Journal of Hazardous Materials 289 (2015) 9–17
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Effect of di-n-butyl phthalate on root physiology and rhizosphere microbial community of cucumber seedlings Ying Zhang a,∗ , Yue Tao a , Hui Zhang a , Lei Wang a , Guoqiang Sun a , Xin Sun a , Kehinde O. Erinle a , Chengcheng Feng a , Qiuxia Song a , Mo Li b a b
School of Resources & Environment, Northeast Agricultural University, Harbin 150030, PR China School of Geography, University of Nottingham, Nottinghamshire NG72RD, UK
h i g h l i g h t s • • • •
Cucumbers were grown with DBP supplementation. DBP had a significant effect on root physiology of cucumber seedlings. The ultrastructural study showed that the organelles were obviously affected by DBP. A period of an ‘on average’ low functional organization was discovered.
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Article history: Received 29 October 2014 Received in revised form 24 January 2015 Accepted 28 January 2015 Available online 11 February 2015 Keywords: DBP Cucumber rhizosphere microbial community Protein content Root activity Ultrastructure
a b s t r a c t The authors investigated the effects of di-n-butyl phthalate (DBP) on root physiology and rhizosphere microbial communities of cucumber seedlings (sativus L. cv Jinyan No. 4). Root protein content and root activity were observed to decrease. From the ultrastructural micrographs, visible impact on the mitochondria, endoplasmic reticulum and vacuole were detected. Moreover, the number of starch grains increased, and some were adhered to other cell components which might be the most direct evidence of DBP causing cellular damage. Results of PCR-DGGE (denaturing gradient gel electrophoresis) indicated that DBP significantly changed the abundance, structure and composition of rhizosphere bacteria when the concentration was higher than 50 mg L−1 . The relative abundances of Firmicutes increased while that of Bacteroidetes decreased. Bacillus was detected as the dominant bacteria in DBP contaminated cucumber rhizospheric soil. The amount of Actinobacteridae and Pseudomonas decreased until it disappeared in the rhizosphere soil when exposed to DBP concentrations higher than 50 mg L−1 . © 2015 Elsevier B.V. All rights reserved.
1. Introduction DBP is one of the important members in the phthalate esters family (PAEs) that has been listed as a priority pollutant by the US Environmental Protection Agency [1]. It is a kind of organic compound that is primarily used as plasticizers in the preparation of polyvinyl chloride (PVC) resins, plastic packing films, adhesives, cosmetics, cellulose materials and insect repellents [2]. Like many other PAEs, DBP is not chemically bonded to the polymer products and it migrates readily from the plastics to the environment [3]. Concerns have been raised about D-BP because it is suspected to have mutagenous, carcinogenous, and endocrine-distrupting effects [4,5].
∗ Corresponding author. Tel.: +86 451 5519 0993; fax: +86 451 5519 1170. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.jhazmat.2015.01.071 0304-3894/© 2015 Elsevier B.V. All rights reserved.
Due to increase in the use of plastic mulching films in agricultural facilities, the content of PAEs in the soil is also on the increase in the agricultural facilities [6]. PAEs play a detrimental role in the physiology of plants by inhibiting plant growth and development [7]. Humans are exposed to PAEs through its entry into the food chain via the contamination of vegetables and food items [8]. It is estimated that the contribution percentages of human exposure to DBP through vegetables, fruits and grains pathways were 42.4%, 2.0% and 7.5% [9]. Yin et al. [10] demonstrated that the decrease of capsaicin content and vitamin C was negatively correlated to the increase the concentration of DBP in capsicum fruit, which suggested that DBP uptake by the plant might be mainly responsible for quality degradation of capsicum fruit. Plant root as medium of absorption and transportation of nutrients, have an important effect on plant growth. Root protein and root activity are used as the general indicators of plant development. Cell ultrastructure could reflect the plants growth whether in good condition.
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Previous researches have proved that microbial community composition show significant differences in the early and late periods of PAEs contamination in the soil [11]. Microbes living in the plant root zone (rhizosphere) exert a profound impact on soil fertility and plant development [12]. Hence, changes in the soil microbial communities may affect functions performed by the microbes, and thus influence overall plant growth and development [13,14]. It means that not only does the contaminant directly affect both the plants and soil microbes, but also plays a detrimental role in the interaction between both components; thus, the further release of DBP into the ecosystem will further increase the level of severity both plants and microorganisms are exposed to. However, since detailed information about the adverse effects of DBP on plant roots and rhizosphere microorganisms are still not available, and thus requires further study. In this study, cucumber was employed as a target plant because it is one of the most widely consumed vegetables and is usually cultivated in greenhouse conditions. Extensive use of plastic films caused a serious threat to agricultural environment, resulting in significant effect on cucumber [6,15]. Different concentrations of DBP were applied to the cucumber seedlings to investigate their effects on root protein content, root activity and root cell ultrastructure. Changes in bacterial species diversity in the cucumber rhizosphere were also studied using DGGE of the PCR-amplified 16S rRNA gene.
2. Materials and methods 2.1. Plant materials and stress treatment Seeds of cucumber sativus L. cv Jinyan No. 4 were planted into planting pots at the horticultural garden of Northeast Agricultural University, Harbin, China. The soil was also sampled in the same
garden. The soil was a black soil with sandy loam texture. The basic properties of the soil: soil water content, 3.74%; TOC, 3.96%; N, 17.1 mg kg−1 ; P, 12.9 mg kg−1 ; K, 301.17 mg kg−1 ; pH, 7.16; No fertilizer was previously added to the soil. The average temperature in the greenhouse was 27 ± 1 ◦ C during the day and 20 ± 1 ◦ C during the night. The relative humidity was kept at 60–70%. Applications of DBP treatment to cucumber plants were made according to the method described by Zhang et al. [16]. 2.2. Root protein content Root tissues (0.5 g) were ground to fine powder using a chilled pestle and mortar. Five milliliter of 50 mM potassium phosphate buffer (pH 7.0), 1 mM ascorbic acid, 0.2 mM ethylenediamine tetraacetic acid and 2% (w/v) polyvinylpyrrolidone were added to the powder. The homogenate was centrifuged at 4 ◦ C for 20 min at 12,000 g. Supernatant was used for protein content assays. An aliquot of the extract was used to determine its protein content by the method of Redmile-Gordone et al. [17] utilizing bovine serum albumin as the standard. 2.3. Root activity Root activity was measured using the TTC (triphenyl tetrazolium chloride) method [18] and expressed as the deoxidization ability (mg g−1 h−1 ). Dehydrogenase was expressed as the deoxidized TTC quantity, which was an index of root activity [19]. Ten milliliter solution of equal quantities of TTC (0.4%) and phosphate buffer was added to root samples (0.5 g) and kept in the dark at 37 ◦ C for 2 h. The reaction was stopped with 1 M H2 SO4 . The roots were ground and transferred into a tube with ethyl acetate to a total volume of 10 ml. The solution was measured at the absorbance of 485 nm.
Fig. 1. The effect of DBP on root physiology of Cucumber sativus L. cv Jinyan No. 4. (a) Protein content. (b) Root activity. Means with different small letters are significantly different from one another under the DBP different concentration treatment, and different capital letters are significantly different from one another under the DBP different treatment times.
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2.4. Observations of root ultrastructure The method of root cell ultrastructure was conducted as described as Zhang et al. [16]. Small root fragments were collected and fixed in 2.5% (v/v) glutaraldehyde solution (pH 7.2) at 4 ◦ C overnight. The samples were washed three times by phosphatebuffered saline solution (PBS) (pH 6.8) and post-fixed with 2% (m/v) osmium tetroxide solution for 4 h in a hood. Then the samples were rinsed with PBS (pH 6.8) and dehydrated through different concentrations of ethanol solutions. Ethanol was replaced by acetone solution twice. Finally, the specimens were embedded in Spurr’s resin for fixing and polymerized at 60 ◦ C. Ultra-thin sections (80 nm) were obtained on copper grids (300 meshes) and double stained with 1.0% (w/v) uranyl acetate followed by 5.0% (w/v) lead citrate. Root tissues were observed with an H-600IV transmission electron microscope (Hitachi, Tokyo, Japan) at 90 kV.
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of template DNA, 2.5 mmol/L each dNTPs mixture. The quality and the size of the PCR products were visualized after electrophoresis on 0.6% (w/v) agarose gel. DGGE was used to assess the diversity of rhizosphere bacterial. Identical amounts of PCR products were loaded in 8% polyacrylamide gels with denaturing gradients ranging from 30% to 60% (100% denaturant contains 7 M urea and 40% formamide). Electrophoresis was performed at a constant voltage of 180 V for 4 h in 1 TAE buffer (40 mM Tris, 20 mM sodium acetate, 1 mmM EDTA, pH 7.4) at 65 ◦ C. Gels were stained with ethidium bromide and illuminated under UV light using a Gel Doc XR Bio-Rad system. Main strips were cut and again recycled to PCR with primers without GC clip. PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd., for sequencing.
2.7. Phylogenetic analysis 2.5. Extraction of total DNA from soil Cucumber rhizosphere soil was collected for DNA extraction at 1, 3, 7, 14 and 21 days after DBP treatment. Samples were collected by shaking the roots by hand to remove the soil adhering to the roots. Total DNA was extracted with a kit of OMEGA Bio-TEK. The DNA was confirmed by agarose gel electrophoresis.
Consensus phylogenetic trees were constructed based on Kimura two-parameter evolutionary distance matrices and neighbor-joining with a Boostrap analysis of 1000 replicates, using MEGA5. Each sequence and their closest relatives, identified in the GenBank database, were used for phylogenetic analyzes [21]. Sequences recovered from DGGE bands were submitted to GenBank under the accession numbers KM571925–KM571942.
2.6. PCR-DGGE 2.8. Statistical analyzes 200 bp fragments of the V3 region of bacterial 16S rRNA gene were amplified by PCR with GC-341F/518R primer [20]. The PCR condition was 10 min at 94 ◦ C followed by 35 cycles of 30 s denaturation at 94 ◦ C, 30 s extension at 72 ◦ C, and final thermal insulation step at 72 ◦ C for 10 min. Each reaction mixture (50 ul) contained 5 U Taq DNA polymerase, 10 × EasyTaq buffer, 10 pmol primers, 1 umol
All the experimental data were expressed with five replicates. Data were analyzed by one-way analysis of variance. Mean values were compared by Duncan’s new multiple range test at the 0.05% level using the SPSS 19.0 software. The data were represented as mean ± SE. DGGE profiles and principal component analysis (PCA)
Fig. 2. Transmission electron microscopy (TEM) micrographic images of Cucumber sativus L. cv Jinyan No. 4. (a) Control plant (without DBP). (b) The plant treated with 30 mg L−1 DBP at 7 d. (c) The plant treated with 50 mg L−1 DBP at 7 d. (d) The plant treated with 100 mg L−1 DBP at 7 d. (e) The plant treated with 200 mg L−1 DBP at 7 d. CW: cell wall. ER: endoplasmic reticulum. M: mitochondria. P: plasma membrane. S: starch grain. V: vacuole.
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Table 1 Number of visible bands (S), Shannon index (H) and evenness index (E) based on DGGE analysis of bacterial communities on DGGE profiles. Time (day)
DBP concentration (mg L−1 ) 1 3 7
14
21
S
0 30 50 100 200
23 23 21 22 24
24 28 25 23 25
24 26 21 22 25
H
0 30 50 100 200
2.79 2.65 2.64 2.76 2.7
2.97 2.96 2.75 2.86 2.78
2.86 2.62 2.63 3.07 3.03
2.73 2.61 2.53 2.62 2.85
2.76 2.59 2.52 2.69 2.8
0 30 50 100 200
0.89 0.85 0.86 0.89 0.85
0.92 0.92 0.9 0.91 0.89
0.88 0.86 0.85 0.93 0.91
0.86 0.89 0.79 0.84 0.88
0.87 0.79 0.87 0.87 0.87
E
25 25 21 23 22
25 21 22 27 27
were analyzed by the Quantity One software (version 4.5) and Canoco for Windows 4.5 software, respectively. 3. Results 3.1. Protein content of cucumber seedling roots The effect of DBP stress on the protein content of cucumber seedling roots is shown in Fig. 1a. The result showed that the protein content of the roots gradually decreased with the extension in time of DBP treatment. But the decrease was more pronounced with increasing stress by DBP. Compared with the controls, the lowest protein contents were found at 200 mg L−1 DBP application on the seventh day. 3.2. Root activity of cucumber seedling Root activity varied with different concentrations of DBP and treatment time (Fig. 1b). The results indicated that, compared to the control group, root activity decreased obviously during the whole treatment time and at all DBP concentrations. The root activity was found to be inhibited significantly when exposed to 100 and 200 mg L−1 DBP; but inhibition was more severe at 200 mg L−1 DBP application.
soils). Analysis of the Pareto–Lorenz (PL) curves derived from DGGE patterns for soil treated with DBP at 7 d had the same result (Fig. 3). PL curves for rhizosphere soils treated with 100 mg L−1 and 200 mg L−1 DBP were closer to the theoretical perfect evenness line (the 45◦ diagonal) than for soil treated with 30 mg L−1 and 50 mg L−1 DBP. As a result, more evenness can be observed in the structure of the studied microbial community of 100 mg L−1 and 200 mg L−1 DBP treated soils. Compared to the control group, rhizosphere bacterial community abundances decreased significantly when DBP was applied at 50 mg L−1 . 3.5. Bacterial community structure PCR-DGGE analyzes showed that application of DBP changed the structures of rhizosphere bacterial communities (Fig. 4a). The DGGE banding patterns were different among the soils treated with different concentrations of DBP. Many bands were commonly detected in all samples. Number of visible bands disappeared when the concentration of DBP was higher than 50 mg L−1 , as observed in bands 3 and 5. PCA analyzes of the DGGE bacterial banding patterns showed that DBP treated soils were different from the control soil (Fig. 4b). Soils exposed to 50, 100 and 200 mg L−1 DBP after 3 days grouped together and had a significant change at 14 days. Application of DBP higher or lower than 50 mg L−1 had different influences on bacterial community structure. 3.6. Bacterial community composition In total, 18 partial sequences of bacterial 16S rRNA sequences were obtained from the control soil and soils treated with DBP. Phylogenetic tree with cut bands (bands 1–18) isolated strains and their closest relatives derived from GenBank data based on 16S rDNA gene analysis was built as shown in Fig. 5. Sequence analysis showed that the major dominants were from the Firmicutes, Actinomycetes, Proteobacteriaand Bacteroidetes family. DBP increased the relative abundances of Firmicutes while that of Bacteroidetes was decreased (Fig. 6). The decrease was enhanced with the extension in time of exposure to DBP treatment but more pronounced with increasing DBP concentration. When the concentration of DBP was higher than 50 mg L−1 the relative abundances of Actinomycetes and -proteobacteria decreased. Band 8 was detected in all samples and its sequence alignment from NCBI BLAST showed 100% similarity to Bacillus toyonensis BCT-7112. Actinobacteridae (band 3) and Pseudomonas (band 5) was detected in rhizosphere soil
3.3. Ultrastructure of cucumber seedling roots Ultrastructural micrographs of cucumber root showed the typical cellular ultrastructure of root-cells with or without DBP application (Fig. 2a–e). After application of DBP, swollen vesicles were detected within the cells and endoplasmic reticulum became blurred. Plasmolysis was observed in some cells, while the plasma membrane was ruptured. Specifically, the mitochondria gradually swelled and inner cristae became invisible until it eventually disintegrated under DBP stress. Moreover, compared with the control group, the number of starch grains increased abundantly and some of them adhered to other cellular apparatus. No arbuscular mycorrhiza was detected in ultrastructural micrographs. 3.4. Bacterial community abundance The results indicated that DBP significantly decreased rhizosphere bacterial community abundances during the whole treatment (Table. 1). Number of visible bands, Shannon index and evenness index were low in DBP treated soils than in the control soil except at the 7th day (in 100 mg L−1 and 200 mg L−1 DBP treated
Fig. 3. Pareto–Lorenz curves derived from DGGE patterns treated with DBP at 7 d. The 45◦ diagonal represents the perfect evenness of a community.
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Fig. 4. Effects of DBP on bacterial community structure. (a) DGGE profiles of partial bacterial 16S rDNA gene sequences. (b) PCA of bacterial community based on DGGE profiles. Letters of A–E represent soil sample treated with DBP after 1, 3, 7, 14 and 21 days, respectively. Figs. of 1–5 represent soil treated with 0, 30, 50, 100 and 200 mg L−1 DBP, respectively.
treated with 30 mg L−1 DBP. However, the species reduced or even disappeared when they were exposed to DBP higher than 30 mg L−1 after 7 days. 4. Discussion Protein synthesis exists in plant growth process; under adverse environmental impact, protein catabolism happens. In the present investigation, protein content was observed to decrease. However, Chinese cabbage leaves proteins were seen to increase under DBP treated [22]. That probably due to DBP exposure caused different injuries on different plants [23]. It means that DBP may have different influence on protein synthesis for different plants. Previous researches have shown that the detrimental effect of DBP stress on the ultrastructure was much [24]. The endoplasmic reticulum has long been considered as the cell’s protein factory, engaging in the biosynthesis, post-translational modification, folding, and trafficking of proteins [25]. Blurred endoplasmic reticulum could be another reason accounting for the decrease in protein content. Among the functions of the root is to absorb moisture, nutrient and fixed plan. Root growth, metabolism and energy change can directly affect the growth of the plant part above ground [26]. Phthalates have oxidative stress effect on plants, the activity of superoxide dismutase, catalase, peroxidase, the content of malondialdehyde and proline have changed after being polluted by DBP [7,27]. Reduced root activity may be caused by the formation of superfluous peroxide and oxygen free radical which damage the cell membrane structure. The roots ultrastructural study showed that mitochondrion was obviously affected. Similar findings were observed in root tip cells of soybean under Al stress [28]. The mitochondria provides the energy for cell activity, it is regarded as the “power house”. Mitochondrial damage was in conformity with the decreased root activity. Enlargement of the vacuoles could be observed in this work. Increase in the size of vacuoles might be helpful in forcing DBP into
a limited area and preventing the circulation of DBP in the cytosol [29]. The vacuole membrane, or tonoplast, has important roles in regulating plant cell metabolism, signaling and stress responses [30]. A large number of starch grains were discovered in the root cells. This increased starch grains were possibly due to the root cells not adapting to DBP and therefore presenting the aging change. Mesophyll cells also showed this appearance when it was exposed to Cd [31,32]. Moreover, we found some starch grains adhere to other apparatus. Perhaps this phenomenon is the most direct evidence that DBP causes cellular damage. The introduction of xenobiotic compounds affects the composition of the indigenous microbial community [33,34]. The adverse effects of PAEs on microorganism communities were not surprising, as di-(2-ethylhexyl) phthalate and diethyl phthalate have been reported to change microorganism communities in soils [35,36]. DBP can affect plant root membrane permeability [37]; thus, it is possible that DBP toxicity might lead to the root leakage of organic compounds that can affect soil microbial communities. Number of visible bands, Shannon index and evenness index are important indices for evaluation of soil microbial diversity. Pareto–Lorenz evenness curves can be constructed based on the DGGE profiles to represent species distribution of a bacterial community [38,39]. Compared with the control, microbial diversity in rhizosphere soil treated with 100 mg L−1 and 200 mg L−1 DBP at 7 d was higher in this research. It is likely that such a condition did not present a well-defined internal structure for species dominance. A relatively long lag phase might be needed to counteract stress due to DBP exposure and the community might have an ‘on average’ low functional organization [40]. PCR-DGGE profiles showed that changes in the abundance and structure of soil microbial communities was not obvious when DBP was added at concentrations lower than 50 mg L−1 . It was likely due to the fact that many microorganisms have developed defense system to pollutants [41]. But if the level of stress is beyond that which the microbes could
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Band 11 (KM 571935) Oceanobacillus kimchii X50 scaffold00001 (NZ CM001792.1) Bacillus toyonensis BCT-7112 (NC 022781.1) Band 8 (KM 571936) Band 3 (KM 571942) Frankia sp. EuI1c (NC 014666.1) Chitinophaga pinensis DSM 2588 (NC 013132.1) Band 2 (KM 571927) Fluviicola taffensis DSM 16823 (NC 015321.1) Pseudoxanthomonas spadix BD-a59 (NC 016147.2) Band 18 (KM 571928) Rhodanobacter sp. 2APBS1 (NC 020541.1) Band 15 (KM 571929) Escherichia coli str. K-12 substr. MG1655 (NC 000913.3) Band 6 (KM 571930) Acinetobacter oleivorans DR1 (NC 014259.1) Band 7 (KM 571931) Agrobacterium sp. H13 -3 (NC 015508.1) Band 12 (KM 571932) Sphingopyxis alaskensis RB2256 (NC 008048.1) Band 4 (KM 571925) Band 5 (KM 571939) Pseudomonas mendocina ymp (NC 009439.1) Band 17 (KM 571941) Band 1 (KM 571926) Thioalkalivibrio sp. K90mix (NC 013889.1) Band 14 (KM 571938) Band 9 (KM 571933) Bordetella petrii DSM 12804 (NC 010170.1) Band 10 (KM 571934) Burkholderia sp. CCGE1001 (NC 015137.1) Frateuria aurantia DSM 6220 (NC 017033.1) Band 13 (KM 571937) Streptococcus agalactiae 2603V/R (NC 004116.1) Band 16 (KM 571940) Ammonifex degensii KC4 (NC 013385.1)
0.2 Fig. 5. Phylogenetic tree with cut bands (bands 1–18) isolated strains and their closest relatives derived from GenBank data based on 16S rDNA gene analysis.
tolerate, then the microbes would be damaged. Notably, DBP stimulated and inhibited certain species of rhizosphere bacteria and decreased the diversity indices of their community. The result indicated that the response of soil bacterial community structure to DBP was time and concentration dependent. Yuan et al. [11] found three of the entire PAE-degrading bacteria were Bacillus strains, which indicates that Bacillus were dominant in the degradation of PAE in mangrove sediment. In this study, band 8 showed 100% sequence similarity to Bacillus toyonensis BCT-
7112. It is possible that DBP can be used as carbon source by the bacteria, therefore, even under the condition of high concentrations of DBP band 8 still existed. On the other hand, Bacillus bacteria are among the most common plant growth promoting rhizobacteria (PGPR) species [42]. It has been suggested that PGPR may promote the plant growth by facilitating the nutrient uptake of plant roots and by producing and supplying growth-promoting substances to the plant roots [43]. Bacillus may protect cucumber by degrading DBP.
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Fig. 6. The relative abundances of the predominant phylogenetic microbial groups in DBP contaminated cucumber rhizosphere soil. (a)–(e) represented samples treated with 0, 30, 50, 100 and 200 mg L−1 of DBP, respectively. Relative abundance is defined as the number of sequences affiliated with that taxon divided by the total number of sequences per sample (%).
Several of the identified microbial species are confirmed to be able to degrade phthalates. The Actinomycetes group, which emerged at higher DEP concentrations, is known to be included among efficient DEP-degrading strains [44,45]. Xu et al. [46] studied n-butyl benzyl phthalate degradation using the Pseudomonas fluorescens B-1. In the present study, large amount of Actinobacteridae and Pseudomonas were detected in rhizosphere soil treated with 30 mg L−1 DBP, but decreased or disappeared in higher DBPtreated rhizosphere soil. It might be these two kinds of bacteria could only degrade DBP at low concentrations. Cartwright et al. [47] reported that Pseudomonas were more sensitive to higher concentrations of DEP. For the change of Actinobacteridae was possibly owing to different structures of pollutants. Application of DBP increased the relative abundances of Firmicutes, which was also found in cucumber rhizosphere soils under p-coumaric acid stress [48]. The reason might be that members of Firmicutes occupy a wide range of habitats; it can resist dehydration and extreme environments [49]. We have not found any
report of DBP-degrading -Proteobacteria sp., or Bacteroidetes sp. The decreases in the relative abundance of -Proteobacteria and Bacteroidetes may be an alteration in the cucumber rhizosphere bacterial community structure as microbial community typing is an effective method for monitoring pollutants [50]. 5. Conclusions DBP do have adverse effect on cucumber root physiology and plant rhizosphere microorganisms. Root protein content and root activity all decreased. Obvious damaged organs in the root cells were observed through ultrastructural micrographs. The microbial community changed with various treatments in the plant rhizosphere. Microbial community expressed obvious changes when DBP was applied at concentrations higher than 50 mg L−1 . Bacillus was detected in all DBP contaminated cucumber rhizospheric soils. Actinobacteridae and Pseudomonas had different responses to different concentrations of DBP. The relative abundances of Firmicutes, Proteobacteria and Bacteroidetes changed under DBP stress.
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Acknowledgments This research was supported by the National High Technology Research and 863 Development Program of China (2012AA101405).
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[24] Fig. A1. The chemical structure of DBP. [25] [26]
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Effect of di-n-butyl phthalate on root physiology and rhizosphere microbial community of cucumber seedlings Ying Zhang a,∗ , Yue Tao a , Hui Zhang a , Lei Wang a , Guoqiang Sun a , Xin Sun a , Kehinde O. Erinle a , Chengcheng Feng a , Qiuxia Song a , Mo Li b a b
School of Resources & Environment, Northeast Agricultural University, Harbin 150030, PR China School of Geography, University of Nottingham, Nottinghamshire NG72RD, UK
h i g h l i g h t s • • • •
Cucumbers were grown with DBP supplementation. DBP had a significant effect on root physiology of cucumber seedlings. The ultrastructural study showed that the organelles were obviously affected by DBP. A period of an ‘on average’ low functional organization was discovered.
a r t i c l e
i n f o
Article history: Received 29 October 2014 Received in revised form 24 January 2015 Accepted 28 January 2015 Available online 11 February 2015 Keywords: DBP Cucumber rhizosphere microbial community Protein content Root activity Ultrastructure
a b s t r a c t The authors investigated the effects of di-n-butyl phthalate (DBP) on root physiology and rhizosphere microbial communities of cucumber seedlings (sativus L. cv Jinyan No. 4). Root protein content and root activity were observed to decrease. From the ultrastructural micrographs, visible impact on the mitochondria, endoplasmic reticulum and vacuole were detected. Moreover, the number of starch grains increased, and some were adhered to other cell components which might be the most direct evidence of DBP causing cellular damage. Results of PCR-DGGE (denaturing gradient gel electrophoresis) indicated that DBP significantly changed the abundance, structure and composition of rhizosphere bacteria when the concentration was higher than 50 mg L−1 . The relative abundances of Firmicutes increased while that of Bacteroidetes decreased. Bacillus was detected as the dominant bacteria in DBP contaminated cucumber rhizospheric soil. The amount of Actinobacteridae and Pseudomonas decreased until it disappeared in the rhizosphere soil when exposed to DBP concentrations higher than 50 mg L−1 . © 2015 Elsevier B.V. All rights reserved.
1. Introduction DBP is one of the important members in the phthalate esters family (PAEs) that has been listed as a priority pollutant by the US Environmental Protection Agency [1]. It is a kind of organic compound that is primarily used as plasticizers in the preparation of polyvinyl chloride (PVC) resins, plastic packing films, adhesives, cosmetics, cellulose materials and insect repellents [2]. Like many other PAEs, DBP is not chemically bonded to the polymer products and it migrates readily from the plastics to the environment [3]. Concerns have been raised about D-BP because it is suspected to have mutagenous, carcinogenous, and endocrine-distrupting effects [4,5].
∗ Corresponding author. Tel.: +86 451 5519 0993; fax: +86 451 5519 1170. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.jhazmat.2015.01.071 0304-3894/© 2015 Elsevier B.V. All rights reserved.
Due to increase in the use of plastic mulching films in agricultural facilities, the content of PAEs in the soil is also on the increase in the agricultural facilities [6]. PAEs play a detrimental role in the physiology of plants by inhibiting plant growth and development [7]. Humans are exposed to PAEs through its entry into the food chain via the contamination of vegetables and food items [8]. It is estimated that the contribution percentages of human exposure to DBP through vegetables, fruits and grains pathways were 42.4%, 2.0% and 7.5% [9]. Yin et al. [10] demonstrated that the decrease of capsaicin content and vitamin C was negatively correlated to the increase the concentration of DBP in capsicum fruit, which suggested that DBP uptake by the plant might be mainly responsible for quality degradation of capsicum fruit. Plant root as medium of absorption and transportation of nutrients, have an important effect on plant growth. Root protein and root activity are used as the general indicators of plant development. Cell ultrastructure could reflect the plants growth whether in good condition.
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Previous researches have proved that microbial community composition show significant differences in the early and late periods of PAEs contamination in the soil [11]. Microbes living in the plant root zone (rhizosphere) exert a profound impact on soil fertility and plant development [12]. Hence, changes in the soil microbial communities may affect functions performed by the microbes, and thus influence overall plant growth and development [13,14]. It means that not only does the contaminant directly affect both the plants and soil microbes, but also plays a detrimental role in the interaction between both components; thus, the further release of DBP into the ecosystem will further increase the level of severity both plants and microorganisms are exposed to. However, since detailed information about the adverse effects of DBP on plant roots and rhizosphere microorganisms are still not available, and thus requires further study. In this study, cucumber was employed as a target plant because it is one of the most widely consumed vegetables and is usually cultivated in greenhouse conditions. Extensive use of plastic films caused a serious threat to agricultural environment, resulting in significant effect on cucumber [6,15]. Different concentrations of DBP were applied to the cucumber seedlings to investigate their effects on root protein content, root activity and root cell ultrastructure. Changes in bacterial species diversity in the cucumber rhizosphere were also studied using DGGE of the PCR-amplified 16S rRNA gene.
2. Materials and methods 2.1. Plant materials and stress treatment Seeds of cucumber sativus L. cv Jinyan No. 4 were planted into planting pots at the horticultural garden of Northeast Agricultural University, Harbin, China. The soil was also sampled in the same
garden. The soil was a black soil with sandy loam texture. The basic properties of the soil: soil water content, 3.74%; TOC, 3.96%; N, 17.1 mg kg−1 ; P, 12.9 mg kg−1 ; K, 301.17 mg kg−1 ; pH, 7.16; No fertilizer was previously added to the soil. The average temperature in the greenhouse was 27 ± 1 ◦ C during the day and 20 ± 1 ◦ C during the night. The relative humidity was kept at 60–70%. Applications of DBP treatment to cucumber plants were made according to the method described by Zhang et al. [16]. 2.2. Root protein content Root tissues (0.5 g) were ground to fine powder using a chilled pestle and mortar. Five milliliter of 50 mM potassium phosphate buffer (pH 7.0), 1 mM ascorbic acid, 0.2 mM ethylenediamine tetraacetic acid and 2% (w/v) polyvinylpyrrolidone were added to the powder. The homogenate was centrifuged at 4 ◦ C for 20 min at 12,000 g. Supernatant was used for protein content assays. An aliquot of the extract was used to determine its protein content by the method of Redmile-Gordone et al. [17] utilizing bovine serum albumin as the standard. 2.3. Root activity Root activity was measured using the TTC (triphenyl tetrazolium chloride) method [18] and expressed as the deoxidization ability (mg g−1 h−1 ). Dehydrogenase was expressed as the deoxidized TTC quantity, which was an index of root activity [19]. Ten milliliter solution of equal quantities of TTC (0.4%) and phosphate buffer was added to root samples (0.5 g) and kept in the dark at 37 ◦ C for 2 h. The reaction was stopped with 1 M H2 SO4 . The roots were ground and transferred into a tube with ethyl acetate to a total volume of 10 ml. The solution was measured at the absorbance of 485 nm.
Fig. 1. The effect of DBP on root physiology of Cucumber sativus L. cv Jinyan No. 4. (a) Protein content. (b) Root activity. Means with different small letters are significantly different from one another under the DBP different concentration treatment, and different capital letters are significantly different from one another under the DBP different treatment times.
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2.4. Observations of root ultrastructure The method of root cell ultrastructure was conducted as described as Zhang et al. [16]. Small root fragments were collected and fixed in 2.5% (v/v) glutaraldehyde solution (pH 7.2) at 4 ◦ C overnight. The samples were washed three times by phosphatebuffered saline solution (PBS) (pH 6.8) and post-fixed with 2% (m/v) osmium tetroxide solution for 4 h in a hood. Then the samples were rinsed with PBS (pH 6.8) and dehydrated through different concentrations of ethanol solutions. Ethanol was replaced by acetone solution twice. Finally, the specimens were embedded in Spurr’s resin for fixing and polymerized at 60 ◦ C. Ultra-thin sections (80 nm) were obtained on copper grids (300 meshes) and double stained with 1.0% (w/v) uranyl acetate followed by 5.0% (w/v) lead citrate. Root tissues were observed with an H-600IV transmission electron microscope (Hitachi, Tokyo, Japan) at 90 kV.
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of template DNA, 2.5 mmol/L each dNTPs mixture. The quality and the size of the PCR products were visualized after electrophoresis on 0.6% (w/v) agarose gel. DGGE was used to assess the diversity of rhizosphere bacterial. Identical amounts of PCR products were loaded in 8% polyacrylamide gels with denaturing gradients ranging from 30% to 60% (100% denaturant contains 7 M urea and 40% formamide). Electrophoresis was performed at a constant voltage of 180 V for 4 h in 1 TAE buffer (40 mM Tris, 20 mM sodium acetate, 1 mmM EDTA, pH 7.4) at 65 ◦ C. Gels were stained with ethidium bromide and illuminated under UV light using a Gel Doc XR Bio-Rad system. Main strips were cut and again recycled to PCR with primers without GC clip. PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd., for sequencing.
2.7. Phylogenetic analysis 2.5. Extraction of total DNA from soil Cucumber rhizosphere soil was collected for DNA extraction at 1, 3, 7, 14 and 21 days after DBP treatment. Samples were collected by shaking the roots by hand to remove the soil adhering to the roots. Total DNA was extracted with a kit of OMEGA Bio-TEK. The DNA was confirmed by agarose gel electrophoresis.
Consensus phylogenetic trees were constructed based on Kimura two-parameter evolutionary distance matrices and neighbor-joining with a Boostrap analysis of 1000 replicates, using MEGA5. Each sequence and their closest relatives, identified in the GenBank database, were used for phylogenetic analyzes [21]. Sequences recovered from DGGE bands were submitted to GenBank under the accession numbers KM571925–KM571942.
2.6. PCR-DGGE 2.8. Statistical analyzes 200 bp fragments of the V3 region of bacterial 16S rRNA gene were amplified by PCR with GC-341F/518R primer [20]. The PCR condition was 10 min at 94 ◦ C followed by 35 cycles of 30 s denaturation at 94 ◦ C, 30 s extension at 72 ◦ C, and final thermal insulation step at 72 ◦ C for 10 min. Each reaction mixture (50 ul) contained 5 U Taq DNA polymerase, 10 × EasyTaq buffer, 10 pmol primers, 1 umol
All the experimental data were expressed with five replicates. Data were analyzed by one-way analysis of variance. Mean values were compared by Duncan’s new multiple range test at the 0.05% level using the SPSS 19.0 software. The data were represented as mean ± SE. DGGE profiles and principal component analysis (PCA)
Fig. 2. Transmission electron microscopy (TEM) micrographic images of Cucumber sativus L. cv Jinyan No. 4. (a) Control plant (without DBP). (b) The plant treated with 30 mg L−1 DBP at 7 d. (c) The plant treated with 50 mg L−1 DBP at 7 d. (d) The plant treated with 100 mg L−1 DBP at 7 d. (e) The plant treated with 200 mg L−1 DBP at 7 d. CW: cell wall. ER: endoplasmic reticulum. M: mitochondria. P: plasma membrane. S: starch grain. V: vacuole.
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Table 1 Number of visible bands (S), Shannon index (H) and evenness index (E) based on DGGE analysis of bacterial communities on DGGE profiles. Time (day)
DBP concentration (mg L−1 ) 1 3 7
14
21
S
0 30 50 100 200
23 23 21 22 24
24 28 25 23 25
24 26 21 22 25
H
0 30 50 100 200
2.79 2.65 2.64 2.76 2.7
2.97 2.96 2.75 2.86 2.78
2.86 2.62 2.63 3.07 3.03
2.73 2.61 2.53 2.62 2.85
2.76 2.59 2.52 2.69 2.8
0 30 50 100 200
0.89 0.85 0.86 0.89 0.85
0.92 0.92 0.9 0.91 0.89
0.88 0.86 0.85 0.93 0.91
0.86 0.89 0.79 0.84 0.88
0.87 0.79 0.87 0.87 0.87
E
25 25 21 23 22
25 21 22 27 27
were analyzed by the Quantity One software (version 4.5) and Canoco for Windows 4.5 software, respectively. 3. Results 3.1. Protein content of cucumber seedling roots The effect of DBP stress on the protein content of cucumber seedling roots is shown in Fig. 1a. The result showed that the protein content of the roots gradually decreased with the extension in time of DBP treatment. But the decrease was more pronounced with increasing stress by DBP. Compared with the controls, the lowest protein contents were found at 200 mg L−1 DBP application on the seventh day. 3.2. Root activity of cucumber seedling Root activity varied with different concentrations of DBP and treatment time (Fig. 1b). The results indicated that, compared to the control group, root activity decreased obviously during the whole treatment time and at all DBP concentrations. The root activity was found to be inhibited significantly when exposed to 100 and 200 mg L−1 DBP; but inhibition was more severe at 200 mg L−1 DBP application.
soils). Analysis of the Pareto–Lorenz (PL) curves derived from DGGE patterns for soil treated with DBP at 7 d had the same result (Fig. 3). PL curves for rhizosphere soils treated with 100 mg L−1 and 200 mg L−1 DBP were closer to the theoretical perfect evenness line (the 45◦ diagonal) than for soil treated with 30 mg L−1 and 50 mg L−1 DBP. As a result, more evenness can be observed in the structure of the studied microbial community of 100 mg L−1 and 200 mg L−1 DBP treated soils. Compared to the control group, rhizosphere bacterial community abundances decreased significantly when DBP was applied at 50 mg L−1 . 3.5. Bacterial community structure PCR-DGGE analyzes showed that application of DBP changed the structures of rhizosphere bacterial communities (Fig. 4a). The DGGE banding patterns were different among the soils treated with different concentrations of DBP. Many bands were commonly detected in all samples. Number of visible bands disappeared when the concentration of DBP was higher than 50 mg L−1 , as observed in bands 3 and 5. PCA analyzes of the DGGE bacterial banding patterns showed that DBP treated soils were different from the control soil (Fig. 4b). Soils exposed to 50, 100 and 200 mg L−1 DBP after 3 days grouped together and had a significant change at 14 days. Application of DBP higher or lower than 50 mg L−1 had different influences on bacterial community structure. 3.6. Bacterial community composition In total, 18 partial sequences of bacterial 16S rRNA sequences were obtained from the control soil and soils treated with DBP. Phylogenetic tree with cut bands (bands 1–18) isolated strains and their closest relatives derived from GenBank data based on 16S rDNA gene analysis was built as shown in Fig. 5. Sequence analysis showed that the major dominants were from the Firmicutes, Actinomycetes, Proteobacteriaand Bacteroidetes family. DBP increased the relative abundances of Firmicutes while that of Bacteroidetes was decreased (Fig. 6). The decrease was enhanced with the extension in time of exposure to DBP treatment but more pronounced with increasing DBP concentration. When the concentration of DBP was higher than 50 mg L−1 the relative abundances of Actinomycetes and -proteobacteria decreased. Band 8 was detected in all samples and its sequence alignment from NCBI BLAST showed 100% similarity to Bacillus toyonensis BCT-7112. Actinobacteridae (band 3) and Pseudomonas (band 5) was detected in rhizosphere soil
3.3. Ultrastructure of cucumber seedling roots Ultrastructural micrographs of cucumber root showed the typical cellular ultrastructure of root-cells with or without DBP application (Fig. 2a–e). After application of DBP, swollen vesicles were detected within the cells and endoplasmic reticulum became blurred. Plasmolysis was observed in some cells, while the plasma membrane was ruptured. Specifically, the mitochondria gradually swelled and inner cristae became invisible until it eventually disintegrated under DBP stress. Moreover, compared with the control group, the number of starch grains increased abundantly and some of them adhered to other cellular apparatus. No arbuscular mycorrhiza was detected in ultrastructural micrographs. 3.4. Bacterial community abundance The results indicated that DBP significantly decreased rhizosphere bacterial community abundances during the whole treatment (Table. 1). Number of visible bands, Shannon index and evenness index were low in DBP treated soils than in the control soil except at the 7th day (in 100 mg L−1 and 200 mg L−1 DBP treated
Fig. 3. Pareto–Lorenz curves derived from DGGE patterns treated with DBP at 7 d. The 45◦ diagonal represents the perfect evenness of a community.
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Fig. 4. Effects of DBP on bacterial community structure. (a) DGGE profiles of partial bacterial 16S rDNA gene sequences. (b) PCA of bacterial community based on DGGE profiles. Letters of A–E represent soil sample treated with DBP after 1, 3, 7, 14 and 21 days, respectively. Figs. of 1–5 represent soil treated with 0, 30, 50, 100 and 200 mg L−1 DBP, respectively.
treated with 30 mg L−1 DBP. However, the species reduced or even disappeared when they were exposed to DBP higher than 30 mg L−1 after 7 days. 4. Discussion Protein synthesis exists in plant growth process; under adverse environmental impact, protein catabolism happens. In the present investigation, protein content was observed to decrease. However, Chinese cabbage leaves proteins were seen to increase under DBP treated [22]. That probably due to DBP exposure caused different injuries on different plants [23]. It means that DBP may have different influence on protein synthesis for different plants. Previous researches have shown that the detrimental effect of DBP stress on the ultrastructure was much [24]. The endoplasmic reticulum has long been considered as the cell’s protein factory, engaging in the biosynthesis, post-translational modification, folding, and trafficking of proteins [25]. Blurred endoplasmic reticulum could be another reason accounting for the decrease in protein content. Among the functions of the root is to absorb moisture, nutrient and fixed plan. Root growth, metabolism and energy change can directly affect the growth of the plant part above ground [26]. Phthalates have oxidative stress effect on plants, the activity of superoxide dismutase, catalase, peroxidase, the content of malondialdehyde and proline have changed after being polluted by DBP [7,27]. Reduced root activity may be caused by the formation of superfluous peroxide and oxygen free radical which damage the cell membrane structure. The roots ultrastructural study showed that mitochondrion was obviously affected. Similar findings were observed in root tip cells of soybean under Al stress [28]. The mitochondria provides the energy for cell activity, it is regarded as the “power house”. Mitochondrial damage was in conformity with the decreased root activity. Enlargement of the vacuoles could be observed in this work. Increase in the size of vacuoles might be helpful in forcing DBP into
a limited area and preventing the circulation of DBP in the cytosol [29]. The vacuole membrane, or tonoplast, has important roles in regulating plant cell metabolism, signaling and stress responses [30]. A large number of starch grains were discovered in the root cells. This increased starch grains were possibly due to the root cells not adapting to DBP and therefore presenting the aging change. Mesophyll cells also showed this appearance when it was exposed to Cd [31,32]. Moreover, we found some starch grains adhere to other apparatus. Perhaps this phenomenon is the most direct evidence that DBP causes cellular damage. The introduction of xenobiotic compounds affects the composition of the indigenous microbial community [33,34]. The adverse effects of PAEs on microorganism communities were not surprising, as di-(2-ethylhexyl) phthalate and diethyl phthalate have been reported to change microorganism communities in soils [35,36]. DBP can affect plant root membrane permeability [37]; thus, it is possible that DBP toxicity might lead to the root leakage of organic compounds that can affect soil microbial communities. Number of visible bands, Shannon index and evenness index are important indices for evaluation of soil microbial diversity. Pareto–Lorenz evenness curves can be constructed based on the DGGE profiles to represent species distribution of a bacterial community [38,39]. Compared with the control, microbial diversity in rhizosphere soil treated with 100 mg L−1 and 200 mg L−1 DBP at 7 d was higher in this research. It is likely that such a condition did not present a well-defined internal structure for species dominance. A relatively long lag phase might be needed to counteract stress due to DBP exposure and the community might have an ‘on average’ low functional organization [40]. PCR-DGGE profiles showed that changes in the abundance and structure of soil microbial communities was not obvious when DBP was added at concentrations lower than 50 mg L−1 . It was likely due to the fact that many microorganisms have developed defense system to pollutants [41]. But if the level of stress is beyond that which the microbes could
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Band 11 (KM 571935) Oceanobacillus kimchii X50 scaffold00001 (NZ CM001792.1) Bacillus toyonensis BCT-7112 (NC 022781.1) Band 8 (KM 571936) Band 3 (KM 571942) Frankia sp. EuI1c (NC 014666.1) Chitinophaga pinensis DSM 2588 (NC 013132.1) Band 2 (KM 571927) Fluviicola taffensis DSM 16823 (NC 015321.1) Pseudoxanthomonas spadix BD-a59 (NC 016147.2) Band 18 (KM 571928) Rhodanobacter sp. 2APBS1 (NC 020541.1) Band 15 (KM 571929) Escherichia coli str. K-12 substr. MG1655 (NC 000913.3) Band 6 (KM 571930) Acinetobacter oleivorans DR1 (NC 014259.1) Band 7 (KM 571931) Agrobacterium sp. H13 -3 (NC 015508.1) Band 12 (KM 571932) Sphingopyxis alaskensis RB2256 (NC 008048.1) Band 4 (KM 571925) Band 5 (KM 571939) Pseudomonas mendocina ymp (NC 009439.1) Band 17 (KM 571941) Band 1 (KM 571926) Thioalkalivibrio sp. K90mix (NC 013889.1) Band 14 (KM 571938) Band 9 (KM 571933) Bordetella petrii DSM 12804 (NC 010170.1) Band 10 (KM 571934) Burkholderia sp. CCGE1001 (NC 015137.1) Frateuria aurantia DSM 6220 (NC 017033.1) Band 13 (KM 571937) Streptococcus agalactiae 2603V/R (NC 004116.1) Band 16 (KM 571940) Ammonifex degensii KC4 (NC 013385.1)
0.2 Fig. 5. Phylogenetic tree with cut bands (bands 1–18) isolated strains and their closest relatives derived from GenBank data based on 16S rDNA gene analysis.
tolerate, then the microbes would be damaged. Notably, DBP stimulated and inhibited certain species of rhizosphere bacteria and decreased the diversity indices of their community. The result indicated that the response of soil bacterial community structure to DBP was time and concentration dependent. Yuan et al. [11] found three of the entire PAE-degrading bacteria were Bacillus strains, which indicates that Bacillus were dominant in the degradation of PAE in mangrove sediment. In this study, band 8 showed 100% sequence similarity to Bacillus toyonensis BCT-
7112. It is possible that DBP can be used as carbon source by the bacteria, therefore, even under the condition of high concentrations of DBP band 8 still existed. On the other hand, Bacillus bacteria are among the most common plant growth promoting rhizobacteria (PGPR) species [42]. It has been suggested that PGPR may promote the plant growth by facilitating the nutrient uptake of plant roots and by producing and supplying growth-promoting substances to the plant roots [43]. Bacillus may protect cucumber by degrading DBP.
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Fig. 6. The relative abundances of the predominant phylogenetic microbial groups in DBP contaminated cucumber rhizosphere soil. (a)–(e) represented samples treated with 0, 30, 50, 100 and 200 mg L−1 of DBP, respectively. Relative abundance is defined as the number of sequences affiliated with that taxon divided by the total number of sequences per sample (%).
Several of the identified microbial species are confirmed to be able to degrade phthalates. The Actinomycetes group, which emerged at higher DEP concentrations, is known to be included among efficient DEP-degrading strains [44,45]. Xu et al. [46] studied n-butyl benzyl phthalate degradation using the Pseudomonas fluorescens B-1. In the present study, large amount of Actinobacteridae and Pseudomonas were detected in rhizosphere soil treated with 30 mg L−1 DBP, but decreased or disappeared in higher DBPtreated rhizosphere soil. It might be these two kinds of bacteria could only degrade DBP at low concentrations. Cartwright et al. [47] reported that Pseudomonas were more sensitive to higher concentrations of DEP. For the change of Actinobacteridae was possibly owing to different structures of pollutants. Application of DBP increased the relative abundances of Firmicutes, which was also found in cucumber rhizosphere soils under p-coumaric acid stress [48]. The reason might be that members of Firmicutes occupy a wide range of habitats; it can resist dehydration and extreme environments [49]. We have not found any
report of DBP-degrading -Proteobacteria sp., or Bacteroidetes sp. The decreases in the relative abundance of -Proteobacteria and Bacteroidetes may be an alteration in the cucumber rhizosphere bacterial community structure as microbial community typing is an effective method for monitoring pollutants [50]. 5. Conclusions DBP do have adverse effect on cucumber root physiology and plant rhizosphere microorganisms. Root protein content and root activity all decreased. Obvious damaged organs in the root cells were observed through ultrastructural micrographs. The microbial community changed with various treatments in the plant rhizosphere. Microbial community expressed obvious changes when DBP was applied at concentrations higher than 50 mg L−1 . Bacillus was detected in all DBP contaminated cucumber rhizospheric soils. Actinobacteridae and Pseudomonas had different responses to different concentrations of DBP. The relative abundances of Firmicutes, Proteobacteria and Bacteroidetes changed under DBP stress.
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Acknowledgments This research was supported by the National High Technology Research and 863 Development Program of China (2012AA101405).
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See Fig. A1. [21]
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[24] Fig. A1. The chemical structure of DBP. [25] [26]
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