subterranean pests.16 However, CP has been reported to have

83

side-effects on sperm activity17 and the hepatic and renal function18 of humans.

84

Prenatal exposure to CP has a strong link with low birth weight, smaller head size of

85

infants, and delayed neurodevelopment of children.19, 20 CP has also been identified as

86

an environmental breast cancer risk factor.21 In China, CP is commonly used to

87

control the larvae of Bradysia odoriphaga Yang et Zhang, which is a destructive 4


Page 5 of 35


88

underground insect pest causing significantly yield losses in Chinese chives

89

production. The larvae often live in the roots and stems of the underground part of

90

Chinese chives, which makes it difficult to control the pest through foliage application

91

of the chemicals. Massive use of CP with drench application to control the pests posed

92

a high risk of CP residues in Chinese chives through root uptake.22

93

The present study focused on an endophytic bacterium (Sphingomonas sp. strain

94

HJY) isolated from the leaves of Chinese chives that showed potential ability for CP

95

degradation. The main objective was to determine the potential ability of HJY

96

colonization to enhance the degradation of CP in Chinese chives. Studies were

97

conducted to screen the Chinese chives endophytes for their CP-degradation ability

98

and degrade CP in vitro, to observe the colonization of the endophyte in the different

99

part of Chinese chives, and to investigate the degradation performance of CP in soil

100

containing plants inoculated with the endophyte. The results will provide a practical

101

way to use plant-endophte interaction to accelerate the degradation of CP residues in

102

Chinese chives which could be a promising approach for the safety of Chinese chives

103

production and consumption. The results could also provide biomaterial for the

104

remediation of CP contaminated soil.

105 106

MATERIALS AND METHODS

107

Chemicals and media. CP (99.9%) was obtained from Dr. Ehrenstorfer GmbH

108

(Augsburg, Germany). All the other reagents used in the study were of analytical

109

grade and purchased from Xilong Chemical Co., Ltd. (Shantou, China). 40% CP EC 5



110

was purchased from Hubei Xianlong Chemical Industry Co., Ltd. (Hubei, China).

111

The mineral salt media (MSM) were prepared by dissolving MgSO4·7H2O (0.4 g),

112

FeSO4·7H2O (0.2 g), K2HPO4 (0.2 g), (NH4)2SO4 (0.2 g), and CaSO4, (0.08 g) in 1 L

113

of water. The pH of MSM was 7.0. The stock solution of CP (20 g/L) was prepared by

114

dissolving CP in acetonitrile and filtering through a membrane (0.22 µm). The

115

enriched media was obtained by adding yeast extract (1.0 g/L) and tryptone (1.0 g/L)

116

to MSM. The glucose medium consisted MSM supplemented with 1% glucose.

117

Luria-Bertani (LB) medium (pH 7.0) contained tryptone (10.0 g/L), yeast extract (5.0

118

g/L), and NaCl (10.0 g/L).

119

Isolation and identification of CP-degrading endophytic bacterium strain

120

HJY. Chinese chives treated with CP to control underground pests during the growth

121

period were collected from the experimental field of Jiangsu Academy of Agricultural

122

Sciences in Nanjing, Jiangsu, China. Plant samples were thoroughly washed with

123

running tap water for 10 min to remove the adhering soil. The samples were separated

124

into roots, stems and leaves and then surface disinfected to eliminate epiphytic

125

bacteria. The protocol of surface disinfection was based on the method described by 23.

126

Briefly, plant tissues were immersed in 75% ethanol for 3 min, followed by 3 rinses

127

with sterile water. Then, the tissue samples were soaked in a sodium hypochlorite

128

solution (2.5%, w/v) for 5 min and then rinsed 5 times with sterile water. The

129

efficiency of surface sterilization was checked by placing 100 µL aliquots of the final

130

rinsing water on LB solid medium and incubated at 30°C for 2 days. After surface

131

sterilization, plant samples were cut into small pieces and ground in a sterilized mortar. 6


Page 6 of 35

Page 7 of 35


132

The ground plant samples was placed onto enriched media containing 100 mg⋅L-1 CP

133

at 30°C for 2~7 days. The endophytic bacteria growing out from the extracts were

134

transferred five times to MSM agar plates containing 10~100 mg⋅L-1 CP as the sole

135

carbon source. The candidate colonies growing on the MSM plates consecutively and

136

developing transparent degradation zones around CP were purified on enriched media

137

plates and stored in enriched media slants for further study.

138

Strain HJY was identified based on morphological, physiological and biochemical

139

tests according to Bergey’s Manual of Determinative Bacteriology

140

gene sequence analysis. The 16S rRNA gene was amplified by PCR using the

141

universal

142

(5’-GGTTACCTTGTT ACGACTT-3’).25 The PCR was performed as follows: 5 min

143

of denaturation (95°C), followed by 30 cycles of 95°C for 1 min, 56°C for 1 min,

144

72°C for 2 min, and extension at 72°C for 10 min. The PCR products were purified by

145

agarose gel electrophoresis and sequenced from both directions at Shanghai Sangon

146

Biotech Co., Ltd. (Shanghai, China). Alignment of 16S rRNA gene sequences from

147

GenBank was performed using ClustalX 1.8.1 with default settings. Phylogenetic and

148

molecular analyses were conducted using MEGA 6.06. Distances were calculated

149

using the Kimura 2–parameter distance model. Unrooted trees were built by the

150

neighbor-joining method. The dataset was bootstrapped 1000 times.

primers

27f

24

(5’-AGAGTTTGATCMTGGCTCAG-3’)

and 16S rRNA

and

1492r

151

Construction of gfp-tagged strain HJY. Shuttle vector pAD43-45 encodes for

152

ampicillin resistance and contains a gfpmut3a gene, which is for high-level

153

constitutive expression of a Green Fluorescent Protein variant 7


26

. The vector was


154

transferred into strain HJY mutant resistant to rifampicin (300 µg/ml) using triparental

155

mating method

156

strain HJY were grown on LB agar containing 100 mg L-1 ampicillin, 25 mg

157

L-1 kanamycin and 300 mg L-1 rifampicin. Fluorescence microscopy was used to

158

observe the expected green fluorescence of 10-15 colonies from each transconjugation.

159

For further confirmation of positive transconjugants, the plasmid pAD43-45 was

160

extracted and the 16SrDNA sequence was also analyzed.

27

with E.coli HB101 (pRK2013) as a helper

28

. Transconjugants of

161

Degradation of CP in liquid cultures by strain HJY. The best growing

162

endophytic bateria from the screening experiment was selected for further

163

CP-degradation verification. Briefly, the isolate of strain HJY was grown in LB

164

medium overnight at 30°C and 150 rpm in a rotary shaker, harvested by centrifugation

165

and washed with autoclaved N-saline (0.85% NaCl in distilled water), and then

166

resuspended in MSM medium adjusted to an OD600 of 0.2. CP was added at a

167

concentration of 20 mg⋅L-1 to 250 mL flask containing 125 mL of MSM medium. The

168

flask was covered with Teflon bottle stopper and shaken at 200rpm on a rotary shaker

169

in the dark at 30°C. The concentration of CP in the MSM solution was measured at

170

different time intervals during incubation with HPLC, and the OD600nm values were

171

measured using an ultraviolet spectrophotometer. Sterilized MSM medium or glucose

172

medium without HJY inoculation was used as controls. Effect of different glucose

173

levels (0.5, 1, 2, or 5%) in the medium on degradation of CP by strain HJY was

174

studied. The pH of the media was adjusted to 7.0 with 1 mol⋅L-1 NaOH and 1 mol⋅L-1

175

HCl prior to disinfection. The experiment was done in triplicates. 8


Page 8 of 35

Page 9 of 35


176

Inoculation and plant experiment. The seed culture of strain HJY was grown in

177

LB media overnight at 30°C and 150 rpm in a rotary shaker. The cells were harvested

178

by centrifugation at 5000g for 15 min at 4°C. The pellets were washed twice with

179

autoclaved N-saline (0.85% NaCl in distilled water) and resuspended in N-saline. The

180

optical density was adjusted to 1.0 OD600nm (compared with sterile N-saline).

181

Inoculation of Chinese chives with strain HJY was conducted by immersing the root

182

in the inoculant solution overnight. Shoots of Chinese chives collected from the field

183

were washed with tap water to remove the adhered soil. The leaves were then cut off

184

and the main roots were cut to about 3 cm long. Thereafter, the roots of Chinese

185

chives were immerged in the inoculant solution for 24 h, and the roots placed in

186

bacterial free solutions were used as controls.

187

Chinese chives were planted in pots filled with 5 kg of non-sterile soil. Plant

188

cultivation was carried out under greenhouse conditions set at 25 °C / 18 °C day/night

189

with 12-h light/dark regime. CP was applied to Chinese chives through two different

190

treatments: root and foliage application. For root application, 40% CP EC was

191

dissolved in tap water to form different concentrations of CP suspension and 10 ml of

192

the suspension were directly applied into the surrounding soil of Chinese chives at

193

four different concentrations (40 mg active ingredient (ai) pot-1, 120 mg ai pot-1, 200

194

mg ai pot-1, and 400 mg ai pot-1). For foliage application, CP was only applied to the

195

above ground part of Chinese chives at two different levels (diluted 100 and 1000

196

times). To avoid any contamination on the soil or root, plastic film was used to cover

197

the plant soil when CP was applied to the above ground part of Chinese chives. All the 9



198

treatments were carried out in triplicate. Chinese chives and the surrounding soil

199

samples were collected to test CP concentrations 30 days after the application for root

200

treatment. For foliage application, Chinese chives samples were collected in different

201

intervals to test the population of HJY (root, stem and leaf) and CP concentrations

202

(aboveground part).

203

Population and visualization of strain HJY within plant tissues. The

204

quantities of strain HJY in Chinese chives roots, shoots and stems were determined by

205

counting the CFUs on plates. Plant samples were surface-sterilized by sequentially

206

immersing in 75% ethanol followed by a 0.1% sodium hypochlorite solution. Then

207

the tissues were rinsed three times with sterile water, ground and grown on LB plates

208

containing 100 mg L-1 ampicillin, 25 mg L-1 kanamycin and 300 mg L-1 rifampicin.

209

The cell counts of strain HJY were taken under UV light.

210

In order to observe the localization and distribution of strain HJY in Chinese chives,

211

the plant tissues were hand cut under aseptic condition and fixed on glass slides using

212

10% (v/v) glycerol (microscopy grade), then the excised tissues were examined using

213

Confocal Laser Scanning Microscopy (UltraVIEWVoX 3D Live Cell Imaging

214

System). Uninoculated plants were used as controls.

215

Analysis of CP in plant and soil samples. The procedure for extraction of CP in

216

plant and soil was adopted from our previous study 29. Shoots: Shoots samples were

217

homogenized with a blender. An aliquot of 5.0 g shoots sample was extracted with 10

218

mL of acetonitrile. After vortexing for 1 min and homogenizing for 2 min, 2.0 g NaCl

219

was added to the mixture then vortex for another min. After centrifugation for 5 min 10


Page 10 of 35

Page 11 of 35


220

at 3802 g, 1 mL of super was dried under a gentle nitrogen gas stream and redissolved

221

in n-hexane for cleanup. The extracts were subjected to florisil cleanup with the

222

procedure as follows: A mass of 0.5 g florisil was placed into a 0.5 cm diameter glass

223

pipettes with frit at the bottom. The column was sequentially conditioned with 5 mL

224

of hexane: acetone (90:10) and 5 mL of hexane. The sample extract was loaded on the

225

top of the column, and the eluent was collected with a 10-mL test tube. The tube

226

contained the sample extract was rinsed with 5 mL of hexane: acetone (90:10) and the

227

solution was loaded on the top of the column. The eluent was collected into the same

228

10-mL test tube. The eluent was dried under a gentle nitrogen stream and dissolved in

229

1 mL of acetone. Then the extract was filtered through a 0.22-µm membrane into a

230

sample vial for analysis. Roots: Root samples were cut into fine pieces. An aliquot of

231

1.0 g root sample was extracted with 5 mL of acetone. After vortexing for 1 min and

232

homogenizing for 2 min, 2.0 g NaCl was added to the mixture then vortex for another

233

min. After centrifugation for 5 min at 3802 g, 1 mL of super was dried under a gentle

234

nitrogen gas stream and dissolved in 1 mL of acetone. Then the extract was filtered

235

through a 0.22-µm membrane into a sample vial for analysis. Soil: An aliquot of 5.0 g

236

soil sample was mixed with 5 g NaCl and extracted with 20 mL of acetonitrile. The

237

mixture was vortexed for 1 min, extracted with ultrasound for 30 min and shaken with

238

a shaker for 2 h. Then the mixture was centrifuged for 5 min at 3802 g. One milliliter

239

of the supernatant was dried under a gentle nitrogen stream and dissolved in 1 mL of

240

acetone. Then the extract was filtered through a 0.22-µm membrane into a sample vial

241

for analysis. 11



242

All samples were analyzed with an Agilent 6890N GC-ECD. The GC-ECD was

243

equipped with a capillary column (HP-5, 30.0 m × 250 µm × 0.25 µm). The flow rate

244

of the carrier gas was 1.0 mL.min- 1. The inlet and detector temperatures were 270 and

245

280 °C, respectively. The temperature program was as follows: 120 °C, 30 °C.min- 1

246

to 230 °C, held for 5 min, 20 °C.min- 1 to 270 °C, held for 2 min. The injection

247

volume was 1.0 µL.

248

Statistical analysis. Independent Samples t-test was used to compare the

249

differences between different treatments. The level of statistical significance was

250

defined at p < 0.05. All statistical analyses were performed in SPSS 16.0 software.

251 252

RESULTS AND DISCUSSION

253

Isolation, characterization and gfp-labeling of strain HJY. The CP-degrading

254

endophytic bacterium Sphingomonas sp HJY was isolated from Chinese chives

255

treated with CP in the early growth period. The colonies of strain HJY were yellow,

256

smooth and wet on LB plates within 36 h at 30°C. The bacterium is Gram-negative

257

with a straight rod-shaped and a single polar flagellum. Endospores were not found.

258

The physiological and biochemical tests showed that HJY was positive for catalase,

259

oxidase, citrate utilization, the V-P test, phenylalanine deaminase, ornithine

260

decarboxylase, arginine dihydrolase and lysine decarboxylase and negative for

261

gluconate and malonate utilization, gelatin liquefaction, nitrate reduction, the M-R

262

test and the indole test. Comparative analyses of the 16S rDNA sequence showed that

263

strain HJY was 98.6% identical to Sphingomonas yabuuchiae strain A1-18 12


Page 12 of 35

Page 13 of 35


264

(NR_028634.1) and 98.1% identical to Sphingomonas pseudosanguinis strain G1-2

265

(NR_042578.1). A phylogenetic tree including strain HJY and related type strains

266

(obtained from EzBioCloud) is presented in Fig. 1. These results indicated that strain

267

HJY belonged to the genus Sphingomonas.

268

Bacteria belonging to the genus Sphingomonas have been extensively studied

269

over the last few decades due to their metabolic versatility. Several Sphingomonas sp.

270

strains isolated from polluted soils/sediments or treated plants showed activity in the

271

degradation of polycyclic aromatic hydrocarbons (PAHs)30,

272

study demonstrates that the endophytic bacterium in Chinese chives belonging to

273

Sphingomonas sp. greatly enhances chlorpyrifos degradation in plants.

31

or pesticides.25 This

274

The plasmid pAD43-45 constitutively expressed gfp in the transconjugants which

275

emitted green fluorescence under fluorescent microscopy (Fig. S1). The cell and

276

colony morphologies were identical between the original strain HJY and the

277

gfp-tagged strain (data not shown). The CP degrading ability and the growth rates

278

were similar, suggesting little growth burden for plasmid on strain HJY. After growth

279

for 20 generations with and without antibiotic pressure, up to 90% of the gfp-tagged

280

cells can grow on LB agar containing the three antibiotics mentioned above,

281

suggesting a considerable stability and suitable for a long-term monitoring of

282

gfp-tagged strain HJY.

283

Biodegradation of CP by strain HJY in culture solution. The growth of strain

284

HJY in liquid MSM spiked with 20 mg⋅L-1 CP as the sole carbon source and the

285

removal of CP were analyzed (Fig. 2 top). As can be seen in Fig. 2 top, the cell 13



286

density increased in the first 3 days and then day 6 to 12, decreased slightly from day

287

3 to 6 and day 12 to 15. The data indicated that strain HJY utilized CP as the sole

288

carbon source. The slight decrease in HJY growth from day 3 to 6 could be attributed

289

to the fast accumulation of CP metabolites, which caused growth inhibition of HJY.25,

290

32

291

medium within 12 days, while less than 10% of CP was removed from the control

292

MSM culture.

More than 90% of CP (initial CP concentration 20 mg/L) was removed from the

293

The growth of strain HJY and the degradation of CP were greatly influenced in

294

the presence of glucose (Fig. 2 bottom). The growth acceleration mainly occurred on

295

the first day of inoculation. Accordingly, the removal of CP from the glucose amended

296

medium was much greater than the medium without glucose. More than 50% of CP

297

was removed on the first day in the medium containing 1% glucose. After a period of

298

fast growth, the cell density of strain HJY maintained a relatively high stationary level.

299

The CP removal maintained a steady rate of approximately 2 mg⋅L-1⋅day-1 until more

300

than 98% of CP was degraded in 6 days. The degradation rate of CP increased along

301

with the addition level of glucose. After a day, approximately 60.7, 53.6, and 48.9%

302

of the CP was removed from the media enriched with 5, 1, and 0.5% of glucose,

303

respectively.

304

Some efficient CP degraders were previously reported. For example, Cupriavidus

305

sp. DT-1 could degrade nearly 100% of 100 mg L-1 CP in 6 h 33 and Pseudomonas sp.

306

Iso 1 could degrade approximately 91% of 500 mg⋅L-1 CP at a rate of 300 mg⋅L-1⋅d-1 34.

307

The relatively low ability of strain HJY to degrade CP was likely due to the nature of 14


Page 14 of 35

Page 15 of 35


308

endophytic bacteria. Strain HJY was isolated from the interior of the plants, which

309

usually did not suffer from high levels of CP stress compared to bacteria from

310

polluted soil or water. Recently, there has been an increase effort to enhance the

311

efficacy

312

plant-endophyte

313

Burkholderia fungorum DBT1 into a hybrid poplar improved the phytoremediation

314

efficiency of polycyclic aromatic hydrocarbons (PAHs)35 and phenolic compounds.36

315

Endophytic bacteria in aquatic plants play an important role in bioremediation of

316

polluted waters.37 CP is commonly used to kill Bradysiao doriphaga22, 38, which is the

317

major insect associated with Chinese chives cultivation, therefore, the use of strain

318

HJY to accelerate the degradation of CP residues in Chinese chives is a promising

319

approach for the safety of Chinese chives consumption and sustainable development.

of

phytoremediation interactions.

of For

contaminated

environments

instance,

inoculation

the

by of

studying endophytic

320

Colonization pattern of gfp-tagged strain HJY in Chinese chives. Seven days

321

after the inoculation, gfp-tagged strain HJY had already colonized in the primary

322

tissues of Chinese chives (Fig. 3F-J). Strain HJY-gfp invaded into the main root and

323

mainly colonized in intercellular space (Fig. 3B). As the time progress, strain HJY-gfp

324

was also visualized in other tissues, such as lateral roots (Fig. 3G), stems epidermis

325

(Fig. 3H), cross-section of stem (Fig. 3I) and leaves (Fig. 3J). Plants without HJY-gfp

326

inoculation served as the controls (Fig. 3A-E). Even though the roots and leaves of

327

Allium tuberosum have the ability of auto-fluorescence, gfp-tagged strain HJY excited

328

brighter green fluorescence making it stand out. No fluorescent bacterium was found

329

on and in control plants (Fig. 3A-E). The colonization of HJY-gfp provided the 15



Page 16 of 35

330

premise for the utilization of HJY-gfp to enhance the degradation of CP resides in

331

Chinese chives. The number of endophytic measured in Chinese chives was between

332

2.0 and 5.0 log CFU.g-1. The highest densities of strain HJY-gfp were found in the

333

roots ranging from 3.37 to 4.60 log CFU.g-1, and the densities decreased in the stems

334

and leaves (Fig. S2).

335

Performances of strain HJY enhance the degradation of CP in Chinese

336

chives. Strain HJY-gfp showed a natural capacity to cope with CP in vitro as well as

337

within

338

concentrations were found between Chinese chives with and without strain HJY-gfp

339

inoculation (Fig. 4A, B), and significant differences of CP residues were even

340

observed in the surrounding soil samples planted with infested and uninfested Chinese

341

chives (Fig. 4C). After inoculated with HJY-gfp for 30 days, the concentrations of CP

342

in inoculated plants were all lower than those in the controls. Moreover, soil

343

containing HJY-gfp inoculated plants underwent a dramatic decrease of CP

344

concentrations. Compared with the controls, up to 70% and 66% of CP was removed

345

from shoots and roots with HJY-gfp inoculation, respectively. Up to 75% of CP was

346

removed from the soil containing plants inoculated with HJY-gfp. The deposit of CP

347

in soil through root treatment did not affect the degradation performance of endophyte

348

strain HJY-gfp in Chinese chives.

349

For foliage treatment, significant differences were only found at the low applied level

350

(diluted 1000 times) from three days after application (Fig. 5B). As can be seen from

351

Fig. 5A, not much reduction of CP residues was found with inoculated Chinese chives

plants.

For

root

treatment,

significant differences

16


of chlorpyrifos

Page 17 of 35


352

at high applied level (diluted 100 times) compared to controls and some was even

353

higher than controls. Meanwhile, the CFUs of strain HJY-gfp was also re-isolated and

354

measured from inoculated Chinese chives. According to the results (Table S1), there

355

was a dramatic increase of CFUs in roots and shoots on the third day after application

356

at low applied level. The highest CFUs in the leaves was found on the seventh day,

357

which was two days later than those in roots and shoots, after application at low

358

applied level. The delay of the sharp increase of CFUs in the leaves indicates that

359

strain HJY-gfp was probably translocated from the lower part of the plant to leaves to

360

help with degrading CP. However, this was not observed with higher applied levels of

361

CP on Chinese chives. It is worthy to note that the total CFUs of strain HJY-gfp in

362

Chinese chives treated with two concentrations of CP was similar; however, the

363

degradation performance came out different. It could be due to the toxicity of high

364

concentration of CP to either some enzyme activity of strain HJY-gfp or some

365

function system of the plants. To determine whether high level of CP inhibit the

366

growth of strain HJY-gfp, strain HJY-gfp was grown in LB medium supplemented

367

with different concentrations of CP. High levels (100 and 1000 mg/kg) of CP did slow

368

down the growth of strain HJY-gfp, however, low level (1 mg/kg) of CP could

369

stimulate the growth of HJY-gfp (Fig. S3).

370

The possible mechanisms of strain HJY enhancing the degradation of chlorpyrifos in

371

Chinese chives could include (1) HJY promotes chlorpyrifos degradation in the

372

environment and thereby reduces the uptake of chlorpyrifos by Chinese chives; (2)

373

HJY inoculants increase the overall degradation potential of endogenous endophytic 17



374

communities by horizontal gene transfer

375

plant detoxification enzymes, such as P450, GST or promote chlorpyrifos-degrading

376

gene expression. Anyhow, the partnership of plant-endophyte could be used to reduce

377

and even eliminate pesticide residues in agricultural products and ensure that residues

378

of pesticides in foods are not present at levels posing a threat to human health.

39

; (3) HJY may influence the activities of

379 380

CONCLUSIONS

381

Our data show that the isolated endophytic bacterium Sphingomonas sp. strain

382

HJY was capable of degrading CP in vitro, meanwhile, strain HJY was successfully

383

colonized in Chinese chives and also enhanced the CP degradation inside the plants.

384

The concentrations of CP with drench application did not affect the interaction of

385

strain HJY and the host at an even high level. However, with foliage treatment, high

386

concentration of CP had an obvious effect on the capability of strain HJY to enhance

387

the degradation of CP in Chinese chives. However, the numbers of CFUs of strain

388

HJY were not much different with high and low levels of foliage treatment. Together,

389

these data indicate that high concentration of CP maybe either inhibit some enzyme

390

activity of strain HJY or physiological activity of Chinese chives, which could affect

391

the interaction of strain HJY and the host.

392

To the best of our knowledge, this is the first report of using a natural

393

chorpyrifos-degrading endophytic bacterium to reduce the residue of chlorpyrifos in

394

Chinese chives, which could probably be used to ensure the safety and quality of

395

agricultural products. Meanwhile, the removal of chlorpyrifos was also improved 18


Page 18 of 35

Page 19 of 35


396

dramatically in the soil containing the inoculated plants. Therefore, interaction of

397

HJY-plant may be also a promising strategy in improving phytoremediation of soil

398

polluted with chlorpyrifos. However, little is known about the mechanisms of HJY

399

enhancing the degradation of CP inside the plants. For better use of plant-endophyte

400

partnership in assuring food safety, further studies are needed to elucidate the

401

mechanisms of strain HJY enhancing the CP degradation in Chinese chives.

402 403 404 405

ACKNOWLEDGMENTS The authors acknowledge the financial support from the Natural Science Foundation of China (NO. 31572032 and 31601660).

406 407 408

CONFLICT OF INTEREST The authors declare no competing financial interest.

409 410

Supporting Information Available: [Table S1 listing the population of strain HJY

411

observed in different tissues of Chinese chives at different time after CP application

412

with foliage treatment. Figure S1 showing fluorescent micrographs of bacterium

413

culture observed under fluorescence microscopy (×20). (A) E.coli top10 with the

414

plasmid pAD43-45; (B) strain HJY, (C) gfp-tagged transconjugant. Figure S2

415

showing the population of strain HJY observed in different tissues of Chinese chives

416

during 30 d experiment period after inoculation. Figure S3 showing the growth curve

417

of HJY in LB medium supplement with different levels of chlorpyrifos.] This material 19



418

is available free of charge via the Internet at http://pubs.acs.org.

419 420 421 422

REFERENCES (1) Popp, J.; Pető, K.; Nagy, J., Pesticide productivity and food security. A review. Agron. Sustainable Dev. 2013, 33, 243-255.

423

(2) Mouron, P.; Heijne, B.; Naef, A.; Strassemeyer, J.; Hayer, F.; Avilla, J.;

424

Alaphilippe, A.; Höhn, H.; Hernandez, J.; Mack, G., Sustainability assessment of crop

425

protection systems: SustainOS methodology and its application for apple orchards.

426

Agric. Syst. 2012, 113, 1-15.

427 428

(3) Wang, Y.; Dai, C. C., Endophytes: a potential resource for biosynthesis, biotransformation, and biodegradation. Ann. Microbiol. 2010, 61, 207-215.

429

(4) Ryan, R. P.; Germaine, K.; Franks, A.; Ryan, D. J.; Dowling, D. N., Bacterial

430

endophytes: recent developments and applications. FEMS Microbiol. Lett. 2008, 278,

431

1-9.

432

(5) Moore, F. P.; Barac, T.; Borremans, B.; Oeyen, L.; Vangronsveld, J.; Van der

433

Lelie, D.; Campbell, C. D.; Moore, E. R., Endophytic bacterial diversity in poplar

434

trees growing on a BTEX-contaminated site: the characterisation of isolates with

435

potential to enhance phytoremediation. Syst. Appl. Microbiol. 2006, 29, 539-556.

436

(6) Weyens, N.; Truyens, S.; Dupae, J.; Newman, L.; Taghavi, S.; Van Der Lelie, D.;

437

Carleer, R.; Vangronsveld, J., Potential of the TCE-degrading endophyte

438

Pseudomonas putida W619-TCE to improve plant growth and reduce TCE

439

phytotoxicity and evapotranspiration in poplar cuttings. Environ. Pollut. 2010, 158, 20


Page 20 of 35

Page 21 of 35


440

2915-2919.

441

(7) Guo, H.; Luo, S.; Chen, L.; Xiao, X.; Xi, Q.; Wei, W.; Zeng, G.; Liu, C.; Wan, Y.;

442

Chen, J., Bioremediation of heavy metals by growing hyperaccumulaor endophytic

443

bacterium Bacillus sp. L14. Bioresour. Technol. 2010, 101, 8599-8605.

444

(8) Wang, Y.; Yang, X.; Zhang, X.; Dong, L.; Zhang, J.; Wei, Y.; Feng, Y.; Lu, L.,

445

Improved plant growth and Zn accumulation in grains of rice (Oryza sativa L.) by

446

inoculation of endophytic microbes isolated from a Zn Hyperaccumulator, Sedum

447

alfredii H. J. Agric. Food Chem. 2014, 62, 1783-1791.

448

(9) Zhang, Y. F.; He, L. Y.; Chen, Z. J.; Zhang, W. H.; Wang, Q. Y.; Qian, M.; Sheng,

449

X. F., Characterization of lead-resistant and ACC deaminase-producing endophytic

450

bacteria and their potential in promoting lead accumulation of rape. J. Hazard. Mater.

451

2011, 186, 1720-1725.

452

(10) Zhu, X.; Ni, X.; Liu, J.; Gao, Y., Application of Endophytic Bacteria to Reduce

453

Persistent Organic Pollutants Contamination in Plants. Clean: Soil, Air, Water 2014,

454

42, 306-310.

455

(11) Zhang, Q.; Xiao, J.; Sun, Q. Q.; Qin, J. C.; Pescitelli, G.; Gao, J. M.,

456

Characterization of Cytochalasins from the Endophytic Xylaria sp. and Their

457

Biological Functions. J. Agric. Food Chem. 2014, 62, 10962-9.

458

(12) Dimitroula, H.; Syranidou, E.; Manousaki, E.; Nikolaidis, N. P.; Karatzas, G. P.;

459

Kalogerakis, N., Mitigation measures for chromium-VI contaminated groundwater –

460

The role of endophytic bacteria in rhizofiltration. J. Hazard. Mater. 2015, 281,

461

114-120. 21



462

(13) Afzal, M.; Khan, Q. M.; Sessitsch, A., Endophytic bacteria: Prospects and

463

applications for the phytoremediation of organic pollutants. Chemosphere 2014, 117,

464

232-242.

465

(14) Barac, T.; Taghavi, S.; Borremans, B.; Provoost, A.; Oeyen, L.; Colpaert, J. V.;

466

Vangronsveld, J.; van der Lelie, D., Engineered endophytic bacteria improve

467

phytoremediation of water-soluble, volatile, organic pollutants. Nat. Biotechnol. 2004,

468

22, 583-588.

469 470

(15) Chishti, Z.; Arshad, M., Growth Linked Biodegradation of Chlorpyrifos by Agrobacterium and Enterobacter spp. Int. J. Agric. Biol. 2013, 15, 19-26.

471

(16) Xu, G.; Zheng, W.; Li, Y.; Wang, S.; Zhang, J.; Yan, Y., Biodegradation of

472

chlorpyrifos and 3,5,6-trichloro-2-pyridinol by a newly isolated Paracoccus sp. strain

473

TRP. Int. Biodeterior. Biodegrad. 2008, 62, 51-56.

474

(17) Meeker, J. D.; Singh, N. P.; Ryan, L.; Duty, S. M.; Barr, D. B.; Herrick, R. F.;

475

Bennett, D. H.; Hauser, R., Urinary levels of insecticide metabolites and DNA

476

damage in human sperm. Hum. Reprod. 2004, 19, 2573-2580.

477

(18) Andreadis, G.; Albanis, T.; Andreadou, E.; Mitka, S.; Eleftheriou, F.;

478

Lampropoulou, D.; Avramidis, N.; Patoucheas, D., Effect of Dimethoate and

479

Chlorpyrifos in Hepatic and Renal Function of People Belonging to Risk Groups in

480

Iraklia Serres (N. Greece). Br. J. Med. Med. Res. 2014, 4, 949-956.

481

(19) Berent, S.; Giordani, B.; Albers, J. W.; Garabrant, D. H.; Cohen, S. S.; Garrison,

482

R. P.; Richardson, R. J., Effects of occupational exposure to chlorpyrifos on

483

neuropsychological function: A prospective longitudinal study. Neurotoxicology 2014, 22


Page 22 of 35

Page 23 of 35


484

41, 44-53.

485

(20) Rauh, V. A.; Garfinkel, R.; Perera, F. P.; Andrews, H. F.; Hoepner, L.; Barr, D.

486

B.; Whitehead, R.; Tang, D.; Whyatt, R. W., Impact of Prenatal Chlorpyrifos

487

Exposure on Neurodevelopment in the First 3 Years of Life Among Inner-City

488

Children. Pediatrics 2006, 118, e1845-e1859.

489

(21) Ventura, C.; Nunez, M.; Miret, N.; Martinel Lamas, D.; Randi, A.; Venturino, A.;

490

Rivera, E.; Cocca, C., Differential mechanisms of action are involved in chlorpyrifos

491

effects in estrogen-dependent or -independent breast cancer cells exposed to low or

492

high concentrations of the pesticide. Toxicol. Lett. 2012, 213, 184-193.

493

(22) Ma, J.; Chen, S. L.; Moens, M.; Han, R. C.; Clercq, P. D., Efficacy of

494

entomopathogenic nematodes (Rhabditida: steinernematidae and Heterorhabditidae)

495

against the chives gnat, Bradysia odoriphaga. J. Pest. Sci. 2013, 86, 551-561.

496 497

(23) Rashid, S.; Charles, T. C.; Glick, B. R., Isolation and characterization of new plant growth-promoting bacterial endophytes. Appl. Soil Ecol. 2012, 61, 217-224.

498

(24) Holt, J. G.; Krieg, N. R.; Sneath, P. H. A.; Staley, J. T.; Williams, S. T., Bergey's

499

Manual of Determinative Bacteriology. 9th ed. Baltimore, M.D., Lippincott Williams

500

and Wilkins, pp. 1994, 626-640.

501

(25) Li, X.; He, J.; Li, S., Isolation of a chlorpyrifos-degrading bacterium,

502

Sphingomonas sp. strain Dsp-2, and cloning of the mpd gene. Res. Microbiol. 2007,

503

158, 143-149.

504 505

(26) Dunn, A. K.; Handelsman, J., A vector for promoter trapping in Bacillus cereus. Gene 1999, 226, 297-305. 23



506

(27) Ditta, G.; Stanfield, S.; Corbin, D.; Helinski, D. R., Broad host range DNA

507

cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium

508

meliloti. Proc. Natl. Acad. Sci. 1980, 77, 7347-7351.

509

(28) Zhang, X.; Lin, L.; Zhu, Z.; Yang, X.; Wang, Y.; An, Q., Colonization and

510

modulation of host growth and metal uptake by endophytic bacteria of Sedum alfredii.

511

Int. J. Phytorem. 2013, 15, 51-64.

512

(29) Ge, J.; Lu, M.; Wang, D.; Zhang, Z.; Liu, X.; Yu, X., Dissipation and

513

distribution of chlorpyrifos in selected vegetables through foliage and root uptake.

514

Chemosphere 2016, 144, 201-206.

515

(30) Coppotelli, B. M.; Ibarrolaza, A.; Dias, R. L.; Del Panno, M. T.; Berthe-Corti,

516

L.; Morelli, I. S., Study of the Degradation Activity and the Strategies to Promote the

517

Bioavailability of Phenanthrene by Sphingomonas paucimobilis Strain 20006FA.

518

Microb. Ecol. 2010, 59, 266-276.

519

(31) Zhong, Y.; Luan, T.; Lin, L.; Liu, H.; Tam, N. F., Production of metabolites in

520

the biodegradation of phenanthrene, fluoranthene and pyrene by the mixed culture of

521

Mycobacterium sp. and Sphingomonas sp. Bioresour. Technol. 2011, 102, 2965-2972.

522

(32) Li, J.; Liu, J.; Shen, W.; Zhao, X.; Hou, Y.; Cao, H.; Cui, Z., Isolation and

523

characterization of 3, 5, 6-trichloro-2-pyridinol-degrading Ralstonia sp. strain T6.

524

Bioresour. Technol. 2010, 101, 7479-7483.

525

(33) Lu, P.; Li, Q.; Liu, H.; Feng, Z.; Yan, X.; Hong, Q.; Li, S., Biodegradation of

526

chlorpyrifos and 3, 5, 6-trichloro-2-pyridinol by Cupriavidus sp. DT-1. Bioresour.

527

Technol. 2013, 127, 337-342. 24


Page 24 of 35

Page 25 of 35


528

(34) Yadav, M.; Srivastva, N.; Singh, R. S.; Upadhyay, S. N.; Dubey, S. K.,

529

Biodegradation of chlorpyrifos by Pseudomonas sp. in a continuous packed bed

530

bioreactor. Bioresour. Technol. 2014, 165, 265-269.

531

(35) Andreolli, M.; Lampis, S.; Poli, M.; Gullner, G.; Biró, B.; Vallini, G.,

532

Endophytic Burkholderia fungorum DBT1 can improve phytoremediation efficiency

533

of polycyclic aromatic hydrocarbons. Chemosphere 2013, 92, 688-694.

534

(36) Ho, Y. N.; Mathew, D. C.; Hsiao, S. C.; Shih, C. H.; Chien, M. F.; Chiang, H.

535

M.; Huang, C. C., Selection and application of endophytic bacterium Achromobacter

536

xylosoxidans strain F3B for improving phytoremediation of phenolic pollutants. J.

537

Hazard. Mater. 2012, 219-220, 43-49.

538

(37) Chen, W. M.; Tang, Y. Q.; Mori, K.; Wu, X. L., Distribution of culturable

539

endophytic bacteria in aquatic plants and their potential for bioremediation in polluted

540

waters. Aquat. Biol. 2012, 15, 99-110.

541

(38) Wang, Y.; Li, H.; Zhao, W.; He, X.; Chen, J.; Geng, X.; Xiao, M., Induction of

542

toluene degradation and growth promotion in corn and wheat by horizontal gene

543

transfer within endophytic bacteria. Soil Biol. Biochem. 2010, 42, 1051-1057.

544

(39) Taghavi, S.; Barac, T.; Greenberg, B.; Borremans, B.; Vangronsveld, J.; van der

545

Lelie, D., Horizontal gene transfer to endogenous endophytic bacteria from poplar

546

improves phytoremediation of toluene. Appl. Environ. Microbiol. 2005, 71,

547

8500-8505.

548 549 25



550 551

Figure captions

552 553

Fig. 1. Phylogenetic tree based on the 16S rDNA sequences of strain HJY and related

554

species. Bootstrap values obtained with 1000 repetitions are indicated as percentages

555

at all branches. The scale bar represents an evolutionary distance of 0.005. GenBank

556

accession numbers are provided in brackets.

557 558

Fig. 2. Degradation dynamics of chlorpyrifos (CP) by strain HJY and its growth curve

559

in MSM medium (top) and medium enriched with different levels of glucose

560

(bottom).

561 562

Fig. 3. Visualization of inoculated endophyte HJY-gfp within plant tissues. After

563

inoculation with HJY-gfp for 7 days, the colonization of HJY-gfp was observed using

564

confocal scanning laser microscopy (488 nm) in plant roots, stems, cross-section of

565

stems and leaves. Bacteria cells with green fluorescence were displayed as bright

566

green rods inside main roots (F), lateral roots (G), stem epidermis (H), cross-section

567

of stem (I) and leaves (J). Plants without HJY-gfp inoculation served as controls (A, B,

568

C, D, E). The red arrows point to the bacterial aggregates. Scale bar = 70 µm.

569 570

Fig. 4. The residues of chlorpyrifos in uninoculated and inoculated Chinese chives

571

shoots (A), roots (B) and plant soil (C) after 30 d growth with root application at 26


Page 26 of 35

Page 27 of 35


572

different concentrations.

573

HJY-inoculated treatment, * p < 0.05, **p < 0.01.)

(t-test was used to analyze the difference between uninoculated and

574 575

Fig. 5. Dissipation patterns of chlorpyrifos in uninoculated and HJY-inoculated

576

Chinese chives after chlorpyrifos was applied on the surface of Chinese chives at two

577

different concentrations (A, diluted 100 times; B, 1000 times).

578

difference between uninoculated and HJY-inoculated treatment, * p < 0.05, **p < 0.01.)

579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 27


(t-test was used to analyze the


594 595

Figure 1.

596 597

T ） 100 Sphingomonas aeria strain R1-3 （KJ572399）

57

598

Sphingomonas zeae strain JM-791T (KP999966) Sphingomonas azotifigens Y39T（AB217471）） Sphingomonas paucimobilis NBRC 13935T（BBJS01000072））

55

100

Novosphingobium capsulatum GIFU11526（（D16147）） Sphingomonas roseiflava MK341T（D84520））

）T（AB071955） Sphingomonas yabuuchiae GTC 868 ） 71 Strain HJY（（KM985687）） 99

98

Sphingomonas pseudosanguinis G1-2T（AM412238）） Sphingomonas parapaucimobilis NBRC 15100T (BBPI01000114)

86

92 Sphingomonas sanguinis NBRC 13937T (BCTY01000091) Sphingomonas endophytica strain YIM 65583T（HM629444）） Sphingomonas desiccabilis CP1DT（AJ871435）） Sphingomonas sp. ODN7T（FJ194436））

94

Stakelama sediminis strain CJ70（（EU099873））

70

0.005

28


Page 28 of 35

Page 29 of 35


599 600

Figure 2

601

602

603 604

Chlorpyrifos concentration (mg/L)

no glucose

0.5% glucose

20

15

10

5

605 606

29


1% glucose


607 608

Figure 3

609

A: Main root (CK)

F: Main root

B: Lateral root (CK)

G: Lateral root

C: Stem epidermis (CK)

H: Stem epidermis

610

611

612

D: Cross-section of stem (CK)

I: Cross-section of stem

613

30


Page 30 of 35

Page 31 of 35


E: Leaf (CK)

J: Leaf

614 615 616

31



617 618

Figure 4

619 620

*

**

621 622 623

*

624 625

** ** **

** 626

**

*

**

627 628 629 32


Page 32 of 35

Page 33 of 35


630 631

Figure 5

632

633

634 635 636 637 638 33



639 640

TOC graphic

641

642

34


Page 34 of 35

Page 35 of 35


Graphic TOC 47x51mm (300 x 300 DPI)


Isolation, Colonization, and Chlorpyrifos Degradation Mediation of the

Recommend Documents