Baseline sensitivity of natural populations and resistance of mutants of Xanthomonas oryzae pv. oryzae to a novel bactericide, zinc thiazole

Authors


Abstract

During 2009–2010, a total of 323 isolates of Xanthomonas oryzae pv. oryzae were obtained from rice with symptoms of bacterial leaf blight (BLB) in four provinces (Zhejiang, Jiangsu, Anhui and Hubei) in China. These isolates were tested for baseline sensitivity to zinc thiazole, a novel bactericide with strong antibacterial activity against Xanthomonas. The sampled pathogenic population had similar sensitivity to zinc thiazole (0·1–16·8 mg L−1) in all four regions and over the whole two-year study period. The baseline sensitivity was distributed as a unimodal curve with a mean EC50 value of 6·79 ± 1·61 mg L−1. The risk of mutation to resistance of zinc thiazole in X. oryzae pv. oryzae was further evaluated in vitro and in vivo. Twelve zinc thiazole-resistant mutants were obtained through ultraviolet (UV) irradiation, culturing on zinc thiazole-amended nutrient agar (NA) plates, and culturing on zinc thiazole-treated rice plants. These zinc thiazole-resistant mutants had resistance factors (RF = EC50 value of a mutant / EC50 value of the wildtype parent of this mutant) of 12·4 to 186·1 with a mean RF value of 44·1. Mutants obtained via UV irradiation, culturing on NA plates and culturing on rice plants had mean RF values of 51·8, 24·5 and 14·4, respectively. All mutants showed decreases in resistance to zinc thiazole after 20 successive transfers on bactericide-free media or 10 successive inoculation–reisolations on bactericide-free rice plants. No significant difference was found in bacterial growth and sensitivity to bismerthiazol between zinc thiazole-resistant mutants and their parents. However, a significant decrease was observed in the pathogenicity of zinc thiazole-resistant mutants compared with their parents, especially for mutants obtained via UV irradiation.

Introduction

Bacterial leaf blight of rice (BLB) caused by Xanthomonas oryzae pv. oryzae is one of the most important rice diseases and is responsible for heavy rice production losses (Mew et al., 1993; Jeung et al., 2006). Under conducive climate conditions, BLB causes at least 10% yield loss in susceptible rice varieties (Chen et al., 2004; Salzberg et al., 2008). When plants are infected at the maximum tillering stage, the yield can be reduced by 20–40% (Ou, 1985). BLB is a vascular disease caused by systemic infection. Early BLB infection can cause up to 50% yield reduction of rice (Osamu, 1973; Ou, 1985). Application of bactericides is an indispensable strategy for control of BLB, especially in regions where susceptible rice cultivars are frequently planted (Zhang et al., 2005, 2010).

Since the 1970s, bismerthiazol (N,N′-methylene-bis (2-amino-5-mercapto-1, 3, 4-thiadiazole) has been the most commonly used bactericide in China. It is mainly used for the control of Xanthomonas diseases such as BLB, rice bacterial leaf streak caused by X. oryzae pv. oryzae, and citrus canker caused by X. campestris pv. citri (Ma et al., 1997; Shen & Zhou, 2002; Huang et al., 2003). However, bismerthiazol-resistant strains of X. oryzae pv. oryzae have been found commonly in rice fields in China (Wang et al., 1998; Shen & Zhou, 2002). Other existing bactericides such as copper-based bactericides and antibiotics are not applicable for the control of BLB because of poor effectiveness, high phytotoxicity, and/or resistance (Shen & Zhou, 2002; Zhang et al., 2010). Therefore, it is urgent to assess and apply alternative bactericides for management of BLB.

Zinc thiazole (IUPAC name: bis (2-amino-5-mercapto-1, 3, 4 thiadiazole) zinc, CAS no. 1202750-31-9; Fig. 1) is a novel bactericide showing strong antibacterial activity against Xanthomonas spp. (Wei et al., 2007, 2008; Li et al., 2008). The bioactivity of zinc thiazole against important plant-pathogenic bacteria was as following: X. oryzae pv. oryzae X. axonopodis pv. citri > X. oryzae pv. oryzicola > Pseudomonas syringae pv. lachrymans > Ralstonia solanacearum > Erwinia carotovora subsp. carotovora > Acidovorax avenae subsp. citrulli (authors' own unpublished data). Zinc thiazole is protected by patents such as ZL001321196, ZL2007101061835 and WO/2008/151513. Zinc thiazole can easily be degraded in different soils with the DT50 (time taken for 50% of the concentration to dissipate) <20 days and is a low-toxic chemical. In acute toxicity studies, zinc thiazole had an oral LD50 of >5000 mg kg−1 and a dermal LD50 of >5000 mg kg−1. Twenty percent zinc thiazole SC has been registered for control of rice bacterial leaf streak and citrus canker in China (registration no. PD20096932) by Zhejiang Xinnong Chemical Co. Ltd. Prior to the wide application of a new chemical bactericide, it is important to establish the baseline level of bactericide sensitivity for the specific pathogen in a representative region (Russell, 2007). However, little is known about the sensitivity baseline and resistance mutation risk of Xanthomonas to zinc thiazole. The objectives of this research were to: (i) determine the baseline sensitivity of natural populations of X. oryzae pv. oryzae to zinc thiazole, (ii) generate and characterize mutants of X. oryzae pv. oryzae resistant to zinc thiazole, and (iii) analyse the cross-resistance relationships between zinc thiazole and other bactericides.

Figure 1.

Chemical structure of zinc thiazole (bis (2-amino-5-mercapto-1, 3, 4 thiadiazole) zinc, CAS no.1202750-31-9).

Materials and methods

Bactericides

Zinc thiazole (20% SC) and bismerthiazol (technical grade 92·5%) were provided by Xinnong Chemical Co. Ltd and Longwan Agrichemical Co. Ltd, respectively.

Isolation of Xanthomonas oryzae pv. oryzae

From 2009 to 2010, rice leaves with BLB symptoms were collected in Zhejiang, Jiangsu, Anhui and Hubei Provinces, China (Table 1). All the rice leaves were surface-sterilized with 0·5% sodium hypochlorite for 3 min, and rinsed in sterile distilled water for 30 s. A leaf extract was prepared by crushing each leaf with a sterile glass rod. The extract was then spread on nutrient agar (NA; polypeptone 5 g, yeast powder 1 g, beef extract 3 g, sucrose 10 g, agar 17 g, distilled water 1 L) plates, and incubated at 28°C for 5–7 days. Single-colony isolation of X. oryzae pv. oryzae was done for each leaf, and a total of 323 isolates were obtained. Each isolate was identified by colony morphology and pathogenicity assay. Zinc thiazole had not been used in the source regions for isolation of X. oryzae pv. oryzae until sampling. Therefore, none of the isolates had ever been exposed to this bactericide.

Table 1. Origin and baseline sensitivity of field isolates of Xanthomonas oryzae pv. oryzae to zinc thiazole
YearProvinceNumber of isolatesCodeEC50 (mg L−1)a
RangeMeanSDb
  1. a

    EC50, effective concentration for 50% of growth inhibition.

  2. b

    SD: standard deviation.

  3. c

    Statistical analysis was conducted with region (province) as a factor and isolates as replications. Mean values followed by the same letter within the same column were not significantly different with LSD (least significant difference) test at = 0·05.

2009Zhejiang65ZJ09-0·2–14·76·32 ac1·56
Jiangsu27JS09-0·9–16·85·77 a1·58
Anhui63AH09-0·2–16·67·06 a1·61
2010Zhejiang40ZJ10-0·1–15·35·99 a0·97
Jiangsu48JS10-0·7–15·26·02 a0·73
Anhui36AH10-0·3–14·47·90 a1·22
Hubei44HB10-1·2–14·08·36 a1·59
Total 323 0·1–16·86·791·61

Inoculation of rice plants with Xanthomonas oryzae pv. oryzae

Rice plants of cultivar Shanyou 63, which is highly susceptible to X. oryzae pv. oryzae infection, were grown in a glasshouse under natural conditions. Xanthomonas oryzae pv. oryzae isolates incubated in NB broth at 28°C to the late logarithmic growth phase were used to inoculate rice plants using the leaf clipping method (Kauffman et al., 1973). Five leaves per plant were inoculated with bacterial suspension (109 colony-forming units (CFU) mL−1), with water inoculated as the negative control. For each isolate, five plants were inoculated and kept in chambers at 30 ± 4°C with >95% relative humidity for 24 h, and then transferred into chambers with conditions for disease development (28°C, 90% relative humidity, 12-h light/dark cycle). Plant symptoms were evaluated by measuring the lesion length of infected leaves 12–15 days after inoculations. The experiments were performed twice.

Baseline sensitivity of Xanthomonas oryzae pv. oryzae to zinc thiazole

The sensitivity of X. oryzae pv. oryzae isolates to zinc thiazole was determined based on in vitro inhibition of bacterial growth (Li et al., 2006). Briefly, each isolate was grown in NB broth at 28°C until the late logarithmic growth phase, and then the suspension was diluted to approximately 107 CFU mL−1. Aliquots (100 μL) of the suspension were added to 25 mL NB in 50-mL Erlenmeyer flasks containing various concentrations of zinc thiazole (0, 0·75, 1·5, 3, 6 and 12 mg L−1). The inoculated flasks were placed on an orbital shaker (28°C, 170 rpm), with bacterial cell density checked at 12 h intervals for the flask with 0 mg L−1 zinc thiazole. When the cell density of the suspension increased to approximately 108 CFU mL−1 in this flask, the optical density (OD600 nm) was measured for bacterial suspensions in all flasks with a WCY-WOG Baoli nephelometer. Each treatment concentration was prepared in triplicate and the experiment was performed twice. A regression equation was derived by correlating the log of inhibitor concentration of zinc thiazole and the probit of inhibition percentage of average OD value of X. oryzae pv. oryzae. The effective concentration for 50% inhibition (EC50) of X. oryzae pv. oryzae was calculated from the regression equation for each of the 323 isolates. A histogram of EC50 was plotted for all these isolates, and the shape of frequency distribution was analysed by examining the EC50 regarding curve shape, range and mean values, as well as the ratio of highest to lowest values.

Generation of zinc thiazole-resistant mutants of Xanthomonas oryzae pv. oryzae

The isolates ZJ09-6, AH10-21, JS10-16 and HB10-33 were randomly selected from the four provinces to generate zinc thiazole-resistant mutants through UV irradiation (method I), zinc thiazole culturing in NA plates (method II) and zinc thiazole culturing in rice plants (method III). In method I, bacterial broth cultures at the logarithmic growth phase (107 CFU mL−1) were treated with UV light (20 W, 254 nm) at 20 cm in vertical distance for 5 min at 28°C, followed by 1 h incubation in the dark to avoid light repairing of DNA damage. Then, 100 μL of the UV-treated bacterial suspensions were poured onto NA plates amended with 50 mg L−1 zinc thiazole, with untreated bacterial suspensions of the same isolates inoculated to control NA plants. Single colonies expanding on the zinc thiazole-containing NA plates were obtained as resistant mutants. In method II, 100 μL of bacterial suspensions were poured onto NA plates amended with 10 mg L−1 zinc thiazole, and then transferred to a series of replicate NA plates with increasing zinc thiazole concentrations at each transfer: 10, 20, 40, 50 and 100 mg L−1. Colonies that survived the highest or final concentration of zinc thiazole were transferred to NA plates amended with 100 mg L−1 zinc thiazole to confirm their resistance. In method III, an isolate was first inoculated onto the rice cultivar Shanyou 63 treated with 0 and 50 mg L−1 zinc thiazole, respectively. After 12–15 days, reisolation was carried out from disease lesions, and the isolates obtained were inoculated onto rice leaves treated with 80 mg L−1 zinc thiazole. The inoculation–reisolation of X. oryzae pv. oryzae isolates was then performed with rice plants treated with 300 mg L−1 zinc thiazole, and zinc thiazole-resistant mutants were selected thereafter. The experiments were performed three times.

Characterization of zinc thiazole-resistant mutants of Xanthomonas oryzae pv. oryzae

Resistance factor

The sensitivity of zinc thiazole-resistant mutants and their sensitive parental isolates were determined as described above. Resistance factor (RF) was calculated for all mutants using the formula RF = EC50R/ EC50P, where EC50R is the EC50 value of the mutant being examined, and EC50P is the EC50 value of the wildtype parent of this mutant. The experiments were performed in triplicate.

Resistance stability

Resistance stability of the mutants was assayed in vitro and in vivo. For the in vitro test, mutants were grown on bactericide-free NA plates in five replicates and transferred to fresh NA plates after 2 days' incubation at 28°C. This procedure was repeated until a 20th transfer had been done. EC50 values were determined for the first and 20th subcultures, and the experiment was performed twice. For the in vivo test, mutants were inoculated on untreated rice plants. After 12–15 days, reisolation was conducted from disease lesions and inoculated to fresh plants. This procedure was repeated until a 10th inoculation had been done, and EC50 values were determined for the first and 10th subcultures. The experiments were performed twice.

Growth ability

The mutants and wildtype parents were grown in NB broth at 28°C until late logarithmic growth phase, and then the suspension was diluted to approximately 107 CFU mL−1. Aliquots (100 μL) of the suspension were added to 25 mL NB in 50-mL Erlenmeyer flasks. After 12 h incubation at 28°C on a reciprocal shaker, the OD600 was measured for bacterial suspensions in all flasks (Huang et al., 2003).

Pathogenicity

Rice plants were inoculated as described above. The lengths of lesions on the inoculated leaves were measured after 12–15 days to assess pathogenicity. The experiments were performed in triplicate.

Sensitivity to bismerthiazol

The sensitivity of X. oryzae pv. oryzae isolates to bismerthiazol, the only bactericide presently applied for BLB management in China, was determined following the same method described for the assay of bacterial sensitivity to zinc thiazole. The experiments were performed twice.

Data analysis

Multiple comparisons (least significant difference, LSD) were used to examine differences in the mean values of EC50 and pathogenicity between zinc thiazole treatments of concentrations. Statistical tests were carried out using spss v. 11.0.

Results

Baseline sensitivity of Xanthomonas oryzae pv. oryzae to zinc thiazole

In this study, a total of 323 single-colony isolates of X. oryzae pv. oryzae were obtained from rice plants with BLB symptoms in four provinces in China. Their sensitivity to zinc thiazole was tested and then compared with respect to geographical origin and over the two-year time period (Table 1). There was no evidence of geographical variation in the sensitivity of X. oryzae pv. oryzae to zinc thiazole, and the sensitivity of tested isolates remained unchanged from 2009 to 2010.

The frequency distribution of the EC50 values of 323 isolates was a unimodal curve (Fig. 2), ranging from 0·1 to 16·8 mg L−1,which represented a range-of-variation factor of 168. The mean EC50 value was 6·79 ± 1·61 mg L−1. There was no resistant subpopulation among the isolates used in the study. Thus, these sensitivity data could be used as a baseline for monitoring the shift of sensitivity of X. oryzae pv. oryzae populations to zinc thiazole.

Figure 2.

Frequency distribution of effective concentration of zinc thiazole for 50% inhibition of growth (EC50) of Xanthomonas oryzae pv. oryzae isolates. In total, 323 isolates were collected from different provinces in China that had never been exposed to zinc thiazole. Points shown are number at specific EC50 value.

Generation of zinc thiazole-resistant mutants of Xanthomonas oryzae pv. oryzae

The proportion of surviving X. oryzae pv. oryzae from UV treatment was approximately 10%. A total of nine resistant mutants, designated ZJ-UR-2, ZJ-UR-3, AH-UR-3, AH-UR-7, AH-UR-12, JS-UR-1, JS-UR-5, HB-UR-4 and HB-UR-11, were obtained via UV irradiation from the wildtype parental isolates of ZJ09-6, AH10-21, JS10-16 and HB10-33. Two mutants, designated ZJ-TR-1 and AH-TR-2, were obtained by culturing on zinc thiazole-amended media from ZJ09-6 and AH10-21, respectively. One mutant, designated HB-PR-1, was obtained from HB10-33 by culturing on zinc thiazole-treated rice plants (Table 2). The 12 zinc thiazole-resistant mutants had RF values of 12·4 to 186·1. Among them, AH-UR-7 showed the highest resistance level (RF = 186·1; Table 2). Mutants from UV irradiation, zinc thiazole culturing in NA plates, and zinc thiazole culturing in rice plants had mean RF values of 51·8, 24·5 and 14·4, respectively.

Table 2. Stability of sensitivity of zinc thiazole-resistant mutants of Xanthomonas oryzae pv. oryzae and their parents during subculture on fungicide-free medium and plants
IsolateEC50 (mg L−1)Resistance Factor (RF)EC50 (mg L−1) at the:RF, FSCa at the:
20th transfer10th inoculation20th transfer10th inoculation
  1. a

    FSC: factor of sensitivity change; FSC = RF at the 20th transfer or 10th inoculation/ the original RF.

  2. b

    Isolates in bold are parents of the isolates listed under them.

  3. c

    Multiple comparisons were performed within a column under each parent. Mean values followed by the same letter were not significantly different with LSD (least significant difference) test at = 0·05.

ZJ09-6 b 2·1 dc2·0c2·3 c
ZJ-UR-267·5 b32·135·8 b47·2 b17·9, 0·5620·5, 0·64
ZJ-UR-395·2 a45·366·1 a58·9 a33·1, 0·7325·6, 0·57
ZJ-TR-135·1 c16·728·1 b33·7 b14·1, 0·4014·7, 0·42
AH10-21 0·9 e1·0 c0·8 e
AH-UR-346·2 c51·321·2 b32·6 c21·2, 0·4140·8, 0·80
AH-UR-7167·5 a186·156·8 a98·0 a56·8, 0·31122·5, 0·66
AH-UR-1277·3 b85·953·2 a49·7 b53·2, 0·6262·1, 0·72
AH-TR-229·1 d32·320·7 b26·2 d20·7, 0·6432·8, 0·81
JS10-16 5·3 b_5·8 c6·1 c
JS-UR-178·6 a14·839·9 a27·5 b6·9, 0·474·5, 0·30
JS-UR-565·7 a12·423·6 b39·0 a4·1, 0·336·4, 0·52
HB10-33 4·2 c4·0 c4·5 c
HB-UR-488·0 a21·038·4 b65·2 a9·6, 0·4614·5, 0·69
HB-UR-1171·4 b17·045·1 b70·9 a11·3, 0·6615·8, 0·93
HB-PR-160·3 b14·451·0 a48·2 b12·8, 0·8910·7, 0·74

Characterization of zinc thiazole-resistant mutants of Xanthomonas oryzae pv. oryzae

Resistance stability

All mutants showed decreases in resistance to zinc thiazole after 20 successive transfers on bactericide-free media or 10 successive inoculation–reisolations on bactericide-free rice plants. The mean RF values were 44·1 for the 12 original mutants. The mean RF value of the 20th subculture on bactericide-free media was 21·8 with a mean factor of sensitivity change (SCZ, the ratio of RF of the 20th transfer or 10th inoculation to the original RF) of 0·54. The mean RF value was 30·9 after 10 successive inoculation–reisolations on bactericide-free rice plants with a mean SCZ value of 0·65 (Table 2).

Growth, pathogenicity and sensitivity to bismerthiazol

As indicated by the turbidity values (Table 3), no significant difference was found in bacterial growth in NB broth between zinc thiazole-resistant mutants and their corresponding parents. A similar phenomenon was observed for the sensitivity of X. oryzae pv. oryzae to bismerthiazol. All 12 zinc thiazole-resistant mutants kept the same EC50 values for bismerthiazol as their wildtype zinc thiazole-sensitive parents (Table 3). However, a significant decrease was detected in the pathogenicity of zinc thiazole-resistant mutants when compared with their parents, especially for those mutants obtained via UV irradiation. The parental isolates had a mean lesion diameter of 11·1 ± 1·09 (±SD) cm and the zinc thiazole-resistant mutants had a mean disease lesion diameter of 7·45 ± 1·63 cm. Among these resistant mutants, nine mutants obtained via UV irradiation had a mean disease lesion diameter of only 6·6 ± 1·23 cm (Table 3).

Table 3. Growth, pathogenicity and sensitivity to bismerthiazol of zinc thiazole-resistant mutants of Xanthomonas oryzae pv. oryzae and their parents
IsolateaPhenotypebTurbiditycLesion diameter (cm)Sensitivity to bismerthiazol (EC50, mg L−1)
  1. a

    Isolates in bold are parents of the isolates listed under them.

  2. b

    R, S: resistant and sensitive to zinc thiazole, respectively.

  3. c

    Turbidity (the optical density at 600 nm) determined with a nephelometer (WCY-WOG, Baoli) represents the growth of Xanthomonas oryzae pv. oryzae.

  4. d

    Multiple comparisons were performed within a column under each parent. Mean values with the same letter were not significantly different with LSD (least significant difference) test at = 0·05.

ZJ09-6 S1052 ad9·8 a31·32 a
ZJ-UR-2R996 a5·7 c31·30 a
ZJ-UR-3R1050 a5·9 c31·35 a
ZJ-TR-1R1078 a7·5 b31·28 a
AH10-21 S1215 a11·6 a22·27 a
AH-UR-3R1210 a7·3 c22·32 a
AH-UR-7R1207 a7·2 c22·04 a
AH-UR-12R1200 a4·5 d2·18 a
AH-TR-2R1215 a9·9 b2·29 a
JS10-16 S1109 a12·3 a6·99 a
JS-UR-1R1116 a7·9 b6·03 a
JS-UR-5R1100 a8·3 b6·11 a
HB10-33S1090 a10·7 a43·56 a
HB-UR-4R1105 a7·6 b43·42 a
HB-UR-11R1000 a7·3 b43·37 a
HB-PR-1R1078 a10·3 a43·62 a

Discussion

Application of bactericides is an indispensable tool for BLB management. As bismerthiazol has been used in China since the 1970s, it is not surprising that bismerthiazol-resistant strains of X. oryzae pv. oryzae are common in rice fields (Wang et al., 1998; Shen & Zhou, 2002). Presently, introduction of novel alternatives that have no cross-resistance with bismerthiazol is urgent. Zinc thiazole is a novel bactericide showing strong antibacterial activity against Xanthomonas spp. (Wei et al., 2007, 2008). In this study, a total of 323 isolates of X. oryzae pv. oryzae were sampled from rice plants with BLB symptoms and tested for baseline sensitivity to zinc thiazole. Their EC50 values ranged from 0·1 to 16·8 mg L−1 with a mean EC50 value of 6·79 ± 1·61 mg L−1. The isolates from four geographical origins showed similar sensitivity levels over a two-year period (2009–2010). As zinc thiazole had not been applied to X. oryzae pv. oryzae in the study area before sampling, the isolates had never been exposed to this bactericide. Therefore, the concentration of 6·79 ± 1·61 mg L−1 could be used as the baseline for monitoring the sensitivity shift of X. oryzae pv. oryzae populations to zinc thiazole (Russell, 2007).

UV irradiation, culturing in zinc thiazole-amended NA plates, and inoculation on zinc thiazole-treated rice plants yielded nine, two and one zinc thiazole-resistant mutants, respectively. All 12 zinc thiazole-resistant mutants showed decreases in the RF after 20 successive transfers on bactericide-free media or 10 successive inoculation–reisolations on bactericide-free rice plants. Significant decreases were also observed in the pathogenicity of zinc thiazole-resistant mutants compared with their parents. These results indicate a potential fitness penalty for the zinc thiazole-resistant mutant, but this should be further investigated using field resistant isolates. The resistance factor and pathogenicity of zinc thiazole-resistant mutants generated by UV irradiation showed greater decreases than those generated by culturing on bactericide-amended media or bactericide-treated plants. This could be partially attributed to the unknown effect of UV mutagenesis on other loci in addition to those of interest here. All 12 zinc thiazole-resistant mutants kept the same EC50 values for bismerthiazol as their wildtype zinc thiazole-sensitive parents. This suggests a lack of cross-resistance between zinc thiazole and bismerthiazol, and difference in mode of action between these two chemicals. A study of the mode of action of zinc thiazole is currently ongoing in this laboratory.

Significant decreases in the RF and the pathogenicity of zinc thiazole-resistant mutants of X. oryzae pv. oryzae were observed. However, one should be very careful about these findings because all mutants were artificially generated. Differences may exist between resistant mutants generated in laboratory conditions and field resistant isolates according to previous results of fungicide resistance study (Russell, 2007). Therefore, appropriate resistance prevention strategies are needed to avoid rapid development of resistance in situ, such as monitoring changes in the sensitivity of bacterial isolates over time (Brent & Hollomon, 2007). The existing bactericides such as copper-based bactericides and antibiotics have the disadvantages of poor effectiveness, phytotoxicity and/or resistance. Future work is needed to examine the potential of zinc thiazole in prevention and control of other bacterial diseases.

Acknowledgements

This research was supported by the Natural Science Foundation of China (31071711) and Public-interest Technology Application Study of Zhejiang Province (2010C32083).

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