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Article Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Biotoxin Tropolone Contamination Associated with Nationwide Occurrence of Pathogen Burkholderia plantarii in Agricultural Environments in China Xiaoyu Liu,○,† Xiaoyan Fan,○,† Haruna Matsumoto,† Yanxia Nie,‡ Zhimin Sha,§ Kunpeng Yi,∥ Jiuyue Pan,⊥ Yuan Qian,† Mengchao Cao,# Yihu Wang,∇ Guonian Zhu,† and Mengcen Wang*,† †

Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, China ‡ Ecology and Environmental Sciences Center, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China § School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China ∥ State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China ⊥ College of Plant Protection, Hunan Agricultural University, Changsha 410128, China # Patent Examination Cooperation Jiangsu Center of the Patent Office, State Intellectual Property Office of the PRC, Suzhou 215163, China ∇ Solution Department, Jiangsu Rotam Chemistry Co., Ltd., Suzhou 215301, China S Supporting Information *

ABSTRACT: Tropolone, a biotoxin produced by the agricultural pathogen Burkholderia plantarii, exerts cytotoxicity toward a wide array of biota. However, due to the lack of quantitative and qualitative approach, both B. plantarii occurrence and tropolone contamination in agricultural environments remain poorly understood. Here, we presented a sensitive and reliable method for detection of B. plantarii in artificial, plant, and environmental matrices by tropolonetargeted gas chromatography−triple-quadrupole tandem mass spectrometry analysis. Limits of detection for B. plantarii and tropolone were 10 colony-forming units (CFU)/mL and 0.017 μg/kg, respectively. In a series of simulation trials, we found that B. plantarii from 10 to 108 CFU/mL produced tropolone between 0.006 and 107.8 mg/kg in a cell-population-dependent manner, regardless of habitat. Correlation analysis clarified a reliable reflection of B. plantarii density by tropolone level with R2 values from 0.9201 to 0.9756 (p < 0.01). Through a nationwide pilot study conducted in China, tropolone contamination was observed at 0.014−0.157 mg/kg in paddy soil and rice grains, and subsequent redundancy analysis revealed soil organic matter to be a dominant environmental factor, having a positive correlation with tropolone contamination. In this context, our results imply that potential ecological and dietary risks posed by long-term exposure to trace levels of tropolone contamination are of concern. including Animalia, Plantae, Monera, and Fungi.11 For instance, it exerts inhibitory effects toward grape polyphenol oxidase and tyrosinase of plants12,13 and is capable of exerting severe cytotoxicity on soil beneficial microbes and mammals at trace levels.14 B. plantarii-produced tropolone not only causes severe damage during agricultural production but also poses varying degrees of risk toward plant−soil-water ecosystems and human health.5,8,15 However, field investigations focused on under-

1. INTRODUCTION Contamination of the natural environment with biotoxins derived from diverse virulent eukaryotes1,2 and prokaryotes3 is an increasing concern worldwide because of their adverse impact on environmental safety and public health.4 The occurrence of Burkholderia plantarii, an important agricultural pathogen first discovered in Japan in 1985, has expanded to the United States, China, and Korea in the last few decades.5−8 B. plantarii is considered to be an increasingly serious biocontaminant in agricultural environments because it is capable of extracellular production of tropolone, a sevenmembered nonbenzenoid biotoxin.9−11 Tropolone is known to pose varying degrees of toxicities toward a wide array of biota © XXXX American Chemical Society

Received: November 18, 2017 Revised: March 1, 2018 Accepted: March 14, 2018

A

DOI: 10.1021/acs.est.7b05915 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology standing the traits of B. plantarii production of tropolone are limited. In natural environments, the detection of pathogens plays a critical role in the prediction and control of biotoxin contamination.3 Conventionally, a specific pathogen has been acquired from hosts and environments through selective media and further subjected to characterization using morphological and biochemical approaches.16 Because of the rapid development of molecular biological techniques, enzyme-linked immunosorbent assay (ELISA)17 and a wide array of nucleic acid-targeted approaches such as nested-polymerase chain reaction (nested-PCR),18 multiplex PCR,19 loop-mediated isothermal amplification (LAMP),20 and quantitative real-time PCR (qRT-PCR),3 have been widely applied to the detection of pathogens in environments or hosts. The first method for specific and rapid detection of B. plantarii was developed by Takeuchi et al. in 1997 through amplification of the 16S−23S rDNA spacer regions, but it was only applied to rice seedlings, with unclear sensitivity and applicability for other rice matrices or natural environments infested with the pathogen.21 Until 2006, a multiplex PCR-based approach using DNA gyrase (gyrB) and σ70 factor (rpoD) as the specific DNA region was developed to detect B. plantarii-infested rice seeds; however, it was only applicable at a high cell density of B. plantarii (1.0 × 108 colony-forming units (CFU)/mL).22 In addition to these biomacromolecule-targeted approaches, the metabolic traittargeted identification of B. plantarii was constructed using high-performance liquid chromatography analysis of the distinct peak despite the unknown structural information regarding the distinct compound produced by B. plantarii.23 Moreover, it was limited to application to pure cultures of B. plantarii. However, these previously established methods were for the characterization of B. plantarii, not for discrimination of B. plantarii from other pathogens or prediction of its occurrence in situ. More importantly, insufficient sensitivity may cause false-negative responses in the precise detection and prediction of relatively low-density B. plantarii population in real environmental and biological matrices, such as soil, water, and host plants. Owing to their excellent quantitative and qualitative performance, high sensitivity, reproducibility, and specificity,24 chromatography combined with various types of mass spectrometry (MS) such as time-of-flight MS (TOF-MS),25 matrix-assisted laser desorption ionization TOF-MS (MALDITOF-MS),26,27 and triple quadrupole tandem MS (QqQ-MS/ MS)28 has been employed to investigate physiological and metabolic traits of microbes. Until recently, these spectroscopic approaches were applied to taxonomic identification of marine and intestinal bacteria and Penicillium fungi based on specific metabolic markers.25,29,30 In terrestrial ecosystems, tropolone derivatives such as stipitatic acid, colchicine, and hinokitiol are biosynthesized in various organisms, including higher plants and fungi, whereas tropolone production only occurs in B. plantarii.31,32 Thus, tropolone functions as an exclusive metabolic and taxonomical marker of B. plantarii, and the development of a tropolone-targeted MS approach is likely to realize the simultaneous elucidation of the occurrence of B. plantarii and tropolone contamination. In this study, a sensitive, rapid and reliable approach to track B. plantarii-originated tropolone in artificial media, plants, and environmental matrices was developed with a focus on B. plantarii occurrence-derived tropolone contamination in diverse simulated systems and real agricultural environments

in China. Our study provides a promising tool with which to predict B. plantarii occurrence in agricultural environments and, more importantly, may serve as an important basis for seasonable prevention of outbreaks and epidemics of B. plantarii as well as the pre-elimination of risk to tropolonecontaminated environments or agricultural products.

2. MATERIALS AND METHODS 2.1. Chemicals and Instruments. Tropolone standard (TCI, purity 98.0%), acetonitrile (ACN), ethyl acetate (EtOAc), and other organic solvent (chromatographic-grade) were purchased from Merck. Agar powder and gellan gum were obtained from Wako. A DisQuE kit containing a 50 mL centrifuge tube, MgSO4, CH3COONa, and primary secondary amine (PSA) was purchased from Waters. Bacterial cell density and relative fluorescence intensity were measured in a relative microplate reader (SpectraMas i3, Molecular Devices), and ultrapure water was purified using a Milli-Q Water Purification System (Merck Millipore). 2.2. Bacterial Strain and Culture Media. B. plantarii ZJ171 previously isolated from rice paddies5 was used as a reference strain of B. plantarii. It was routinely grown in MA media (NH4H2PO4, 1 g; KCl, 0.2 g; MgSO4·7H2O, 0.2 g; glucose, 10 g; distilled water, 1 L [pH adjusted to 6.2]) at 25 °C in the dark.33 To prepare the solidified media, 1.5% agar powder (Solarbio, Beijing) was supplemented into the MA medium. 2.3. Extraction and Purification of Tropolone. To extract and purify tropolone from artificial media and rice matrices, the QuEChERS method34 was employed using the DisQuE Kit with minor modification as follows. Solid matrices (10 g), including sheared rice seedling, smashed rice seeds, paddy soil, and crushed MA agar media cultures were supplemented with 10 mL of ultrapure water and subjected to homogenization. The resulting homogenates of the solid matrices and the liquid matrices (10 mL) including paddy water and MA media cultures (removal of B. plantarii cells by at 10000g centrifugation) were both adjusted to pH 4.0 and mixed (10 mL of ultrapure water supplemented for the solid samples) with 50 mL of acetonitrile and 1 g of NaCl in a 250 mL centrifuge bottle. The resulting mixtures were subjected to homogenization for 1 min. After centrifugation at 4000 rpm for 1 min, the aliquot of the upper layer (25 mL) was pipetted into a 50 mL DisQuE centrifuge tube preloaded with 6 g of MgSO4, and 1.5 g of CH3COONa. Each mixture was shaken drastically for 1 min and centrifuged at 4000 rpm for 1 min, and then the supernatant was concentrated by a rotatory evaporator at 45 °C for further purification.35 The resulting concentrates were redissolved with EtOAc (1 mL) and pipetted into a 2 mL clean-up tube filled with 50 mg of PSA and 150 mg of MgSO4. After vigorous shaking for 30 s, the tube was centrifuged at 12 000 rpm for 1 min and then filtered through a 0.22 μm filter, and the resulting filtrates were subjected to analysis of tropolone by gas chromatography (GC)−QqQ-MS/MS. 2.4. Qualification and Quantification of Tropolone. The stock solution of tropolone was prepared by dissolving tropolone standard in EtOAc at 1000 mg/L, which was further diluted in each blank matrix at a series of concentrations (0.001, 0.01, 0.1, 1.0, and 10.0 mg/kg) to generate working standard solution for quantitative analysis of tropolone in various matrices (external standard method). All resulting solutions were stored in amber bottles at 4 °C. B


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using the resultant water. In these simulant seedling beds prepared, 50 mL of B. plantarii cell suspension (from 10 to 106 CFU/mL) was evenly sprayed on the soil surfaces to generate B. plantarii infestation, while 50 mL of sterile water was applied to the control group. Each treatment consisted of 10 replicates. Incubation was done in the green house (25 °C), and sampling was conducted from 0 to 22 d for soil and water phases, respectively. One portion diluted in sterile water was used for viable cell number measurement as previously reported10,41 and the other portion was subjected to analysis of tropolone per the method currently developed. To investigate the traits of tropolone production by B. plantarii in rice seedlings, a B. plantarii-free seedling bed was prepared as described above. The tropolone-free rice seeds (O. sativa cv. Xiushui09) were surface-sterilized and incubated until germination and then subjected to inoculation of B. plantarii from 0 to 106 CFU/mL per a previous report,10 and then sowed into the B. plantarii-free seedling beds. After 10 days of growth, the seedlings were collected and subjected to measurement of viable cell number of B. plantarii and tropolone production. Meanwhile, observation of disease severity was assessed using a scale of 0−III (0 = healthy, I = stem inhibition below 10% and root inhibition below 50%, II = stem inhibition of 10%−20% and root inhibition of 50%−100%, and III= stem inhibition above 20% and root inhibition of 100%), which was established based on a previous nursery survey with an unlabeled randomized block design. To investigate the traits of tropolone production by B. plantarii in rice seeds, spikelets of rice plants (Oryza sativa cv. Xiushui09) cultivated in the green house were inoculated with B. plantarii from 10 to 106 CFU/mL by spraying application (10 mL cell suspension per plant)22 or 10 mL sterile water (control), with 10 replicates for each treatment. At the ripening stage, the rice grains collected from the spikes were surfacesterilized and then smashed, from which 20 g were equally divided into two portions. One portion diluted in sterile water was used for viable cell number counting and the other portion was subjected to analysis of tropolone. To investigate the traits of tropolone production by B. plantarii in artificial media, overnight MA cultures of B. plantarii ZJ171 were inoculated into 10 mL of fresh MA agar plate at a serial cell density from 10 to 108 CFU/mL. After being cultured statically for 3 days at 25 °C in dark, the culture was homogenized with the blender (Ultra-Turrax T18, IKA, Germany) for 3 min and subjected to measurement of viable cell number of B. plantarii and tropolone production. 2.7. Pilot Study on B. plantarii-Derived Tropolone Contamination in a Paddy Environment. To analyze the occurrence of B. plantarii in the real paddy environment, a pilot study was carried out in 2016 in 16 dominant rice production regions in China including Anhui, Fujian, Guangdong, Guangxi, Guizhou, Hainan, Heilongjiang, Hubei, Hunan, Jiangsu, Jiangxi, Jilin, Liaoning, Sichuan, Yunnan, and Zhejiang provinces. In each province, 10 sampling sites (Figure S1) were selected for collection of 100 paddy soil samples (10 samples per site, 0−10 cm in-depth) and 100 rice grain samples (the portion of whole spikelet) at the senescence stage of rice plants. For each sampling site, the 5-point sampling method was applied, and 500 g was representatively separated from the evenly mixed sample was stored at −20 °C until the determination of tropolone and verification of B. plantarii. Measurement of soil pH (ratio of soil to water, 1:2.5 dry wt/v) was done using a pH meter with a glass electrode (Mettler Toledo FE-20), and an

For the detection of tropolone, separation was performed on a GC−QqQ-MS/MS (Agilent 7000C) installed with a capillary column (Agilent HP-5MS, 30 m × 0.25 mm internal diameter, with a 0.25 μm film thickness) using helium (99.999%) as the carrier gas at a constant flow of 1.5 mL min−1 and nitrogen (99.999%) as the collision gas at rate of 1.0 mL/min. The temperature of injector was set at 300 °C, and the injection volume was 1 μL in the splitless mode. The oven temperature was raised from 50 °C at a rate of 10 °C/min to 100 °C and held for 1 min and then raised to 250 °C at a rate of 30 °C/min and held for 3 min. MS/MS was operated in electron ionization mode using electron impact ionization (EI, 70 eV) and detector voltage of 1.1 kV with a mass range of 50−200 m/z. Multiple reaction monitoring (MRM) transitions were 122.0 > 94.0 (quantifier) and 122.0 > 66.0 (qualifier), both with collision energy at 30 V. The temperature of transfer line and ionization source was maintained at 250 and 230 °C, respectively. The software Agilent 7000 Mass Hunter was applied for data acquisition and processing. 2.5. Method Verification. The analytical method was verified through systematical evaluation of limit of detection (LOD), limit of quantification (LOQ), matrix effect (ME), recovery, and intra- and interday precision as follows.36−38 The LODs in various matrices were determined by considering a signal-to-noise ratio of 3, and the LOQs were determined by considering a signal-to-noise ratio of 1038. ME evaluation was conducted by diluting tropolone with EtOAc and acetonitrile extracts of matrices (tropolone-free samples) redissolved in the same volume of EtOAc, respectively. Using the two sets of calibration curves obtained, ME was calculated per the equation below: ME = [slope(matrix) − slope(solvent)]/slope(solvent) (1)

,where slope (matrix) and slope (solvent) represent the slopes of the calibration curves of the matrix and solvent standards, respectively. Recovery test was carried out by fortification of the tropolone-free samples with tropolone working solutions at five spiking levels (0.005, 0.05, 0.5, 5.0, and 50.0 mg/kg) for 5 replicates. These fortified samples together with control sample (fortified with the same volume of solvent only) were subjected to extraction, purification, and analysis using the modified DisQuE kit and GC−QqQ-MS/MS as described above. Intraday precision test was conducted by comparing standard deviation of the recoveries of five replicates in the same day for the method repeatability, and the interday precision test was determined by analyzing fortified samples in three alternate days for the method reproducibility.39,40 Intra- and interday precisions tests were also done at 5 spiking levels including 0.005, 0.05, 0.5, 5.0, and 50.0 mg/kg. 2.6. Traits of Tropolone Production by B. plantarii in Different Matrices. To investigate the traits of tropolone production by B. plantarii in the rice-seedling-growing environment, simulant seedling bed was prepared as follows. The soil and water (tropolone-free) were collected from a seedling nursery in the rice experiment station of Zhejiang University. After the water was filtered through no. 101 filter paper (Advantec, Tokyo, Japan) and the soil was completely dried under 60 °C for 24 h and sieved (10 mesh), both samples were subjected to sterilization. In each polyvinyl chloride box (length of 40 cm, width of 30 cm, and depth of 5 cm), the resultant soil was placed and adjusted to 80% moisture content C


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Figure 1. GC−QqQ-MS/MS analysis of tropolone. Tropolone was detected at 7.65 min in total ion chromatograms with characteristic fragments at m/z (A) 94.0 and (B) 66.0.

For the liquid matrices, supplementation of 10% NaCl into the aqueous phase significantly enhanced acetonitrile-based recovery for tropolone, whereas it was not applicable for methanol and acetone (Table S2). However, the recoveries of tropolone were still only between 69.5% and 82.2% (Table S2). The pKa value of tropolone is 7.12 ± 0.10 (ACD/Laboratories software V11.02), suggesting that the weak dissociation may limit the recovery of tropolone from the aqueous phase. Accordingly, we found that the recoveries was significantly increased with the decrease of pH, but this effect was when they were below 4.0 (p > 0.05; Figure S2). Hence, an acidic condition was critical to maintenance of tropolone in the molecule instead of the ion. The validity of the analytical method was systematically confirmed using ME and various recovery tests. The sevenpoint-calibration curves43 in solvent and six matrices (sheared rice seedlings, smashed rice seeds, paddy soil, crushed MA agar, paddy water, and MA media) were constructed and compared. Tropolone gave correlation coefficients (R2) between 0.991 and 0.996 in solvent and matrices (Table S3). Ion-suppression effects of −5.5% and −9.3% occurred in rice seedling and paddy soil, respectively, while enhancement effects between 6.4% and 9.7% were observed in the remaining matrices (Table S3). According to the evaluation criterion for ME level (Kmellar et al., 2008), the MEs calculated in these matrices were at a moderate level. Based on the modified QuEChERS and the constructed calibration curves, the recoveries of tropolone spiked into the matrices at 5.0 μg/kg to 50.0 mg/ kg were between 81.2% and 108.5%, with relative standard deviations (RSD) of 5.0−13.8% (Table S4). Referring to China’s National Standard (NY/T 788-2004), all recoveries acquired were between 75.0% and 115.0% with an RSD of 0.05), except that methanol and acetonitrile showed a relatively higher recovery for paddy soils (Table S2). D


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Figure 2. Traits of tropolone production by B. plantarii in the simulation trial. (A) Tropolone production and (B) corresponding viable cell number were simultaneously measured in the soil-water phase; correlation analysis of tropolone production and viable cell number of B. plantarii was done in the (C) paddy soils and (D) rice seedlings.

in various matrices, and the LOQs were 0.063−0.315 μg/kg. These findings indicated that the high sensitivity of the GC− QqQ-MS/MS currently developed ensures the effective monitoring of trace levels of tropolone in diverse matrices. 3.2. Tropolone Production Indication of B. plantarii Occurrence and Population Density in Diverse Habitats. In nature, B. plantarii lives in soil and aquatic environments or the inside of rice plants and some other nonhost plants once it is successfully established in an ecological niche in certain agricultural environments.8,10 To date, few studies have focused on the traits of tropolone production and its association with the B. plantarii population in either environmental or plant matrices.10 When B. plantarii was initially inoculated into the paddy soilwater two-phase system at 103 CFU/mL, a larger proportion of tropolone production by B. plantarii was found in the soil phase, in contrast to a significantly lower level of tropolone (p < 0.05) in the water phase, throughout the entire incubation period (Figure 2A). In the soil phase, tropolone production was initially observed at 0.038 mg/kg after 2 days and then reached the maximum level of 8.61 mg/kg after 10 days, after which it stabilized from 14 to 22 days (p > 0.05, Figure 2A). Interestingly, the cell population of B. plantarii living in the soil phase increased from day 1 to 10 (p < 0.05), while it reached a relatively stable level between day 14 and 22 (p > 0.05, Figure 2B), which seemed to be associated with the contemporary tropolone production level in the soil phase. This was further demonstrated by inoculation of a gradient cell population of B. plantarii into the soil phase, in which tropolone production showed a good linearity with the log 10 value of viable cells (Figure 2C), giving the linear equation: y = 8.66x − 23.3 (R2 = 0.9756, p < 0.01). These findings clearly

indicated the cell-population-dependent manner of tropolone production by B. plantarii in the soil phase. In the water phase, tropolone production was initiated after day 4 and maintained at 0.021−0.046 mg/kg between day 6 and 22 (Figure 2A) despite there being no significant differences among sampling intervals (p > 0.05). We also noticed that the population of B. plantarii living in the water was significantly lower than that in the soil phase within 1 day and drastically decreased to a nondetectable level after 2 days (p < 0.05, Figure 2B). These findings implied that tropolone diffused from the soil phase was responsible for the minor portion of tropolone detected in the water phase after 4 days and that the soil phase was likely to be a preferable habitat for B. plantarii. Hence, tropolone production in the soil phase was deemed to reflect the occurrence and general population of B. plantarii in the environmental matrices of rice paddies. In the biological matrices (rice seedlings and rice seeds), the highest tropolone production was detected at 65.07 mg/kg in rice seedlings inoculated with B. plantarii at 106 CFU/mL, while tropolone was only produced at 1.26 mg/kg in seedlings inoculated with B. plantarii at 10 CFU/mL (Figure 2D). Similarly, a good linearity between tropolone production and the log 10 value of viable cells was observed in rice seedlings (Figure 2D), giving the linear equation y = 20.2x − 56.56 (R2 = 0.9702, p < 0.01). Moreover, in these B. plantarii-infected rice seedlings, the cell-population-dependent tropolone production was also positively correlated with the severity of seedling blight symptoms, such as root growth inhibition (Figure S3A−D) despite there being no typical symptoms of seedling blight observed in the rice seedlings inoculated with tropolone at 1.26 and 2.72 mg/kg (Figure S3E,F) relative to the control (Figure S3G). The level of tropolone produced in situ was considered a critical factor determining the development of disease E


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Environmental Science & Technology symptoms,33 and the initial threshold concentration of tropolone causing disease incidence of seedling blight was known to be 3.0 mg/kg; thus, the rice seedlings infected with B. plantarii at a relatively lower level did not show typical symptoms. However, neglect of these B. plantarii-infected symptom-free rice seedlings during rice cultivation may lead to disruption of the rice grain quality because B. plantarii is likely to migrate inside the rice plant and subsequently accumulate tropolone in the spikelets.8,44,45 Accordingly, accumulation of tropolone by B. plantarii in the rice seeds was found to increase with increasing population density of viable B. plantarii present in the spikelets, with the maximum level of tropolone being 104.42 mg/kg (Figure 3). Interestingly, in matrices with

promoted by abundant carbon and nitrogen sources in environmental habitats,46 such as carbohydrate- and amino acids-enriched spikelets. Hence, tropolone produced by B. plantarii was a convincing indicator of detection of B. plantarii occurrence that also reflected the general population of B. plantarii in not only environmental matrices but also plant matrices. 3.3. Tropolone Contamination and B. plantarii Occurrence in Paddy Environments and Rice Grains in China. During the seedling period, tropolone was detectable in a large proportion of paddy environments in China with the exception of Hainan province (Table 1). To avoid false positives for B. plantarii, the tropolone-detectable samples were also subjected to screening of B. plantarii through tropoloneenriched media. Colonies grown under such tropoloneenriched conditions showed a monomorphology and the amplicons of their 16S rRNA genes were 99.8−100.0% homologous with the sequence of the reference strain B. plantarii ZJ117 (Table 1). Moreover, the 16S−23S rRNA spacer region amplicon appeared in Heilongjiang, Jilin, Liaoning, Jiangsu, and Sichuan provinces, while the gyrB amplicon was only observed in Heilongjiang and Sichuan provinces (Table 1). These findings indicate the accuracy of the tropolone-targeted approach, providing an ideal alternative to the previous DNA marker-targeted monitoring of B. plantarii, particularly in real environmental samples. Across all rice-production regions harboring B. plantarii in China, the highest tropolone level and detection rates were observed in the Heilongjiang and Jilin provinces, respectively, while the lowest were both found in Guangxi province (Table 1). RDA was employed to investigate the level of environmental factors affecting B. plantarii occurrence and tropolone contamination, and it revealed five influencing environmental variables highly involved (Table S6 and S7). Heilongjiang, Jilin, and Liaoning were all enclosed by an ellipse, indicating that the B. plantarii occurrence in the three northern provinces was significantly different from that of other provinces and that these differences could be identified by the high tropolone levels (Figure 4). Among the environmental factors (Table S6 and S7), OM content exhibited the greatest influence on

Figure 3. Correlation analysis of tropolone production and the viable cell number of B. plantarii in rice grains and artificial media.

enriched glucose and ammonium nitrogen, such as MA solid culture, we also found that tropolone was produced at a similar level (from 3.01 to 107.79 mg/kg) with increasing cell density of B. plantarii (Figure 3). A good linearity between tropolone production and the log 10 value of viable cells of B. plantarii was shown, with equations of y = 31.8x − 83.4 (R2 = 0.9671, p < 0.01) and y = 11.9x − 7.09 (R2 = 0.9291, p < 0.01) for rice seeds and MA media, respectively (Figure 3). These findings suggest that B. plantarii produced tropolone in an innate population-dependent manner but would be exogenously

Table 1. Occurrence of B. plantarii in Paddy Environment in 16 Rice-Cultivation Regions in China regions Anhui Fujian Guangdong Guangxi Guizhou Hainan Heilongjiang Hubei Hunan Jiangsu Jiangxi Jilin Liaoning Sichuan Yunnan Zhejiang

tropolone (mg/kg, mean ± SD)

detection rate (%)

16Sa (±)

16S identity (%)

16S-23Sb (±)

gyrBc (±)

± ± ± ± ±

0.051 0.015 0.009 0.004 0.009

± ± ± ± ± ± ± ± ± ±

0.044 0.011 0.009 0.049 0.011 0.055 0.054 0.020 0.014 0.018

12.0 6.0 7.0 4.0 9.0 0.0 15.5 7.5 4.5 13.0 8.5 21.0 18.5 11.5 8.0 9.5

+ + + + + − + + + + + + + + + +

100.0 100.0 100.0 100.0 100.0 99.8 100.0 100.0 100.0 99.8 99.9 99.9 100.0 100.0 100.0

− − − − − − + − − + − + + + − −

− − − − − − + − − − − − − + − −

0.038 0.025 0.017 0.009 0.024 UD 0.055 0.030 0.017 0.042 0.025 0.049 0.045 0.031 0.014 0.026

PCR amplicon for 16S rRNA gene. bPCR amplicon for 16S−23S rRNA spacer region gene. cPCR amplicon for β-subunit polypeptide of DNA gyrase gene. + , positive result; −, negative result; UD, undetectable (below 0.005 mg/kg).

a

F


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In addition, mature rice grains in these regions were randomly collected and analyzed for tropolone. The results revealed tropolone in rice grains at 0.013−0.157 mg/kg with a detection rate of 2.0−15.0% (Table 2), which is similar to the pattern shown in the paddy environment (Table 1). Moreover, amplicons of the 16S rRNA genes of tropolone resistant colonies from the tropolone-positive samples also showed 99.9−100% homology with the sequence of the reference strain, B. plantarii ZJ117 (Table 2), indicating consistency between the tropolone-containing samples and occurrence of B. plantarii. Although the detection ratio of the amplicon of gyrB and the 16S−23S rRNA spacer region gene increased in the rice grains (Table 2) in contrast to the paddy environment (Table 1), false negatives in a total of 16 regions still remained at 43.8% and 56.3%, respectively (Table 2). However, this could be remedied by supplemental application of the currently developed tropolone-targeted method (Tables 1 and 2). Furthermore, it was not surprising that tropolone was detected in the rice grains at a relatively higher level than in the paddy environment (Tables 1 and 2), similar to the simulation trials (Figures 2 and 3). This could be explained as follows: (1) the active amino acids metabolism in spikelets was likely to provide the sufficient precursors for tropolone biosynthesis in B. plantarii;46,47 and (2) the active uptake of water via roots occurred during the blooming and filling stages,48 which simultaneously leads to absorption and deposition of watersoluble tropolone from the B. plantarii-infested paddy environment. Taken together, the general occurrence of B. plantarii was first revealed through the relative abundance of tropolone contamination across the rice production regions in China (Figure 5), which strongly suggests that constant attention should be paid to monitoring of the B. plantarii occurrence dynamics and tropolone contamination in agricultural environments, particularly in north China. Overall, the results of this study demonstrated that tropolone produced by B. plantarii was precisely quantified using the modified QuEChERS method coupled with GC−QqQ-MS/ MS to clarify B. plantarii occurrence in host plant and agricultural environments. This approach could be directly applicable to field prediction of pathogen occurrence. More

Figure 4. RDA analysis of tropolone contamination in diverse regions in association with environmental factors. Red arrow lines indicated different environmental variables; blue arrow lines indicated tropolone and related index; black hollow circles showed samples from diverse regions; and data from the Jilin, Heilongjiang, and Liaoning provinces were enclosed by an ellipse, which indicates that tropolone and the related index from these part were significantly different from other regions.

tropolone levels (p < 0.01) and followed by pH (Figure 4 and Table S7). Therefore, rice paddies in northern China (such as Heilongjiang province) possessing a relatively high content of OM are likely to provide ideal habitats and sources of B. plantarii; accordingly, such systems are in favor of the survival and spread of this pathogen in agricultural environments, as well as contamination with its associated biotoxins.

Table 2. Occurrence of B. plantarii in Rice Grains in 16 Rice-Cultivation Regions in China regions Anhui Fujian Guangdong Guangxi Guizhou Hainan Heilongjiang Hubei Hunan Jiangsu Jiangxi Jilin Liaoning Sichuan Yunnan Zhejiang

tropolone (mg/kg, mean ± SD) 0.074 0.038 0.027 0.013 0.036 UD 0.157 0.048 0.035 0.080 0.034 0.105 0.091 0.046 0.024 0.041

± ± ± ± ±

0.103 0.023 0.018 0.005 0.006

± ± ± ± ± ± ± ± ± ±

0.117 0.008 0.020 0.091 0.014 0.028 0.110 0.029 0.024 0.021

detection rate (%)

16Sa (±)

16S identity (%)

16S−23Sb (±)

gyrBc (±)

10.5 5.0 5.5 15.0 2.0 0.0 8.5 4.5 2.5 3.0 3.5 2.5 6.5 7.0 3.0 6.5

+ + + + + − + + + + + + + + + +

100.0 99.9 100.0 100.0 100.0 − 99.9 99.9 100.0 100.0 99.9 99.9 99.9 100.0 99.9 100.0

+ + − − − − + + − + − + + + − −

+ − − − − − + − − + − + + + − −

PCR amplicon for 16S rRNA gene. bPCR amplicon for 16S−23S rRNA spacer region gene. cPCR amplicon for β-subunit polypeptide of DNA gyrase gene. + , positive result; −, negative result; UD, undetectable (below 0.005 mg/kg).

a

G


Article


Figure 5. Profile of tropolone contamination by Burkholderia plantarii in paddy environments in China. NDR indicates the nondominant riceproduction regions.

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importantly, the nationwide pilot study revealed the general occurrence of B. plantarii and tropolone contamination, which indicates that further attention should paid to the risks posed by environmental exposure to biotoxin-contaminated environments or dietary intake of biotoxin-contaminated foods.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-571-88982517; fax: +86-571-88982517; e-mail: [email protected]. ORCID

Mengcen Wang: 0000-0001-7169-6779

ASSOCIATED CONTENT

S Supporting Information *

Author Contributions ○

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b05915. Figures showing the distribution of the sampling sites, the effect of pH on recovery for tropolone, and the growth of rice seedlings inoculated with gradient cell density of B. plantarii. Tables showing primer pairs used for amplification, a comparison of extraction efficiency for tropolone, the linearity and ME of tropolone in diverse matrices, recoveries of tropolone in diverse matrices, recovery experiments with intraday and interday variability, meteorological and environmental parameters in 16 rice cultivation regions in China, and correlation coefficients between environmental factors and tropolone and detection rate. (PDF)

X.L. and X.F. equally contributed to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (grant no. 31501684), Zhejiang Provincial Natural Science Foundation of China (grant no. LQ16C140001), Zhejiang Provincial Key Research and Development Program of China (grant no. 2015C02019), and the National Key R&D Program of China (grant no. 2017YFD0202100). We also appreciate Dabeinong Funds for Discipline Development and Talent Training in Zhejiang University. Special thanks go to the local agricultural experiH


Article


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ment stations that participated in the environmental sampling of this study.

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