bioRxiv preprint doi: https://doi.org/10.1101/2021.04.14.439927; this version posted April 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.
1 Isolation, screening, degradation characteristics of a quinclorac-degrading bacteria D and its
2 potential of bioremediation for rice field environment polluted by quinclorac
3 HUANG Siqi PAN Jiuyue TUWANG Mancuo LI Hongyan MA Chenyi CHEN Mingxue LIN
4 Xiaoyan*
5 Rice Quality Supervision and Testing Center, Ministry of Agriculture and Rural Affairs, China Rice Research Institute,
6 Hangzhou 310006, China
7 Running Head: Identification of quinclorac-degrading bacteria
8 Keywords: Quinclorac, Cellulosimicrobium cellulans sp. D, degradation characteristics, field
9 effect evaluation
10 Abstract: Quinclorac (QNC) is a highly selective, hormonal, and low-toxic herbicide with a long
11 duration. And the growth and development of subsequent crops are easily affected by QNC
12 accumulated in the soil. In this paper, a QNC-degrading strain D was isolated and screened from
13 the rice paddy soil. Through morphology, physiological and biochemical tests and 16Sr DNA gene
14 analysis, strain D was identified as Cellulosimicrobium cellulans sp. And the QNC degradation
15 characteristics of strain D were studied. Under the optimal culture conditions, the
16 QNC-degrading rate was 45.9% after culturing for 21 days. The QNC-degrading efficiency of
17 strain D in the field was evaluated by a simulated pot experiment. The results show that strain D
18 can promote the growth of rice and QNC-degrading effectively. This research could provide a
19 new bacterial species for microbial degradation of QNC and lay a theoretical foundation for
20 further research on QNC remediation .
21 Importance
22 At present, some QNC-degrading bacteria have been isolated from different environments, but
23 there are no reports of Cellulosimicrobium cellulans sp. bacterial that could degrade QNC. In this
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24 study, a new QNC-degradation strain was selected from the paddy soil. The degradation
25 characteristics of strain D were studied in detail. The results shown that strain D had a
26 satisfactory quinclorac-degrading efficiency. Two degradation products of QNC by strain D were
27 identified by HPLC-Q-TOF/MS: 3-pyridylacetic acid (138.0548 m/z) and 3-ethylpyridine (108.0805
28 m/z), which have not been reported before.The strain D had a potential ability of
29 quinclorac-degrading effectively in the quinclorac-polluted paddy field environment.
30 Introduction
31 Quinclorac (QNC) is a highly selective hormone and low toxicity herbicide, which is mainly used
32 to control barnyardgrass in rice fields (1). It was widely used because of its good herbicidal effect,
33 wide application period, small application dosage and long-lasting time. According to "Southern
34 Pesticides" report, QNC was one of the top 10 pesticides used in rice herbicides in China, and its
35 annual saled can reach 41.21 million US dollars (2). However, due to the long-lasting usage of
36 QNC, it is easy to accumulate in the soil and affect the growth and development of subsequent
37 crops (3, 4). In Hunan Province, Jiangxi Province, Guangdong Province and other regions of China,
38 the tobacco-rice rotation cropping model was often adopted. When the spray amount of QNC
39 exceeding 25% of the recommended application dose in the rice season, it was harmful to
40 subsequent crops, increased the deformity rate of tobacco leaves and reduced the yield of
41 tobacco (5-7); the residual phytotoxicity of QNC also seriously threatened solanaceae crops,
42 umbelliferae crops and chenopodiaceae crops. After the application of QNC, crops such as
43 potatoes, peppers, carrots, celery, spinach, beets, etc. were sensitive to this herbicide. They
44 would not be planted until at least two years later (8-10). Moreover, the residual QNC in the
45 environment could affect the growth and development of animals adversely, caused abnormal
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46 development or reproductive abnormalities, and further affected the diversity and abundance of
47 natural organisms (11). With the wide application of QNC in rice production, its harmed to
48 animals and plants has attracted more and more attention. Therefore, it is necessary to find a
49 practical and efficient method to degrade QNC residues and solve the environmental pollution
50 problem caused by QNC residues.
51 The current degradation methods of QNC are mainly chemical degradation and biodegradation.
52 Chemical methods mainly include photodegradation and electrodynamic soil cleaning.
53 Photodegradation was achieved by using titanium dioxide to catalyze the photodegradation of
54 QNC in paddy field water and ultrapure water. TiO2 P25 was used to degrade QNC completely in
55 ultrapure water within 40 minutes under the irradiation of 1100 W xenon lamp, however, it took
56 130 minutes for QNC to be completely degraded in paddy water (12, 13). The
57 degradation-products of photodegradation became more complex, and QNC was not yet fully
58 mineralized. The light absorption range of TiO2 P25 is limited to the ultraviolet region of 320-400
59 nm, and ultraviolet light only accounts for 5% of the entire solar spectrum. Therefore, most of
60 the experiments of photocatalytic degradation of pollutants were carried out under the
61 irradiation of high-power xenon lamps or ultraviolet lamps. It required a lot of electrical energy,
62 which limited large-scale experiments. Electrokinetic soil cleaning (14) is one of the most
63 promising technologies for remediation of soil polluted with pesticides. However, during the
64 electrochemical process, the initial pH value of the soil would change, which led to degradation
65 of agricultural soil. In addition, during the electric soil cleaning process, pollutants were removed
66 from the soil and then dissolved in the medium of water. Therefore, additional treatment was
67 required to eliminate pollutants in the water, and this process was tedious and complicated. Due
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68 to the diverse metabolic pathways of microorganisms, extensive substrates and few by-products,
69 microorganisms have broad application prospects in pesticide degradation.
70 At present, some QNC-degrading bacteria have been isolated from different environments.
71 Bordetella sp. HN36 could degrade not only QNC, but also quinoline, phthalic acid, phenol and
72 catechol (15) . The pot experiment shown that Alcaligenes sp. J3 had obvious repairing effect
73 on phytotoxic tobacco (16). Results of the bioremediation experiment of strain Pantoea QC06
74 shown that it also had a significant repair effect on tobacco phytotoxicity, and had certain
75 practical applications for degrading QNC residues in tobacco fields (17). Arthrobacter sp. MC-10
76 could degrade QNC residues in contaminated soil within 7 days (18). Li Yingying et al. shown that
77 the degradation product of this strain Mycobacterium sp. F4 was
78 3-chloro-7-hydroxyquinine-8-carboxylic acid or 7-chloro-3-hydroxyquinine-8-carboxylic acid, and
79 the degradation products was non-phytotoxic to tobacco (19). Lang et al. screened out
80 Streptomyces sp. AH-B through circulating fluidized bed culture and enrichment , the main
81 degradation product were 3-chloro-7-methoxy-8-quinoline carboxylic acid, 3-chloro-7-methyl-8-
82 Quinoline carboxylic acid, 3-chloro-7-oxyethyl-8-quinoline-carboxylic acid, and
83 3,7-dichloro-6-methyl-8-quinoline carboxylic acid (20). Research by Zhou Ting et al. found that
84 the QNC-degrading efficiency of Stenotrophomonas maltophilia J03 in MSM medium was 33.5%
85 after 21 days, and it could effectively alleviate the harm of QNC to tobacco plant growth (21).
86 The QNC-degrading bacteria screened in most of the existing studies are used to remediate
87 phytotoxic tobacco, and there were few studies on the remediation of QNC residual pollution in
88 paddy soil. The purpose of this study is to screen out QNC-degrading bacteria adapted to the
89 paddy field environment, with the hope that it will be widely used in QNC residue-contaminated
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90 paddy fields.
91 Until now, there are no reports of Cellulosimicrobium cellulans sp. bacterial that could degrade
92 QNC. Current research shown that Cellulosimicrobium cellulans sp. bacterial could not only
93 decompose cellulose, alkane powder, gelatin, xylan, and even paraffin, but also fixed nitrogen
94 (22); Liang et al. isolated and screened out the cellulosimicrobium cellulans DGNK-JJ1, which
95 could effectively dissolve potassium. Provided effective strains for the production of
96 potassium-dissolving bacterial fertilizers and contributed to the development of ecological
97 agriculture (23); Ferrer et al. shown that Cellulosimicrobium cellulans sp. bacterial could be used
98 as biocontrol strains for plant diseases (24). These results revealed that this kind of
99 microorganism could be used in fertilizers, plant protection and extraction of protective agents,
100 etc., and had a excellent biological research prospect and development value. And this study
101 found that Cellulosimicrobium cellulans sp. bacterial had a new characteristic of satisfactory
102 QNC-degrading effect. Firstly, in this study, a strain of QNC-degrading bacteria D was isolated
103 and screened from the paddy soil. It was identified as a Cellulosimicrobium cellulans sp. bacteria.
104 Then through experiments, the degradation effect, degradation characteristics and degradation
105 products of this strain were determined. Finally, the actual application of the strain was
106 evaluated by pot simulation test.
107 Results and discussion
108 Identification of strain D
109 In our experiment, a strain that could degrade QNC effectively was obtained through isolation
110 and screening, and named as strain D. The colony was round, umbilical, light yellow, moist,
111 smooth surface and neat edges. The cell was rod-shaped, swollen cysts or spores, thicker capsule,
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112 chain-like arrangement, and gram positive. Its physiological and biochemical properties were
113 shown in Table S1. The glucose, D-ribose, and contact enzyme tests are positive, and the
114 raffinose test is negative. This result was consistent with the description of Cellulosimicrobium
115 cellulans sp.bacterial in the "Berger’s Bacterial Identification Manual" ( 8th edition).
116 Table S1 Physiological and biochemical characteristics of strain D
117 Tests Results Tests Results
β-galactosidase + Mannitol - 118
Arginine - Inositol - 119 Lysine - Sorbitol - 120 Ornithine + L-Rhamnose - Citric acid - sucrose + 121
Hydrogen sulfide - Melibiose - 122 Urease - Amygdalin + 123 lactose - Arabinose + Indole + Oxidase - 124
V. P. + D-ribose + 125 gelatin - Raffinose - 126 glucose + Catalase + teste 127
128
129 Note: "+" means positive and "-" means negative.
130 The 16S rRNA sequence of strain D was uploaded to NCBI for nucleotide sequence comparison，
131 and constructed a phylogenetic tree. Fig. S2 is the 16S rDNA phylogenetic tree of strain D. It can
132 be seen from the Fig. S2, strain D (1384 bp, GenBank accession no. MW404399) and
133 Cellulosimicrobium cellulans DSM 43879 (GenBank accession no. NR_119095) are clustered in
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134 the same branch on the 16S rRNA phylogenetic tree, with homology is 99.93%. Therefore, strain
135 D was identified as Cellulosimicrobium cellulans strain.
136 Cellulosimicrobium cellulans strain DSM 43879（NR 119095） 88 D （MW404399） 99 137 Cellulosimicrobium funkei strain W6122（NR 042937） 67 83 Cellulosimicrobium aquatile strain 3bp（ NR 146008） 138 61 Cellulosimicrobium marinum strain RS-7-4（NR 146662） 40 Cellulosimicrobium terreum strain DS-61（NR 044070） 139 Luteimicrobium subarcticum strain R19-04（ NR 112891） 140 Luteimicrobium album strain RI148-Li105（NR 108122） 58 Luteimicrobium xylanilyticum strain W-15（NR 126237） 141 Isoptericola dokdonensis strain DS-3（NR 043779） 100 142 Isoptericola cucumis strain AP-38（NR 151863） 76 143 Krasilnikoviella flava strain CC 0387（NR 115102）
144 0.0050
145 Fig. S2 Phylogenetic tree of the 16S rDNA of strain D
146 Degradation characteristics of QNC by strain D
147 The effect of initial pH on the degradation of QNC by strain D
148 It can be seen from Fig. S3(A) that there was a poor QNC-degrading effect by strain D in an
149 environment with strong acidity (pH=4) and strong alkaline (pH=10). When the pH of the culture
150 solution was 6, strain D had the best degradation effect in a weakly acidic environment, and the
151 degradation rate was 31.6%. The chemical structure of QNC contains easily hydrolyzable carboxyl
152 groups, with a pH of 4.35, which is weakly acidic (25). QNC is easy to dissociate in alkaline
153 conditions, and acidic conditions can inhibit its dissociation. The suitable pH value for rice growth
154 is 6-7, so QNC is not easy to dissociate naturally in the paddy soil. The strains selected in this
155 study had a better degradation effect than other conditions when the pH value was 6-7. And
156 they are very suitable for degrading QNC in rice fields under natural environment.
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A B
40 40
)
) %
% 30 30
20 20
10 10
Degradation Degradation rate ( Degradation( rate 0 0 D 4 6 7 8 10 -- 20 mg/L 50 mg/L 100 mg/L 200 mg/L 500 mg/L ph QNC Concentration C D
40 40
)
) % % 30 30
20 20
10 10
Degradation Degradation rate ( Degradation Degradation rate (
0 0 1% 2% 5% 7% 10% -- 0 0.1% 0.5% 1% 2% 15# E Inoculation amount F Yeast extract content 40
50
) ) % % 30 40
30 20
20
10 Degradation Degradation rate ( Degradation Degradation rate ( 10
0 0 Urea Peptone NH4CL (NH4)2SO4 NH4NO3 20℃ 25℃ 30℃ 35℃ 40℃ N Source T 157
158 Fig. S3 Factors affecting QNC degradation. A: Effect of initial pH on QNC-degradation by strain D;
159 B: Effect of initial concentration of QNC on QNC-degradation by strain D ; C: Effect of
160 inoculation on the QNC-degradation effect by strain D ; D: Effect of external nitrogen source on
161 QNC-degradation by strain D ; E: Effect of yeast extract content on the QNC-degradation effect by
162 strain D; F: Effect of temperature on QNC-degradation by strain D.
163 The effect of initial QNC on the degradation efficiency of QNC
164 The degradation effect of strain D in the MSM liquid medium with the initial concentration of
165 QNC at 20, 50, 100, 200, 500 mg·L-1 was shown in Fig. S3(B). When the concentration of QNC was
166 50 mg·L-1, strain D had the best degradation effect, and the degradation rate was 33.8%. As the
167 carbon source of the strain, QNC had a great influence on the degradation effect of strain D.
168 Within a certain range of 20-50 mg·L-1, the degradation rate increased with the increase of QNC
169 concentration. But when the concentration was too high, the degradation rate of QNC was at a
170 lower level. The reason might be that the high concentration of physiological toxicity of QNC
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171 produces inhibited the reproduction and metabolism of the strain D. Studied by Lv Zhenmei (26)
172 shown that QNC concentrations exceeding a certain limit would cause oxidative stress to
173 microorganisms.The optimal initial mass concentration of QNC reported is in the range of 5-1000
174 mg·L-1. The Stenotrophomonas maltophilia J03 (21) 21d can degrade 33.5% of QNC at the initial
175 mass concentration of 5 mg·L-1.. And the Burkholderia cepacia WZ1 (26) 5 d can degrade 90% of
176 QNC. But degrading bacteria with low or high optimal QNC concentration are not suitable for
177 common QNC contaminated farmland (375 g·hm-2·a-1). The optimal QNC concentration of strain
178 D isolated by us was closer to the QNC concentration in seriously polluted paddy soil, which is
179 suitable for heavily polluted paddy fields.
180 The influence of the inoculum size on the degradation efficiency of QNC
181 The influence of the inoculation amount on the degradation efficiency of QNC was shown in Fig.
182 S3(C). The growth of microorganisms would be affected by the amount of inoculum, which in
183 turn affected the growth and reproduction of strains. If the amount of inoculation was little, the
184 bacterial culture would have a longer delay time, and then as the amount of inoculation
185 increased, the degradation rate of the strain would show an upward trend. Strain D had the best
186 degradation-effect when the inoculation amount was 7%, and it could continuously and stably
187 degrade QNC. The degradation rate of QNC could reach 32.4% after 21 days of culture. When the
188 inoculum amount exceeded 7%, the degradation rate shown a downward trend, which may be
189 due to the inhibition of competition between bacteria, resulting in insufficient nutrients and
190 affecting the degradation rate of QNC.
191 The effect of N source on the degradation efficiency of QNC
192 The type and content of nutrients affect the degradation ability of microorganisms. By
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193 optimizing the type of N source, the degradation efficiency of strain D could be effectively
194 improved. The effect of different N sources on the degradation efficiency of QNC was shown
195 in Fig. S3(D). It could be seen that the degradation of QNC was different when the culture
196 medium contained different kinds of nitrogen sources. The promotion effect of different N
197 sources on degradation of QNC by strain D was: (NH4)2SO4>NH4NO3>NH4Cl>Urea>Peptone.
198 Therefore, (NH4)2SO4 was selected as the optimal N source. When using (NH4)2SO4 as the N
199 source, strain D had the best QNC-degradation effect with the degradation rate of 38.3% in
200 21 days. When other conditions were the same, the nitrogen source was (NH4)2SO4, the
201 strain degradation rate could be increased by 5.9% to 15.8%. Moreover, (NH4)2SO4 is an
202 excellent nitrogen fertilizer, suitable for general soil and crops. It can make branches and
203 leaves grow vigorously, improve fruit quality and yield, and enhance crop resistance to
204 disasters. It can be used as base fertilizer, top dressing and seed fertilizer, widely used in rice
205 production.
206 The effect of yeast extract content on the degradation efficiency of QNC
207 Yeast extract is rich in protein, amino acids, peptides, nucleotides, B vitamins, and trace elements.
208 It can not only be used as a supplement for N and C sources, but also contains vitamins and
209 micronutrients that are beneficial to the growth of microorganisms (27). Appropriate addition of
210 yeast extract could promote the growth and reproduction of microorganisms and improve the
211 degradation efficiency. The effect of the content of yeast extract on the degradation of QNC by
212 strain D was shown in Fig. S3(E). When the content of yeast extract was 0.1%, strain D had the
213 best degradation effect, with a degradation rate of 35.1%. When yeast extract was not added
214 and when the amount of yeast extract was too much, the QNC-degradation effect of strain D was
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215 not as good as the addition of 0.1% yeast extract. When the amount of yeast extract was 2%, the
216 degradation effect of 21d was the worst. Perhaps the large amount of yeast extract changed the
217 degradation-target of strain D.
218 The effect of temperature on the degradation of QNC by strain D
219 Temperature is an important physical factor affecting the growth and survival of microorganisms.
220 Temperature mainly affected the QNC-degrading effect of strain D by affecting the growth,
221 reproduction and metabolism of microorganisms. The optimal temperature of QNC degrading
222 bacteria had been reported to be between 25℃ and 35℃. In the same other culture conditions, at
223 different temperatures, the degradation of QNC by strain D was shown in Fig. S3(F). It could be
224 seen from the figure that as the culture temperature increased, the ability of strain D to degrade
225 QNC gradually increased. When the culture temperature reached 30℃, the degradation effect
226 was the best, and the degradation rate was 45.9%. Then the degradation effect decreased with
227 increasing temperature. Strain D had a degradation rate of 31.4%-45.9% in an environment
228 between 25 ℃ and 35 ℃, which shown that the strain could adapt to the temperature during rice
229 planting and growth.
230 QNC Degradation of optimal culture conditions
231 In the optimal culture conditions, the initial pH was 6, the initial QNC concentration was 50
232 mg·L-1, the inoculation amount was 7%, the yeast extract content was 0.1%, and the nitrogen
233 source was (NH4)2SO4, the culture temperature was 30℃, and cultured for 21d. The degradation
234 of QNC by strain D and the growth curve were shown in Fig. S4. Strain D could degrade 45.9% of
235 QNC after 21 days of culture. Strain D entered the logarithmic growth phase after 3 days of
236 culture, and entered the stable phase after 18 days. QNC could only be degraded by light and
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237 microorganisms under natural conditions. The QNC-degrading bacteria screened in most of the
238 existing studies were used to repair phytotoxic tobacco, and there were few studies on the repair
239 of QNC residual pollution in paddy soil (17, 28). The purpose of this study was to screen out
240 QNC-degrading bacteria adapted to the paddy field, with the hope that it will be widely used in
241 QNC residue-contaminated paddy fields. The degradation effect of QNC-degrading bacteria was
242 related to many factors, including temperature, pH value, initial substrate mass concentration,
243 inoculum amount, and the degradation ability of the strain, etc (29). In this study, a single factor
244 experiment was used to optimize the degradation conditions of strain D. Finally, it was found that
245 the degradation conditions of strain D were similar to the basic paddy field environment, and
246 strain D could adapt to the paddy field environment.
Content OD
0.5 5
0.4
4
0.3 600
OD Content (mg) Content
3 0.2
0.1
2 0 5 10 15 20 25 247 t (d)
248 Fig. S4 The effect of on the optimal conditions on the QNC-degradation of strain D and growth
249 curve
250 Analysis of degradation products of QNC by strain D
251 The QuEChERS method was used to extract QNC and its degradation products from MSM
252 medium at different culture stages. QNC and its degradation products were detected by
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253 HPLC-Q-TOF/MS. Two possible QNC metabolites had been detected. The mass spectrum and
254 chromatogram of the detected QNC and the two degradation products were shown in Fig. S5.The
255 main degradation products confirmed by mass spectrometry were 3-Pyridylacetic acid (138.0548
256 m/z) and 3-Ethylpyridine, (108.0805 m/z). Due to the lack of standards, degradation products
257 have not been quantified. These products had not been reported in previous studies on the
258 QNC-degradation, and the degradation pathways and enzymes involved are still in study. These
259 compounds were probably new metabolites of QNC degraded by the strain D. Of course, further
260 tests were needed to verify our hypothesis.
261
262
263
264
265 Fig. S5 The mass spectrum and chromatogram of QNC and QNC degradation products. A: The
266 mass spectrum of QNC; B: The mass chromatogram of QNC; C: The mass spectrum of
267 3-Pyridylacetic acid; D: The mass chromatogram of 3-Pyridylacetic acid; E: The mass spectrum of
268 3-Ethylpyridine; F: The mass chromatogram of 3-Ethylpyridine.
269 The effect evaluation of strain D on the degradation of QNC in simulated rice fields conditions
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270 In a potted simulated paddy field environment, the recommended dosage and twice the dosage
271 were applied for soil restoration experiments. In this experiment, part of the soil was sterilized,
272 and added QNC-degrading bacteria to explore the field bioremediation effect of QNC-polluted. In
273 the 28-day pot experiment, the degradation of QNC in the soil in each treatment group was
274 shown in Fig. S6. Experiments on normal soil shown (Fig. S6A and S6B) that strain D can
275 effectively accelerate the degradation of QNC in normal soil. When the QNC concentration was
276 750 g·hm-2·a-1 in the environment, the degradation effect was better. Experiments on sterilized
277 soil shown (Fig.S6C and S6D) that the degradation rate of QNC in the control group without
278 degradation bacteria was significantly slower than that with degradation bacteria. Similarly, in an
279 treatment with a QNC concentration of 750 g·hm-2·a-1, strain D had a stronger degradation-ability
280 of degrading 75.45% of QNC within 21 days. It could be seen that the strain D was very suitable
281 for the seriously polluted rice field environment. By comparing the degradation of QNC in the
282 experimental group in the normal soil and the sterilized soil (Fig.S6E and S6F), it could be found
283 clearly that QNC was degraded faster in the normal soil. There were two possible reasons, First,
284 in the sterilized soil, only the degradation-bacteria D played a degradation-effect, while
285 exogenous additive QNC-degrading strains in the normal soil also played a certain role in
286 promoting the degradation of QNC. When exogenous additive QNC-degrading strains and
287 degradation bacteria D had a synergistic effect, the degradation effect of QNC would be better.
288 Second, after the soil was sterilized, its physical and chemical properties may be changed, and
289 the degradation bacteria couldn't perform better degradation in this condition. Whether the soil
290 was sterilized or not, the degradation bacteria D could accelerate the degradation of QNC, which
291 indicated that degradation bacteria D could adapt to the paddy field environment, effectively
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292 degraded QNC, and repaired QNC-contaminated rice fields.
A 1000 B C 800 600 nCK-1 nCK-2 sCK-1 700 nT-2 nT-1 800 500 sT-1 600 500 600 400
400 300 400 300 200 200 200 100
100 (µg/kg) Concentration
Concentration (µg/kg) Concentration Concentration(µg/kg)
0 0 0 -100 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (d) Time (d) Time (d) D E 800 F nT-2 1000 sCK-2 nT-1 1000 sT-2 700 sT-2 900 sT-1 600 800 800 500 700 600 400 600 300 400 500 200 400
Concentration (µg/kg) 200 100 Concentration (µg/kg) Concentration (µg/kg) Concentration 300 0 0 200 -100 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (d) Time (d) Time (d) 293
294 Fig. S6 The degradation of QNC in the soil. A: In normal soil, the degradation of the
295 recommended dosage; B: In normal soil, the degradation of 2 times the recommended dosage; C:
296 In sterilized soil, the degradation of the recommended dosage; D: In sterilized soil, the
297 degradation of 2 times the recommended dosage; E: Degradation of the experimental group with
298 the recommended dosage in sterilized and normal soil; F: Degradation of the experimental group
299 at 2 times the recommended dose in sterilized and normal soil.
300 The effect evaluation of strain D on the growth of rice plants in simulated rice fields conditions
301 Sensitive dicotyledons may be significantly affected by high concentrations of QNC, such as
302 chlorosis, necrosis, and growth inhibition (30). Although rice had a relatively high resistance to
303 QNC, a higher risk would be brought to the growth of rice seedlings if QNC was used excessively.
304 This study explored the effects of strain D on rice physiology under QNC stress through pot
305 simulation experiments. In the pot simulation experiment, the application of
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306 degradation-bacteria had obvious effects on the plant. After spraying 7d, when the QNC
307 concentration in the pot was at the recommended dose (375 g·hm-2·a-1) (Fig. S7A and S7B) , the
308 rice plant height of the normal experimental groups were 20.5% higher than that of the control
309 group. The fresh stem weight of the sterilized experimental group was 64.3% heavier than that in
310 the sterilized control group. When the QNC concentration in the pot was 2 times the
311 recommended dose (750 g·hm-2·a-1) (Fig. S7C and S7D), only the sterilized and normal
312 experimental groups had significant differences in the dry root weight of rice from the control
313 group. There were obvious differences in the roots of plants, but not in other parts. The reason
314 may be that rice root exudates interacted with rhizosphere soil microorganisms to promote root
315 growth (31). After 21 days, when the QNC concentration in the pot was 2 times the
316 recommended dose (750 g·hm-2·a-1) (Fig.8C and 8D), compared with the control group, the rice
317 plant height, root length and stem fresh weight of the sterilized and normal experimental groups
318 were significantly different after 21 days. However, this situation did not appear on the 7th day of
319 the test. The reason might be that high concentrations of QNC had a greater impact on plant
320 physiology, and QNC-degrading bacteria needed more time to adapt to the environment before
321 they could begin to play a role. And the root dry weight of the normal experimental group was
322 41.4% heavier than that of the sterilized experimental group. This situation was consistent with
323 the previous phenomenon of faster degradation in the normal experimental group. Through the
324 pot experiment, it could be seen that the QNC-degrading bacteria D had a significant
325 bioremediation effect on QNC-polluted soil. It showed that in the QNC-polluted paddy field
326 environment, degrading bacteria D could promote the degradation of residual QNC and reduce
327 its phytotoxicity to rice.
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Fresh stem weight Plant height Stem dry weight) Root length 60 A B Fresh root weight Root dry weight) 1.8 a a 1.6 a 50 1.4 a
b b ）
） 1.2
g （ cm 40 b
1.0 b （
0.8 Weight
Length 30 0.6 a a a 10 b 0.4 a a a b b a 0.2 b a b b c b 0 0.0 sCK-1 sT-1 nCK-1 nT-1 sCK-1 sT-1 nCK-1 nT-1 Group Group C Plant height D 1.0 Fresh stem weight Root length Stem dry weight 50 Fresh root weight Root dry weight
0.8 ）
） 40
0.6
cm
cm
（ （
30 0.4
Weight Length
10 0.2
a b a b 0 0.0 sCK-2 sT-2 nCK-2 nT-2 sCK-2 sT-2 nCK-2 nT-2 Group Group 328 329 Fig. S7 The growth of rice plants in simulated rice fields conditions in 7d. Note that different
330 letters indicate significant differences at the 0.05 level. A : plant height and root length of
331 experimental group and control group at recommended dose (375 g·hm-2·a-1); B: fresh stem
332 weight, stem dry weight, fresh root weight and root dry weight of experimental group and
333 control group at recommended dose (375 g·hm-2·a-1); C: plant height and root length of
334 experimental group and control group at 2 times recommended dose (750 g·hm-2·a-1); D: fresh
335 stem weight, stem dry weight, fresh root weight and root dry weight of experimental group and
336 control group at 2 times recommended dose (750 g·hm-2·a-1).
337
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Fresh stem weight Plant height B 70 A 10 Stem dry weight Root length a Fresh root weight a Root dry weight a b a 60 c 8 b
） 50 b
） 6
g
cm
（ （ a 40 a
20 4 Weight
Length a b c b a a b 10 2 b a a c c b a 0 0 sCK-1 sT-1 nCK-1 nT-1 sCK-1 sT-1 nCK-1 nT-1 Plant height Group D Group Fresh stem weight 70 C Root length a a Stem dry weight 12 Fresh root weight b Root dry weight 60 b c 10 b
50 b ）
） 8
cm
cm
（ （ 40 a 6 a
a c a Length
20 Weight b c 4 b b 10 a 2 a a b b b b a 0 0 sCK-2 sT-2 nCK-2 nT-2 sCK-2 sT-2 nCK-2 nT-2 Group Group
338 339 Fig. S8 The growth of rice plants in simulated rice fields conditions in 21d. Note that different
340 letters indicate significant differences at the 0.05 level. A : plant height and root lenght of
341 experimental group and control group at recommended dose (375 g·hm-2·a-1); B: fresh stem
342 weight, stem dry weight, fresh root weight and root dry weight of experimental group and
343 control group at recommended dose (375 g·hm-2·a-1); C: plant height and root height of
344 experimental group and control group at 2 times recommended dose (750 g·hm-2·a-1); D: fresh
345 stem weight, stem dry weight, fresh root weight and root dry weight of experimental group and
346 control group at 2 times recommended dose (750 g·hm-2·a-1).
347 Since it was discovered that QNC is mainly degraded by microorganisms in nature, many kinds of
348 QNC degrading bacteria had been screened out, but the researchers only studied its degradation
349 characteristics, etc. There were no relevant reports on the bioremediation of QNC-contaminated
350 rice fields by QNC-degrading bacteria, and no reference basis for the practical application of
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351 degrading bacteria in the future. In this experiment, the high-efficiency degrading bacteria
352 selected for QNC were used and potted simulated rice fields were used to study the
353 bioremediation of QNC-contaminated rice fields. The test results shown that the
354 QNC-degradation bacteria D could be adapted to the rice field environment and the rice field soil
355 bacteria could degrade QNC cooperatively. The good repair effect can obviously reduce the
356 negative influence of QNC on rice plant physiology, showing a excellent potential broad prospect
357 for application.
358 materials and methods
359 Materials
360 QNC standard (98%, Shanghai Yuanye Biological Company); QNC Wettable powder (50%, Jiangsu
361 Kuaida Agrochemical Co., Ltd.); LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, pH
362 7.0, 1000 mL distilled water; LB solid medium: 10 g tryptone, 5 g yeast extract, 10 g Nacl, 20 g
363 agar, pH 7.0, 1000 mL distilled water; MSM liquid medium: MgSO4·7H2O 0.2g, CaCl2 0.02g,
364 KH2PO4 1.0g, Na2HPO4 1.0g, FeCl3 0.05g, NH4NO3 1.0g, 1000 mL distilled water; rice paddy soil
365 and rice seeds are all acquired from China Rice Research Institute.
366 Screening of QNC-degrading strains
367 5g paddy soil sample was added to 100mL MSM liquid medium with QNC of 100 mg·L-1. After
368 incubating at 30°C and 180 rpm for 7 days, 5 ml of the medium was transferred to new MSM
369 liquid medium with QNC of 200 mg·L-1 for cultivation. After 7 days, repeated the transfer until the
370 concentration of QNC in MSM medium reached 500 mg·L-1. The final culture medium was
371 gradient diluted and inoculated on LB solid medium. After culturing at 30°C for 48 hours, a single
372 colony was picked and streaked on the LB solid medium to obtain single colonies. Subsequently,
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373 different single colonies were selected and cultured in MSM liquid medium with 100 mg·L-1 QNC
374 for 21 days, and then QuEChERS-liquid chromatography-tandem mass spectrometry was used to
375 extract QNC and determine the residual concentration of QNC (32) to select the candidate
376 dominant strain.
377 Identification of QNC-degrading strains
378 The selected strain was inoculated on LB solid medium and cultured at 30°C for 48 hours to
379 observe the morphological characteristics of the colony. Gram stain was employed to identify
380 the gram-positive or gram-negative of the strain and the morphology of bacteria was observed
381 under a scanning electron microscope. Physiological and biochemical kits (Qingdao Haibo
382 Biological Co., Ltd.) were used for physiological and biochemical identification. "Berger’ Bacterial
383 Identification Manual" was referred for the identification. After strain D were activated, the
384 BigDye Teminator V3.1 Cycle Sequencing Kit (ABI, USA) was used to extract the total DNA of
385 strain D. 1% agarose gel electrophoresis was used to detect DNA extraction efficiency. A couple
386 of bacterial universal primer (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' / 1492R:
387 5'-TACCTTGTTACGACTT-3') was used. Amplification reaction system was: DNA 1 μL, BigDye 8 μL,
388 primer (3.2 pmol·L-1) 1 μL, sterile deionized water 10 μL. Reaction conditions was as followed:
389 firstly, pre-denaturation at 96 ℃ for 1 min; secondly, denaturation at 96 ℃ for 10 s, annealing at
390 50 ℃ for 5 s, extension at 60 ℃ for 4 min, 25 cycles; finally, storage at 4 ℃. Amplification products
391 were submitted to Shanghai Shangya Biotechnology Co., Ltd. for sequencing. The sequencing
392 results were uploaded to the NCBI database, and the homology comparison was performed by
393 Blast in GenBank (http:/www.ncbi.nlm.nih.gov). The phylogenetic analysis was carried out by
394 MEGA7.0 software, and the phylogenetic tree was constructed by the Neighbor-Joining method.
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395 The degradation characteristics of QNC by strain D
396 Preparation of bacteria suspension
397 Strain D was inoculated into 100 ml liquid LB medium, incubated at 30°C, 180 rpm for 48 hours,
398 centrifuged at 8000 rpm for 5 minutes, and then poured off the upper layer of LB. The
399 precipitated strain was washed twice with sterile physiological saline solution, and then
400 resuspended the pellet in MSM liquid medium (OD600=1.0) for later use.
401 Effects of different culture conditions on the degradation rate of QNC
402 The effects of pH, initial QNC, inoculum amount, yeast extract content, N source and
403 temperature on QNC degradation were investigated. The pH of the culture solution was adjusted
404 to 4, 6, 7, 8, 10 respectively, and culture was performed at 30 ℃. MSM liquid medium was made
405 up with QNC concentration of 20, 50, 100, 200, and 500 mg·L-1, respectively,cultured at 30 ℃. The
406 initial Inoculation amount of the culture solution was set to 1%, 2%, 5%, 7% and 10%,
407 respectively, cultured at 30 ℃. Add 0, 0.1%, 0.5%, 1% and 2% of yeast extract to investigate the
408 effect on QNC degradation. Urea, peptone, (NH4)2SO4, NH4NO3 and NH4CL was added
409 individually to cultures at 30 ℃ to assess the effect of the N source. The medium was cultured at
410 20 ℃, 25 ℃, 30 ℃ ,35 ℃ and 40 ℃ to assess the effect of temperature. Samples were taken at 3, 7,
411 12, 15, 18 and 21 days after inoculation to determine the concentration of QNC and calculate
412 the degradation rate. Three parallels were set for each treatment, and the treatment without
413 inoculation was set as the control.
414 Degradation of QNC by strain D in optimal culture conditions
415 The MSM liquid medium with a QNC concentration of 50 mg·L-1 was prepared, adjust the pH to 6.
416 The N source of MSM medium was (NH4)2SO4, added with 0.1% yeast extract powder, and 7%
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417 bacterial suspension. The culture condition of MSM medium was 30 ℃, and 180 rpm. Samples
418 were taken at 3, 7, 12, 15, 18 and 21 days after inoculation to determine the concentration of
419 QNC and calculate the degradation rate. And the OD(600) value of the bacterial suspension was
420 determined by using an ultraviolet spectrophotometer (UV2600，Shimadzu，Japan) . Three
421 parallels were set for each treatment, and the treatment without bacteria was set as the control.
422 Determination of degradation products
423 Strain D was cultured in optimal conditions for 21d, and samples were taken at 3, 7, 12, 15, 18
424 and 21 days after inoculation. The QuEChERS method was used to extract QNC and its
425 degradation products. The degradation products of QNC were detected by high performance
426 liquid chromatography tandem quadrupole-time-of-flight mass spectrometer (HPLC-Q-TOF/MS,
427 Agilent, USA). The HPLC conditions were as follows: Xselect HSS T3 column (American waters, 2.1
428 mm × 150 mm × 3.5 μm); column temperature, 45°C; aqueous solution containing 0.1% formic
429 acid (v/v) (A) and acetonitrile (B) as the flow Phase, gradient elution is 0-5 min, 10%-40% B; 5-11
430 min, 40%-95% B; 11-15 min, 95% B; 15-15.1 min, 95%-10% B ; 15.1-21 min, 10% B; flow rate 300
431 μL·min-1, injection volume is 5 μL. Mass spectrometry conditions: The electrospray source used in
432 the mass spectrometer is ESI, using positive and negative ion full scan modes, the mass scan
433 range m/z 50-1200, and the mass scan rate is 4 spectra·s-1. The parameters of positive and
434 negative ion scanning are set as follows: drying gas temperature 350℃, drying gas flow rate 8
435 L·min-1, sheath gas temperature 280℃, sheath gas flow rate 11 L·min-1, atomizing gas pressure 40
436 psig, fragmentation voltage 130 V, cone The skimmer voltage is 40 V, and the RF voltage is 750 V.
437 Positive ion mode: capillary voltage 4000 V, nozzle voltage 500 V; negative ion mode: capillary
438 voltage 3500 V, nozzle voltage 1000 V.
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439 The evaluation of strain D on the degradation of QNC and the growth of rice plants in
440 simulated QNC-contaminated rice fields
441 Pre-treatment for pot experiment
442 Soil: Paddy soil was dried and sieved (2mm), and part of the soil was sterilized; Strains: strain D
443 was inoculated in LB liquid medium, cultured at 30°C, 180 rpm for 48 h, then washed twice with
444 physiological saline, resuspended in steriled water to obtain a bacterial suspension for later use;
445 Pesticide preparation: According to the recommended dosage of QNC and 2 times the
446 recommended dosage, the mother liquor of QNC with an active ingredient of 375 g·hm-2·a-1 and
447 an effective ingredient of 750 g·hm-2·a-1 was prepared for use; Rice seeds: the rice seeds were
448 sterilized and soaked to accelerate germination, and cultivated at the two-leaf one-heart stage in
449 an artificial incubator. Rice seedlings of uniform growth were selected for transplanting.
450 Pot experiment
451 Selected rice seedlings that grow consistently and robust were transplanted into potting buckets
452 (25cm×20cm) with 6 kg of soil in each bucket. After 7 days of transplanting, 8 treatments were
453 carried out respectively: nCK-1: normal soil, sprayed QNC according to the recommended dosage
454 (375 g·hm-2·a-1). nT-1: normal soil, sprayed QNC according to the recommended dosage, added
455 the bacterial suspension and mixed evenly to make the concentration of 3.38×108 CFU·g-1; nCK-2:
456 normal soil, applied twice of the recommended dosage (750 g·hm-2·a-1). nT-2: normal soil, apply
457 2 times of the recommended dose, and added the bacterial suspension and mixed evenly to
458 make the concentration of 3.38×108 CFU·g-1; sCK-1: sterilized soil, applied the recommended
459 dose. sT-1: sterilized soil, applied QNC according to the recommended dose, added the bacterial
460 suspension and mixed evenly to make the concentration of 3.38×108 CFU·g-1; sCK-2: sterilized
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461 soil, applied twice of the recommended dose. sT-2: sterilized soil, apply 2 times of the
462 recommended dose, added the bacterial suspension and mixed evenly to make the
463 concentration of 3.38×108 CFU·g-1. Each treatment was repeated for three times. After
464 application, random multi-point sampling was used at 0, 3, 7, 14, 21, and 28d to collect rice
465 plants and soil samples. Soil samples were employed to detect QNC residues. The plant height,
466 root length, stem dry weight, stem fresh weight, root dry weight and root fresh weight of rice
467 plant samples collected at 7d and 21d were measured respectively.
468 Data availability
469 The complete 16S rDNA sequence of Cellulosimicrobium sp D have been deposited in GenBank
470 under the accession no. NR_119095.
471 Acknowledgements
472 This work was founded by the Special Fund for the Construction of Modern Agricultural Industrial
473 Technology System (CARS-01-47), Collaborative Innovation Task of the Science and Technology
474 Innovation Project of the Chinese Academy of Agricultural Sciences: Product Creation and
475 Demonstration Application of Quick Test Product Quality and Safety of Agricultural Products
476 (CAAS-XTCX2019024).
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