Isolation of a Pseudomonas putida strain that degrades p -hydroxybenzoic acid from the soil of a Panax ginseng field

Background: Panax ginseng is a valuable herbal medicine which has been applied in eastern Asia to maintain their physical vitality for millennia. However, continuous cropping leads to the accumulation of allelochemicals, which subsequently reduces the quality and yield of P. ginseng , and arable soils for P. ginseng cultivation are getting scarce. Methods: In this study, a strain highly efficient degrading p -hydroxybenzoic acid was isolated from the rhizosphere soil of a ginseng cultivation field. Subsequently, its draft genome was sequenced, and the key genes involving the p -hydroxybenzoic acid-degrading pathway were identified by bioinformatics software. Results: Based on its biophysical/biochemical characteristics, analysis of the 16S rRNA sequence and gyrB gene authentication, strain LD6 was identified as Pseudomonas putida LD6. The initial p hydroxybenzoic acid concentration in minimal salt medium was 100 mg•L -1 , and after 8 d of bacterial culture in the laboratory, the degraded reached 97.35%. The p -hydroxybenzoic acid content was decreased by 35.21%, and the ginseng death rate was decreased by 38.46% in the rhizosphere soil of P. ginseng inoculated with strain LD6. The draft genome sequence consisted of 5,765,634 bp, and the genome comprised 5186 protein-coding genes, and the pobA , catB , pcaG , UbiA , and UbiX genes were annotated, and may play important roles in p -hydroxybenzoic acid degradation. Conclusion: Isolation and manipulation of functional bacteria is an effective strategy to alleviate ginseng continuous cropping problems and enhance the quality and yield of P. ginseng .

ginseng rhizosphere soil and inhibit the growth of ginseng seedlings, and it was found in abundance in our test field which was secreted by the roots of ginseng. Eliminating the allelochemicals is an effective method to alleviate the continuous cropping obstacles and increase crop productivity of P.

ginseng.
Microbial degradation is an important and effective strategy for the bioremediation of environmentally toxic substances [9]. As one example, Wu et al. [10] isolated a Bacillus sp. strain that can efficiently biodegrade di-n-butyl phthalate in a shallow aquifer environment. Microbial degradation of allelochemicals also occurs in the exudates produced during the growth of medicinal plants. Zhao et al. [11] isolated five bacterial strains possessing autotoxin-degrading activity from non-rhizospheric soil in which 6-year-old ginseng plants were grown, and the strains were used to degrade autotoxins in the planting base of P. ginseng. Therefore, there is an urgent need to develop remediation with toxic substance-degrading bacteria to eliminate continuous cropping problems in P. ginseng plantation. However, little is known regarding how ginseng-associated bacteria degrade toxic substances, and thus, their degradation mechanisms require elucidation.
Microbial degradation would be helpful and remediation strategies could be developed to alleviate poor growth of P. ginseng caused by autotoxins. During the course of an investigation of a bacterial community in the rhizosphere soil obtained from a P. ginseng field, a p-hydroxybenzoic aciddegrading strain was isolated and designated strain LD6. Phenotypic, physiological, and phylogenetic analyses based on 16S rRNA gene sequences were performed, and LD6 was identified as a P. putida.
In the current study, the strain degradation rate of the p-hydroxybenzoic acid from the bacterial culture and rhizosphere soils of P. ginseng was analyzed, and the draft genome sequence and degradation mechanism of P. putida LD6 were analyzed.

Materials and reagents
Rhizosphere soil adhering to the roots of P. ginseng was collected from a ginseng-cultivated field in Jilin, China (E: 126.85°, N: 42.39°) (Fig. S1 in Additional file 3). The ingredients used to create the mineral salt medium (MSM) have been previously described by Wang et al. [12]. The genomic DNA of bacterial strain was extracted and purification by Genomic DNA Purification Kit (Tiangen, Beijing, China). The p-hydroxybenzoic acid (99% purity) was procured from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultra-high performance liquid chromatography (UPLC) grade formic acid and methanol were obtained from CNW Technologies GmbH (Dusseldorf, Germany). Other chemical reagents were analytical grade and were purchased from local suppliers.

Strain isolation and culture conditions
The rhizosphere soil was dispelled by gently shaking the roots of P. ginseng, and then the soil was collected and homogenized through a 2-mm sieve and stored at 4°C for use. Next, one gram of fresh, intensively cultivated soil was homogenized in 100 ml MSM inorganic culture solution that contained allelochemicals (100 mg•L -1 ) as the sole carbon source. In order to dilute and reduce possible carbon sources from soils, incubation and transfer were performed three times at 30°C for 5 days, each time on a rotary shaker operating at 150 r•min -1 . The final enriched bacteria were isolated following serial dilution with distilled water. Petri plates with separated single colonies were stored at 4°C for the following experiments [11]. One of the purified strains, designated LD6, displayed superior efficiency in the degradation of p-hydroxybenzoic acid, and was selected for further characterization. The isolate was deposited at -80°C in the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

Strain LD6 growth and p-hydroxybenzoic acid degradation
For the determination of the degradation efficiency of p-hydroxybenzoic acid, a single colony of strain LD6 was inoculated in 3 ml MSM (in a 15-ml plastic tube with cover), and incubated at 30°C with shaking (120 r•min -1 ) until the OD600 of the culture solution reached 0.5. The culture solution was transferred to 100 ml MSM, with an initial concentration of p-hydroxybenzoic acid at 100 mg•L -1 , and bacterial strain at 1.4×10 7 CFU•ml -1 in 250-ml flasks, and incubated at 30°C with shaking at 120 r•min -1 . The OD600 of the culture solution, the autotoxin content, and the degradation in soil were analyzed after 0, 0.5, 1, 1.5, 2, 4, 6, and 8 d of incubation [11]. Except for isolation and identification experiments, other tests were repeated at least three times and with three parallel 5 treatments.

Phenotypic and biochemical characteristics of LD6
The Gram reaction of strain LD6 was determined as previously described [13]. The bacterial morphology was determined by incubation on LB medium for 1 d, and then observation and recording using a Hitachi S-3400N scanning electron microscope. Growth was assessed in LB agar (LB, Difco) at 25°C. Catalase and oxidase utilization was determined as previously described [14]. The effect of pH on growth was analyzed in R2A broth using the three buffer tests (acetate buffer 5.0-5.5, phosphate buffer 6.0-8.0, and Tris buffer 8.5-10.0). To determine salt tolerance, the bacteria were grown on R2A agar with 0.5%-10% (w•v -1 ) NaCl for 7 days. Physiological characteristics were determined according the method of Park et al. [15].

16S rRNA sequencing and phylogenetic analysis
The 16S rRNA gene of strain LD6 was amplified from bacterial genomic using the 27F and 1492R universal bacterial primers. Purified PCR products were analyzed using a 3730 XL sequencer (Applied Biosystems). The 16S rRNA gene sequences of other species were downloaded from GenBank and compared with the sequence of strain LD6 using the Clustal X program [16].
Phylogenetic trees based on the neighbor-joining algorithm were constructed using MEGA v4.0 software [17]. The evolutionary distances for strain LD6 were calculated using the maximum composite likelihood method [18].

Influences of strain LD6 on the replanting soil of P. ginseng after inoculation
To assess the degradation efficiency of LD6, we implemented a field experiment from June to August in the replant soil plantation base of P. ginseng. Three biological replicate blocks were used, and each block contained one field plot (1.2 m×5 m) with an initial p-hydroxybenzoic acid concentration of 1 mg•kg -1 . Two hundred 2-year old uniform seedlings were planted in each plot on May 1. Successively, 10 mL (10 7 CFU mL −1 ) of P. putida culture was added to the rhizosphere of uniform P. ginseng plants on June 1. The plot without the addition of cultures containing P. putida acted as a control (CK). After 60 d of inoculation, soil samples from each plot were collected and subsequently used for the analysis 6 of the p-hydroxybenzoic acid degradation rate and the death rate of P. ginseng [4].

UPLC analytical methods
A reaction mixture containing p-hydroxybenzoic acid and LD6 was incubated in MSM at 30°C for 8 d.

DNA preparation and genomic sequencing
Total genomic DNA was extracted using a T-Guide Bacteria Genomic DNA Kit (OSR-M502, TIANGEN, China), and purified by RNase-free DNase I (Takara, Japan). The DNA was sheared using a S220 focused ultrasonicator (Covaris, USA). Purified genomic DNA was analyzed using a hybrid sequencing strategy that combined the PacBio RS II and Illumina HiSeq sequencing platforms as previously described [20].

Bioinformatics analysis
After sequencing, the filtered low-quality reads were assembled with SOAPdenovo software to generate scaffolds. The cleaned reads were assembled using Velvet (version 1.2.07) and PrInSeS-G (version 1.0.0. beta) to correct sequencing errors [21]. Open reading frames (ORFs) were predicted and translated using MetaGene Annotator [22]. The nucleotide sequence was compared to the NCBI database (http://www.ncbi.nlm.nih.gov/). The tRNA and rRNA analyses were performed using tRNAscan-SE v.1.4 and RNAmmer, respectively [23]. Protein-coding gene prediction and gene annotation were conducted using RPSBLAST and Prokka, respectively [24]. Gene prediction and assembly were performed using Glimmer, and gene functions were annotated by BLASTP against the 7 Clusters of Orthologous Groups (COG) database. A genomic overview of annotation information was created using Circos [25]. The genome alignment was performed by Progressive Mauve [26].

Nucleotide sequence accession number
The 16S rRNA gene sequence for strain LD6 determined in the present study was deposited in GenBank (GenBank accession no. MF588953). The gyrB sequence was submitted to GenBank (GenBank accession no. MF588954). Full genome sequences of P. putida KT2440 (NC_002947.4) and P. putida F1 (CP000712.1) were downloaded to GenBank.

Phenotypic characteristics
In this study, morphological analysis of LD6 using scanning electron microscopy (SEM) indicated that the bacteria were consistent with the genus of Pseudomonas. Phenotypically, strain LD6 was shown to be short and rod-like in shape, an obligate aerobe that is oxidase-and catalase-positive, and Gramstrain-negative, and has a single polar-inserted flagellum (Fig. 1). Other phenotypic features of strain LD6 are summarized (Table S1 in Additional file 1).

Phylogenetic analysis
A complete 16S rRNA nucleotide sequence of 1198 bp was obtained. The phylogenetic position using the neighbor-joining method revealed that strain LD6 was allocated to the genus Pseudomonas and clustered with P. putida E16 (KC820813.1) with high bootstrap values of 100% (Fig. 2). The gyrB gene sequence (GenBank accession no. MF588954, 1149 bp) further supported the identity of LD6 as P. putida.

Strain LD6 growth and p-Hydroxybenzoic acid degradation
A degradation experiment was conducted in mineral salt medium (MSM) using allelochemicals, which it indicated that LD6 could use p-hydroxybenzoic acid as the sole carbon source in 50 mL of MSM (Fig.   3). The concentration of p-hydroxybenzoic acid in the MSM markedly decreased after inoculum in 1 d (71.33%), and 97.35% of the p-hydroxybenzoic acid was effectively degraded by strain LD6 after incubation for 8 d (Fig. 3b). The amount of p-hydroxybenzoic acid sharply decreased, accompanied by an increase in the density of the bacteria, indicating that the LD6 strain was highly effective in degrading p-hydroxybenzoic acid. The pH and salt data of experiments has not been decreased obviously. When a dual culture assay was used to screen microbial antagonists against p-hydroxybenzoic from the fresh rhizosphere soils of 2-year-old ginseng seedlings, the strain LD6 decreased the p-hydroxybenzoic content by 35.21%, and the ginseng death rate was decreased by 38.46% after 60 d ( Fig. 3c and 3d). These data indicate that the continuous cropping obstacle of P.
ginseng could be solved through the use of microbiologic control.

Genomic sequence of LD6
A draft genome of P. putida was sequenced based on its specific p-hydroxybenzoic aciddegrading characteristics. The genomic sequence of P. putida was deposited in the National Center for Biotechnology Information (NCBI) database (GenBank accession no. NPKG00000000). The linear, double-stranded genome of LD6 consists of a 5,765,634 bp draft genomic sequence with 62.54% G+C content (Fig. S2). The coding sequences (CDSs) are 5,066,091 bp in length. The total number of predicted genes is 5271, which covers 5186 (98.39%) protein-coding genes and 85 RNA encoding genes, as well as 69 tRNA encoding genes and 16 rRNA (5S, 23S, and 16S) encoding genes ( Table 1).
The 4310 (83.11%) predicted genes were annotated and distributed in the 21 clusters of orthologous group (COGs) categories. The function of these predicted proteins included information storage and processing, cellular processes and signaling, and metabolism. The numbers of genes in the important categories were approximately 498 for amino acid transport and metabolism (11.55%), 387 for transcription (8.98%), 288 for inorganic ion transport and metabolism (6.68%), and 282 for energy production and conversion (6.54%) ( Table S2 in Additional file 1, Fig. S3 in Additional file 3).
We conducted comparative genomics analysis among three strains: P. putida LD6, P. putida KT2440, 9 and P. putida F1 (Fig. S4 in Additional file 3). Block outlines surround a region of the genome that is presumably homologous and internally free from genomic rearrangement. Regions outside blocks lack detectable homology among the input genomes. These three genomes were aligned with Progressive Mauve using default parameters. The genomic synteny between strain P. putida LD6 and P. putida KT2440 was higher. However, the genomic synteny between P. putida LD6 and P. putida F1 was much lower. The alignment analysis demonstrated that P. putida LD6 exhibited high affinity with P. putida KT2440 by Mauve software.

Discussion
Ginseng is an herbaceous, perennial medicinal plant that requires continuous cultivation in fixed plots for 5-6 years. During this long growing process, allelochemicals accumulation, soil-borne diseases, and deterioration of soil physicochemical properties pose threats to the health of P. ginseng. Because successful replanting of P. ginseng requires 30 years of soil restoration, arable soil for P. ginseng cultivation is becoming scarce [5]. The inability to successfully replant P. ginseng and enable the crop to thrive is termed the continuous cropping obstacle. Allelochemicals are considered the primary cause of this problem, and the use of microorganisms to control these allelochemicals is an effective method [27]. In this study, the bacterial strain LD6 was isolated from the rhizosphere soil of ginseng, and it degraded p-hydroxybenzoic acid at a high rate, which indicates that microbes was a method to solve the continuous cropping obstacle of P. ginseng. Based on the morphological, biochemical, 16S rRNA and gyrB gene, as well as previously published studies, strain LD6 was confirming as P. putida [28][29]. However, the fact that p-hydroxybenzoic acid content in soil samples from the field experiment decreased about 35.21% in 60 days. It's indicate that the positive effect of added P.
putida on ginseng wasn't higher, the influence factors should research in the future.
P. putidais a widely distributed Pseudomonas bacterium, and possesses the ability to degrade the nicotine from tobacco as well as the p-coumaric acid from rice [30][31]. Bertani et al. [32] found a phydroxybenzoate hydroxylase gene (pobA) of P. putidaWCS358 involved in the catabolism of phydroxybenzoic acid. Our results indicated that P. putida has the ability to degrade p-hydroxybenzoic acid from P. ginseng planting soil. If additional genes or enzymes involved in p-hydroxybenzoic acid biodegradation were characterized, this strain could be exploitable as inoculum used degradation of p-hydroxybenzoic acid, and this strain could be developed and applied to autotoxin degradation agents. However, a single microbial species is unlikely to completely degrade all of the phydroxybenzoic acid in P. ginseng fields. For any bacteria with promising degradation capabilities, the stability in farmland soil and ability to quickly grow should also be studied.
Bacterial genome sequencing analysis could offer the most important information for the identification of species' unique genes and pathways to provide powerful genetic evidence to support the exploitation of functional bacteria for desirable abilities [33]. In this study, the pobA gene that catalyzed the conversion of p-hydroxybenzoic acid into 3,4-dihydroxybenzoic acid, and the catB and pcaG genes that catalyzed 3,4-dihydroxybenzoic acid into 3-carboxy-cis,cis-muconate, were annotated in the KEGG metabolic pathway of the strain LD6 genome (Fig. S5 in Additional file 3).

Conclusions
In this study, a highly effective p-hydroxybenzoic acid-degrading bacterium was isolated from rhizosphere soil of P. ginseng. Based on morphological, physiological, 16S rDNA and gyrB gene authentication, the bacterial strain LD6 was identied as P. putida. The P. putida metabolized phydroxybenzoic acid completely at higher concentrations, suggesting that P. putida represents a novel tool for removing allelochemicals from rhizosphere soil of P. ginseng. And the draft genome sequence of P. putida LD6 was determined and analysis, the results will helped to reveal useful basic information regarding the p-hydroxybenzoic acid degradation mechanism of P. putida. Isolation and manipulation of functional bacteria is an effective strategy to alleviate ginseng continuous cropping problems and enhance the quality and yield of P. ginseng.