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Article

Endophytic Bacteria in Ricinus communis L.: Diversity of Bacterial Community, Plant−Growth Promoting Traits of the Isolates and Its Effect on Cu and Cd Speciation in Soil

by
Qian Li
,
Qingling Fu
,
Jun Zhu
,
Yuxin Sun
,
Huan He
and
Hongqing Hu
*
College of Resource and Environment, Huazhong Agricultural University, Wuhan 430070, China
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(2), 333; https://doi.org/10.3390/agronomy13020333
Submission received: 23 December 2022 / Revised: 18 January 2023 / Accepted: 21 January 2023 / Published: 23 January 2023
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Abstract

:
Ricinus communis L. shows certain tolerance to and good accumulation ability with heavy metals. Endophytic bacteria−enhanced phytoremediation is an effective method to improve heavy metal extraction efficiency. Here, for better application of castor in phytoremediation, the Illumina high−throughput sequencing was carried out to reveal the endophytic bacterial community in the tissues of castor grown in two locations, and traditional microbial cultivation was used to isolate endophytic bacteria from castor. The dominant bacterial phyla were Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes and Acidobacteria, and Proteobacteria was absolutely dominate in all castor tissues. There were significant differences in the composition of endophytic bacterial communities between castor grown in two sites, with obvious variation in the relative abundance of the dominant phylum. The samples from two sites also had their own unique dominant bacterial genera. The analysis of alpha diversity illustrated that the diversity and species richness of endophytic bacteria community in different parts of castor sampled in Tonglushan mining area were lower than those in Gangxia village, Yangxin county. In total, there were 44 endophytic bacteria strains isolated from the tissues of castor, of which 42 strains possessed three or more growth−promoting properties. Most of these isolates were tolerant to Cu or Cd to varying degrees. Eight isolates were selected for further Cu mobilization and soil incubation experiments. Strains TR8, TR18, TR21, YL1, YS3 and YS5 could well solubilize Cu2(OH)2CO3 in medium. Strain YS3 had the best effect on increasing soil DTPA−Cu and DTPA−Cd contents by 8.4% and 6.9%, respectively. Inoculated endophytic isolates were conducive to the conversion of heavy metal forms from insoluble to relatively unstable, and could increase available phosphate content in soil (10.8–29.2%). Therefore, the plant growth−promoting endophytes screened from castor have great application prospects and can provide important support for the microbial−assisted phytoremediation of heavy metal−contaminated soil.

1. Introduction

Plant endophytic bacteria are microorganisms that colonize healthy plant tissues by symbiosis. In the long−term evolution process, they have established a good interaction relationship with host plants, and play a critical role in regulating the growth and development of host plants. As an important element of the plant micro−ecosystem, plant endophytes are an indispensable part of plants, and host plants also provide a stable living environment for them [1,2]. Previous studies indicated that the inoculated endophytic bacteria with growth−promoting properties could mobilize mineral elements and increase their bioavailability in the rhizosphere, which could facilitate the uptake of these nutrients, promote the growth and development of the plant, and also alleviate the toxicity of various abiotic stresses to the plant [3,4,5,6]. Moreover, as one of the important components of microbial resources, understanding the community and structure of plant endophytes is helpful to better understand the interaction between plants and endophytes. For different plants, the community composition of endophytes is often different, depending on the plant species, growth cycle and living environment [7,8,9].
Ricinus communis L. is an annual or perennial plant of the Euphorbiaceae family. The oil contents of castor seeds can reach 41–64% [10], and it is a raw material of industrial, medical and chemical products. Studies have shown that castor plants can grow normally in soil contaminated by various heavy metals because they have a certain tolerance to and good accumulation ability with different heavy metals such as Pb, Cd, Cu and Zn [11,12,13,14,15]. The castor plant has broad prospects in the phytoremediation of heavy metal−contaminated soil with the advantages of low soil fertility requirement, large biomass and high economic value. In addition to the ability of the plant to take up and translocate of pollutants, plant biomass is one of the decisive factors affecting the phytoremediation efficiency [16,17]. Bauddh and Singh [18] found that the biomass of castor bean is significantly greater than that of Indian mustard on Cd−contaminated soil; although the content of Cd in various parts of Indian mustard is higher than that in castor beans, the total amount of Cd extracted by the roots and shoots of Indian mustard from soil is significantly lower than that of castor bean plants.
Because of the limited ability of castor to translocate heavy metals to the aboveground part, it is necessary to explore effective methods to improve the accumulation of heavy metals in the above−ground parts of castor for utilizing castor to remediate heavy metal−contaminated soil. Under abiotic stress condition, it is one of the effective methods to strengthen the resistance of host plants to heavy metals, improve soil nutrient status and increase the bioavailability of soil heavy metals, and then improve the phytoremediation efficiency of heavy metals by host plants through inoculating exogenous endophytic bacteria with growth promoting properties [19,20,21]. At present, little is known about the community structure and composition of endophytic bacteria in castor, and it is still a question whether the plant growth promoting endophytic bacteria isolated from castor can be used to enhance the extraction of heavy metals by host plants. Therefore, the objectives of this study are: (1) to analyze the endophytic bacterial community structure and composition of castor which were grown in two regions by Illumina high−throughput sequencing; (2) to isolate endophytic bacteria with plant growth−promoting properties from the tissues of castor; (3) to explore the effects of the isolated endophytic bacteria on the mobility of soil heavy metal activity and soil available phosphorus.

2. Materials and Methods

2.1. Materials

Samples of castor were collected from Tonglushan Mining area, Daye (30°4′49″ N, 114°56′13″ E) and Gangxia village, Yangxin county, Huangshi (29°48′40″ N, 115°25′53″ E), Hubei province, China, respectively. Tonglushan is a copper mine with more than 3000 years of mining history, and the soil is mainly contaminated by Cu and Cd. Six healthy castor plants were randomly selected from two sampling sites and placed in sterile sample bags, then stored on the ice surface. After being brought back to the laboratory, the plants were immediately placed in a refrigerator at 4 °C, and the endophytic bacteria were isolated within 48 h. The soil surrounding the castor roots was collected, and the basic properties of the soil were measured and presented in Table 1.
The plant samples were washed with tap water and rinsed 5 times with deionized water, then washed with sterile water for 30 s. The plants were surface sterilized using 70% ethanol for 2 min, then immersed in 2.5% NaClO (containing 0.1% Tween 80) for 5 min followed by transferring to 70% sterile ethanol for 30 s, and finally washed 3 times using sterile water. The plant samples were divided into two parts. One part was frozen with liquid nitrogen and quickly stored in a refrigerator at −80 °C for Illumina high−throughput sequencing, in which the root, stem and leaf samples from Tonglushan were named TR, TS and TL, respectively, and the samples from Gangxia village were named YR, YS and YL, respectively. The other part was used to isolate the endophytic bacteria immediately.

2.2. Illumina High−Throughput Sequencing of Endophytic Bacterial Community of the Castor

The total DNA of plant samples was extracted using FastDNA® SPIN Kit (MP Biomedicals, Santa Ana, CA, USA), and the concentration and purity of total genomic DNA were assessed. Then, the extracted DNA was amplified via two pairs of primers and two rounds of PCR [22,23,24]. The details of amplification were presented in Table S1. The amplified PCR products of each sample were tested by 2% agarose gel electrophoresis. The products were gel−extracted by AxyPrepDNA gel extraction kit (Axygen, San Francisco, CA, USA) and quantified by the QuantiFluor™ −ST Blue Fluorescence Quantification System (Promega, Madison, WI, USA), and mixed proportionally according to the required sequencing volume of each sample. The paired−end sequencing of purified amplicons was performed on Illumina’s Miseq PE250 platform (2 × 250 bp) (Shanghai Majorbio Bio−pharm Technology Co., Ltd., Shanghai, China). All raw sequences were submitted to NCBI Sequence Read Archive (SRA) (BioProject ID: PRJNA893461).
The paired−end reads obtained by Miseq sequencing were assembled by Flash software (v1.2.11), while sequence quality was controlled and filtered using Fastp software (v0.19.6). Then, the sequences were clustered at 97% identity into operational taxonomic units (OTUs), and the OTUs obtained by clustering were analyzed by RDP classifier (v2.11), for which the comparison database was SILVA (v138). The rarefaction curves for OTUs were used to determine the sample sequencing depth, and Simpson, Chao1 and Coverages indices were used to characterize the diversity and richness of microbial community (Mothur, V.1.30.2). Based on Bray–Curtis distances, non−metric multidimensional scaling (NMDS) analysis was conducted using the R language (v3.3.1) vegan package to compare the differences in community composition among samples. Analysis of similarities (ANOSIM) was used to detect the significance of differences between groups.

2.3. Isolation of Endophytic Bacteria

The surface−sterilized plant tissues were fully ground together with 5 mL sterilized PBS buffer (pH 7.4), and 100 μL suspension was spread on an LB agar plate with the method of gradient dilution coating (10−1, 10−2, 10−3). The stems of the castor were cut into slices with a sterilizing blade and were spread on LB agar plates. All plates were incubated at 28 °C for 3–5 d, and single colonies with different forms were picked and purified on LB agar plates. All details of the media used in the study were listed in Supplementary Materials Table S2.

2.4. Plant Growth−Promoting Properties of the Isolates

IAA production of isolates was measured in SMS medium containing 500 mg/L tryptophan and incubated at 30 °C for 2 d. After centrifugation, 1 mL of supernatant was mixed thoroughly with 2 mL of Salkowski’s reagent and colored in the dark for 30 min at room temperature, in which the appearance of pink indicated the production of IAA, then the absorbance of the mixture was measured by colorimeter at 530 nm [25].
Siderophore secretion was determined according to the method described by Schwyn and Neilands [26]. Then, 1.5 mL of cell−free supernatant from incubation in SMS liquid medium for 48 h was mixed with an equal volume of Chrome azurol S (CAS) detection solution. The mixture was colored in the dark for 1 h at room temperature, then the absorbance was measured by colorimeter at 630 nm (λ) and the non−inoculated SMS liquid medium was used as the control (λ0). The ability to produce siderophores is represented as (λ0 − λ)/λ0 and the higher the value, the higher the capacity for siderophore production.
Aminocyclopropane−1−carboxylate (ACC) deaminase activity was analyzed using the protocol described by Penrose and Glick [27]. In brief, the isolates were inoculated in DF medium and cultured at 30 °C for 24 h. After centrifugation at 8000× g for 10 min at 4 °C, cell pellets were collected and resuspended with an equal volume of ADF medium, then incubated to induce the activity of ACC deaminase of bacteria at 30 °C for 24 h. After that, the cell pellets were harvested and washed by 5 mL 0.1 mol/L Tris−HCl (pH 7.6) twice and stored at −20 °C for measurement of ACC deaminase activity. The total protein of bacterial cells was measured using the Bradford method. The unit of ACC deaminase activity is the amount of 1 µmol α−ketobutyrate generated by the ACC deaminase catalyzing ACC per hour (µmol α−KA/h).
The ability of nitrogen fixation was determined using Ashby’s nitrogen−free agar medium. After subculturing 5 times, if the isolates were still growing on this medium, they were regarded as having stable nitrogen fixing activity.
NBRIP agar medium was used to estimate the capacity of inorganic phosphate solubilization of isolates according to the halos around the colonies. The strains possessing phosphate solubilization were inoculated in NBRIP liquid medium and cultured at 30 °C for 4 d. The supernatant was collected by centrifugation at 8000× g for 10 min at 30 °C, and then the pH and phosphorus concentration were measured. The concentration of soluble phosphate was determined by the molybdenum blue method [28].

2.5. The Tolerance of the Isolates to Cu and Cd

The tolerance of the isolates was determined in LB agar plate contained increasing concentrations of Cu2+ (80 mg/L–1200 mg/L) or Cd2+ (10 mg/L–250 mg/L). The plates were incubated at 30 °C for several days and the strains growing on it were recorded.

2.6. Molecular Identification of the Isolates

Based on the plant growth−promoting capacity and heavy metal tolerance of these isolates, eight endophytic bacteria were selected for further experimentation. The 16S rDNA gene of the eight endophytic bacteria was amplified using universal primers 27F and 1492R. The detailed reaction system and procedure of PCR amplification are shown in Table S3. The PCR products were sequenced by Tsingke Biotechnology Co., Ltd. (Wuhan, China). ContigExpress was used for splicing the sequence, and the inconsistent parts on both ends were removed. The splicing sequences were submitted to NCBI database (Accession numbers: OP310027-OP310034, accessed on 31 August 2022). The sequences were blasted with the model strains at the database using EzBioCloud (https://www.ezbiocloud.net/, accessed on 31 August 2022) [29] and the 16S rDNA sequences of model strains with the highest homology were obtained according to the homology between the isolates and model strains. The phylogenetic tree was constructed by the maximum−likelihood method using Mega software (v11.0).

2.7. Cu Mobilization Assay

The ability of Cu mobilization of the above−mentioned eight strains was analyzed according to the method of Sheng et al. [25]. Briefly, the bacterial suspension of the tested strains was inoculated in 10 mL sterilized SMS medium containing 10 mg Cu2(OH)2CO3 and a medium with an equal volume of sterilized water was used as the control. These cultures were incubated at 30 °C with a rotation of 180 r/min. The samples were collected at different times. After centrifugation at 10,000× g for 10 min at 4 °C, Cu2+ concentration and pH values in the supernatant were determined.

2.8. Impact of the Endophytic Bacteria on Heavy Metal Speciation and Available Phosphate in Soil

Soil used for the incubation experiment was collected from Yangxin county, Hubei province (29°48′40″ N, 115°25′53″ E), China. The basic physicochemical properties of the soil were as follows: pH 7.88, OM 19.7 g/kg, Cu 380.6 mg/kg, and Cd 1.16 mg/kg. The tested soil was air−dried, ground and screened with a 10−mesh sieve. Then, 40 g soil was added an appropriate amount of deionized water (maintaining 40% of the field capacity) to activate the soil microorganisms and incubated at 28 °C for 7 d. Next, the bacterial suspension (about 2 × 108 CFU/mL) of the eight isolates was added to the soil with the volume of 6 mL, respectively. The addition of bacterial suspension was divided into two parts, of which 4 mL was added on the first day and 2 mL on the eleventh day during incubation, respectively, maintaining the soil water content within 60–70% of the field capacity. The same amount of sterile deionized water was supplemented as the control (CK). Three replicates were set for each treatment. The soil samples were collected after culturing at 28 °C for 20 d. Soil available Cu and Cd were extracted by 0.005 mol/L DTPA (pH 7.3 ± 0.2) with a soil/water ratio of 1/2. Heavy metal speciation in soil was using BCR sequential extraction procedure [30]. Cu content in solution was measured by Flame Atomic Absorption Spectrometry (FAAS), and Cd content in solution was measured using Graphite Furnace Atomic Absorption Spectrometry (GFAAS). Soil available phosphate was extracted by 0.05 mol/L NaHCO3 (pH 8.5) shaking at 25 °C for 30 min, and determined by the molybdenum blue method.

3. Results

3.1. The Composition and Diversity of Endophytic Bacteria in Ricinus communis L.

As shown in Table 2., the number of OTUs in the castor samples collected from Yangxin was 1124 (YR), 1061 (YS) and 902 (YL), respectively, and the Chao1 index was 654.8, 678.2 and 443.6, respectively, which were higher than those in the samples collected from Tonglushan mining area; Simpson index was 0.09 (YR), 0.19 (YS) and 0.20 (YL), respectively, which were lower than values of samples collected from Tonglushan mining area. These results indicated the diversity and species richness of the endophytic bacterial community in castor collected from Gangxia were greater than that collected from Tonglushan mining area, in which those in root samples were higher than those in stem and leaf samples.
The NMDS analysis (Figure 1A) showed that there were significant differences in the composition community of endophytic bacteria among all sample groups (r = 0.58, p = 0.001). To further test the inter−group differences in the endophytic bacterial community structure of the same plant tissue from two regions, the ANOSIM analysis was conducted based on the Bray–Curtis distance (Table S4). The results showed that there were significant differences between TR and YR (r = 0.50, p = 0.034), and TS and YS (r = 0.42, p = 0.034), indicating that there were significant differences in the endophytic bacterial composition of plants growing in the two soils. Moreover, ANOSIM analysis of different parts of plants in the same area also revealed that there were significant differences in the structure of endophytic bacterial community between tissues.
As given in Figure 1B., there were five phyla with relative abundance ≥1% in each sample, at phylum level, which are Proteobacteria, Bacteroidota, Actinobacteriota, Firmicutes and Acidobacteria. Among them, Proteobateria was the most dominant phyla with the relative abundance ranging from 69.8% to 97.0%, and its relative abundance in tissues was ordered as leaf > stem > root. The relative abundance of Proteobacteria in TR, TS and TL (82.5%, 90.7% and 97.0%) was higher than that in YR, YS and YL (69.8%, 82.3% and 84.3%), respectively. The relative abundance of Bacteroidota between the two groups showed an opposite trend, and the relative abundance of TR and TS (3.26%, 1.14%) was significantly lower than that of YR and YS (11.9%, 10.3%). The relative abundance of Actinobacteriota and Acidobacteria in TR and TL was lower than that in YR and YL, respectively, but there was no significant difference in the relative abundance of TS and YS samples.
At genus level (Figure 1D), the bacterial genera in the six samples mainly included Sphingomonas, Ralstonia, Burkholderia−Caballeronia−Paraburkholderia, Pseudomonas, Delftia and Escherichia−shigella. Moreover, unclassified_f_Rhizobiaceae (3.1%), Aquamicrobium (1.3%), Moheibacter (1.3%) are the TR characteristic advantage genera; Acidothermus and norank_f_Xanthobacteraceae were found in TR (0.92%, 1.4%) and YR (2.8%, 3.5%). Tenacibaculum, norank_f_Saprospiraceae, unclassified_f_Flavobacteriaceae and unclassified_o_ Chitinophagales mainly existed in YR, YS and YL, and the relative abundance of Tenacibaculum decreased from underground parts to aboveground.

3.2. Isolation and Plant Growth−Promoting Characteristics of Endophytic Bacteria

A total of 44 endophytic bacterial strains were isolated from tissue of castor, including 22 strains from the root, 13 strains from the stem and 9 strains from the leaf. The growth−promoting traits and heavy metal tolerances of isolates are given in Table 3. It is shown that all the isolates had the ability to produce IAA (4.0–140.1 mg/L), among which 18 strains produced more than 30 mg/L IAA. A previous study showed that when the microorganisms with ACC deaminase activity is no lower than 20 nmol α−KA/(mg·h), it is sufficient for the promotion of plant growth [27]. In this study, there were 30 strains of endophytic bacteria which could generate the ACC deaminase via using ACC, and the ACC deaminase activity of those strains was 11.6–233.9 µmol α−KA/(mg·h). There were 42 isolates that could grow normally in Ashby’s nitrogen−free plate after subculturing 5 times, suggesting that these strains had potential nitrogen fixation ability. Moreover, there were 18 endophytic bacteria that could dissolve insoluble tricalcium phosphate, among which 10 isolated strains could increase soluble phosphate content in culture medium over 300 mg/L. The maximum content of soluble phosphate in NBRIP liquid medium inoculated strain TR16 could reach 495.5 mg/L. Furthermore, the tolerance of the isolates to Cu and Cd indicated that most of the endophytic isolates were resistant to both metals. The Cu2+ tolerance concentration of 17 isolates were up to 500 mg/L, of which strain TR16 could tolerate Cu2+ with the highest concentration of 1100 mg/L. The Cd2+ concentration tolerance of these strains ranged from 10 mg/L to 200 mg/L.

3.3. Characterization of Endophytic Bacterial Isolates

Eight strains with different growth−promoting characteristics selected from the above 44 isolates were identified using 16S rDNA sequencing, and their taxonomy was determined by sequence Blast analysis and phylogenetic tree construction. According to the results of sequence Blast (Table 4) and phylogenetic tree (Figure S1), strain TR4, TR18 and TR21 located in the same branch and belong to Enterobacter. Strain TR4 and TR18 were closely related to the model strain Enterobacter kobei DSM 13645T, while TR21 was closely related to Enterobacter huaxiensis EB−24T. The top−hit strain of TR8 was Buttiauxella agrestis ATCC 51602T and TR8 was identified as Buttiauxella. Strain TR16 was characterized as Acinetobacter, which had the highest homology with the model strain Acinetobacter pittii CIP 70.29T and was much closer on the phylogenetic tree. Strain YL1 had the highest similarity of 99.2% with Bacillus altitudinis 41KF2bT and was assigned to Bacillus genera. Strain YS3 and Proteus myxofaciens ATCC 19692T were clustered into an independent branch on the phylogenetic tree and YS3 was identified as Proteus. Strain YS5 had the closest genetic distance with the model strain Metabacillus idriensis SMC 4352−2T and was characterized as Metabacillus.

3.4. The Mobilization of Cu by Endophytic Bacteria

At the initial cultivation (12 h and 24 h), Cu2+ concentration in the supernatant of inoculated treatments decreased to varying degrees (9.8–53.7%) compared with that of in the uninoculated treatment (Figure 2A), which could be attributed to the adsorption capacity of these strain for heavy metals (Sheng et al., 2008). In the eight treatments, inoculated endophytic bacteria, strains TR21, TR8, YL1, YS3 and YS5, showed obvious mobilization ability with Cu2(OH)2CO3 (Figure 2A). After 144 h of cultivation, the concentration of Cu2+ in the supernatant of these five treatments reached 84.1 mg/L, 84.3 mg/L, 100.4 mg/L, 108.2 mg/L and 98.9 mg/L, respectively. Strain TR16 did not possess the ability to mobilize Cu2(OH)2CO3 in medium. With the increase in culture time, the pH value of the supernatant decreased, but was still higher than that of the CK. In comparison to CK treatment (42.7 mg/L), the Cu2+ concentration in strain TR16 treatment was 16.7 mg/L, which decreased by 60.9%. Furthermore, the pH value of TR4 treated supernatant gradually decreased and then stabilized with the culture time, while the concentration of Cu2+ in supernatant was lower than that in the control (CK1).

3.5. The Inoculation of Endophytic Bacteria on DTPA−Cu/Cd and Speciation in Soil

The effect of endophytic bacteria inoculation on DTPA extractable Cu and Cd in soil are shown in Figure 3. The inoculation of the eight endophytic bacteria could affect the bioavailability of soil Cu and Cd to varying degrees. Compared with the CK, strain YS3 treatment significantly improved the bioavailability of soil Cu and Cd most greatly, in which the content of DTPA extractable Cu and Cd increased by 8.4% and 6.9%, respectively. In addition to the treatment of strain TR4 and TR16, other inoculation strains also increased the content of DTPA−Cu in soil to some extent, but they had no significant effect on the content of DTPA−Cd in soil.
As given in Figure 4, Cu in soil mainly existed in the fraction of the reducible and the residual, while Cd mainly existed in the fraction of the acid extractable, the reducible and the residual. For Cu (Figure 4A), except for inoculation of strain YS5, other inoculation treatments had no significant effect on acid extractable Cu content. Compared with the control, YS5 treatment significantly increased acid extractable Cu and reducible Cu content in soil by 4.4% and 4.1%, respectively, but oxidable Cu content did not change significantly, indicating that inoculation of YS5 strain was conducive to the transformation of soil residual Cu to acid extractable Cu and reducible Cu, thus improving the bioavailability of soil Cu. In addition, strain YL1 and YS3 treatments also significantly increased the content of reducible Cu in soil.
For Cd (Figure 4B), in the treatments of strain TR8, TR16, YR8, TR21, YL1 and YS3, the content of acid extractable Cd, reducible Cd and oxidable Cd in soil increased by 12.5–58.1%, 7.4–33.0% and 102.7–141.1%, respectively, while the content of residual Cd decreased by 27.0–57.9%. The content of extractable Cd, oxidizable Cd and residual Cd in soil treated with strain TR4 had no significant change, but the content of reducible Cd significantly increased by 22.8%, while the content of residual Cd significantly decreased by 13.0%. YS5 treatment significantly increased the content of acid extractable Cd (23.3%) and oxidizable Cd (120%) in soil, and significantly decreased residual Cd content (21.7%), but had no significant effect on reducible Cd content. Therefore, the treatment of these eight endophytic bacteria strains was conducive to the transformation of soil heavy metal Cd from more stable form to unstable forms, such as acid extractable, reducible or oxidizable forms.

3.6. The Inoculation of Endophytic Bacteria on Soil Available Phosphate

The effects of endophytic bacteria inoculation on soil available phosphate are shown in Figure 5. Compared with the no−inoculation treatment, the inoculation of eight endophytic bacteria increased the soil available phosphorus content to a certain degree. Except for the YL1 treatment, the soil available phosphorus content in soil significantly increased in the other seven treatments. Compared with the 12.8 mg/kg available phosphate content of the CK, the content of soil available phosphate increased by 11.4%, 19.3%, 16.6%, 13.0%, 29.2%, 15.7% and 10.8% in the treatments with TR4, TR8, TR16, YR8, TR21, YS5 and YS3, respectively. Among them, TR21 treatment had the best effect on increasing the content of soil available phosphate, reaching 16.5 mg/kg.

4. Discussion

4.1. Differences in Diversity of Endophytic Bacterial Community of Castor Grown on Two Sites

Soil is one of the main sources of plant endophytic bacteria. Due to different physical and chemical properties, the microbial community composition in various soils is also different, which in turn affects the community structure of plant endophytic bacteria [1,31,32]. Metabolites secreted by different plant roots can recruit specific microorganisms and induce them to colonize on the root system, then part of these microorganisms can enter the plant tissue by invading the root system [2,33,34]. The plant periderm zone is an important selection barrier to control the composition of plant endophytic bacterial communities [35]. Wang et al. [9] reported that soil type and Cd level would affect the composition of the endophytic bacterial community in Sedum alfredii Hance. In the present study, Illumina Hiseq high−throughput sequencing was used to study the community structure of endophytic bacteria in various tissues (roots, stems and leaves) of the castor grown in two soils (mine soil and farmland soil). It was found that the diversity and species richness of endophytic bacteria in various tissues of plants growing in farmland (Gangxia village, Yangxin county) were greater than those in mine (Tonglushan, Daye city) soil. These should be attributed to the different land use patterns, physical and chemical properties, heavy metal pollution levels and distribution of the two sampling sites [36]. The sampling site of Tonglushan mining area is a long−term abandoned mine site polluted heavily by heavy metals and has low vegetation cover with a harsh environment. The sampling site of Gangxia village belongs to farmland. Although the site is also contaminated by various elements to a certain extent, long−term crop planting and agronomic practices can provide a better living environment for the growth and reproduction of soil microorganisms [37,38]. The concentration of heavy metals in soil plays a critical role in regulating the structure of the plant endophyte community. High concentrations of heavy metals lead to a decline in the diversity and species richness of soil microbial community structures, thus affecting the composition of endophyte communities in plants [39]. Deng et al. [32] also showed that the bacterial community diversity and species richness in rhizosphere soil of Elsholtzia haizhouensis growing in Tonglushan mining area were lower than those growing in Macheng city, of which soil pH, nitrogen sources and heavy metals were the important factors affecting the composition of microbial communities.
Through Illumina Hiseq high−throughput sequencing, it was found that the main phyla of endophytic bacteria were Proteobacteria, Bacteroidota, Actinobacteriota, Firmicutes and Acidobacteria in the tissue of the castor grown on the two sites. Notably, the relative abundance of each phylum was different to varying degrees, of which the relative abundance of Bacteroidetes and Actinobacteria in the samples of two sites represented a diametrically opposite trend (Figure 1B,C). The results of ANOSIM analysis based on Bray Curtis distance (Table S4) also proved that there were significant differences in the composition of endophytic bacterial community in the castor grown on the two regions. Lv et al. [40] studied the composition of root endophytic bacterial community of five pea varieties grown in the same soil and found that the root endophytic bacteria of these five pea varieties had similar community compositions. Compared with the seed endophytic bacterial community, the root endophytic bacteria community composition was much closer to the soil bacterial community composition. Durand et al. [34] found that there was no difference in the community structure of endophytic bacteria in the seeds of two species of Noccaea caerulescens collected from two different regions. However, pot experiments indicated that the composition of endophytic bacteria communities in the roots, stems and leaves of Noccaea caerulescens grown in two soils was similar at the phylum level, but the relative abundance was significantly different, which is consistent with our present result. Moreover, the samples from the two places also had different specific dominant genera at genera level (Figure 1D). Consequently, it is suggested that the endophytic bacterial community composition of the same plant is affected obviously by the soil environment in which the plant is growing, which means the soil conditions are one of the driving factors to shape the endophytic bacterial community in plants.

4.2. Cu Mobilization of Eight Endophytic Bacteria Isolates in Medium

Recently, plant growth−promoting endophytic bacteria have attracted widespread attention in strengthening plant remediation of contaminated soil [41,42,43]. A previous study reported that the castor grown in Tonglushan mining area could grow normally on soil with severe Cu pollution, and accumulated more Cu in the tissues with strong tolerance to Cu [12]. In this study, 44 strains of endophytic bacteria were isolated from the tissues of the castor. Basically, most of the strains possessed three or more kinds of growth−promoting properties, and had different degrees of tolerance to Cu and Cd. These results suggest that the castor is a high−quality material source for screening endophytic bacteria with heavy metal resistance, and its endophytic bacteria contained a variety of good characteristics with great development potential. Therefore, it is of great significance to isolate endophytic bacteria with growth promoting properties from the castor growing in various heavy metal−contaminated areas to find new functional microbial resources that can be used for phytoremediation.
The bioavailability of heavy metal is one of the important factors affecting the extraction efficiency of plants [44], in which carbonate bound heavy metals in the form of precipitation or coprecipitation are very sensitive to any change in soil pH [45]. Hence, the present study investigated the mobilization capacity of eight endophytic bacteria to insoluble Cu by using Cu2(OH)2CO3 as the experimental material. The results demonstrated that in addition to strains TR4 and TR16, the strains TR8, TR18, TR21, YL1, YS3 and YS5 had strong mobilization capacity to Cu2 (OH)2CO3 (Figure 2), and their capacity is obviously related to the change in pH in the culture solution, which should be attributed to the low molecular weight organic acids produced by the strain during the culturing process. Yang et al. [46] compared the total amount of organic acids secreted by two strains with different mobility for CdCO3; the results showed that the total amount of organic acids secreted by Pseudomonas fluorescens GI with better Cd mobilization was much higher than that of Bacillus cereus HL, which was the reason why the two strains had significantly different Cd solubilization capacities. They further calculated the amount of CdCO3 that can be solubilized by the concentration of secreted organic acid by using the linear fitting curve of low molecular organic acid to CdCO3 mobility, and found that the fitting amount of dissolved Cd was basically equivalent to the actual amount of Cd solubilized by the strains. Li et al. [47] revealed that the addition of K+ could significantly enhance the solubilization of Agrobacterium sp. to CdCO3 by inducing the secretion of tartaric acid, 3−hydroxybutyric acid, fumaric acid and succinic acid, and the pH of the culture solution was also reduced to varying degrees due to the addition of K+. Moreover, microorganisms also can release protons through the assimilation of NH4+, thus reducing the pH of the medium. Furthermore, although the Cu2+ concentration in the supernatant of the medium inoculated strain TR4 was still lower than that of the non−treated medium after 6 days of incubation, and the pH value decreased sharply, which might be related to the adsorption capacity of the strain itself for heavy metal ions.

4.3. Effect of the Eight Endophytic Isolates on Soil Cu and Cd Speciation

According to soil incubation experiment, it was further found that these eight endophytic bacterial strains had different influence on the availability of heavy metals in soil, of which YS3 had the best effect on increasing the content of DTPA−Cu and DTPA−Cd in soil. In the Cu mobilization experiment, strain YS3 also had a strong solubilizing ability on Cu2(OH)2CO3. Moreover, the eight strain treatments also increased the content of available phosphate in soil. It is well known that soil pH value is the principal factor that affects the activity and fraction change of heavy metal in soil, and the metabolites secreted by plant endophytic growth−promoting bacteria, such as low molecular organic acids and siderophores, can reduce soil pH and activate soil heavy metals [25,48,49]. The eight isolates used in the present study had the growth promoting traits of producing IAA, siderophores or solubilizing phosphate. Wu et al. [50] showed that the inoculation of Serratia Marcescens with plant growth−promoting properties mobilized soil Cd, which was one of the direct reasons for the improvement in plant extraction efficiency, and it also increased the bioavailability of phosphate in soil. Liu et al. [51] revealed that inoculated endophytic bacteria Bacillus cereus reduced soil pH, increased soil extractable Cd and available P. Except for TR8 and TR16, the other treatments reduced soil pH value to varying degrees compared with uninoculated soil (Figure S2), which may be a reason for the increased availability of heavy metals in soil in this study. Furthermore, according to the analysis of the chemical speciation of soil heavy metal, the inoculation of endophytic bacteria could promote the transformation of soil heavy metals Cu or Cd from a stable residue form to a relatively unstable acid extractable form, reducible form and oxidizable form. The bioavailability of heavy metals in soil is closely related to their chemical speciation in soil. The inoculation of microorganisms affects the composition of chemical forms of heavy metals in soil. Wang et al. [52] reported that the inoculation of two endophytic bacteria could promote the transformation of soil residual heavy metals to other three forms (acid extractable, reducible and oxidable), in which the content of soil oxidable V, Cr and acid extractable Cd was significantly increased.

5. Conclusions

A total of 44 strains of endophytic bacteria with great growth−promoting characteristics were isolated from the tissue of castor plants and most of the isolates were tolerant to various concentration of Cu2+ and Cd2+. Eight endophytic bacteria with different growth−promoting properties were selected from the isolated strains for molecular identification. Among them, six strains were found to be able to solubilize Cu2(OH)2CO3 in the medium, in which strains TR21, TR8, YL1, YS3 and YS5 could increase the concentration of Cu2+ in the supernatant, reaching 84.1 mg/L, 84.3 mg/L, 100.4 mg/L, 108.2 mg/L and 98.9 mg/L, respectively. Inoculating the soil contaminated by heavy metals with these eight endophytic bacteria to could not only improve the bioavailability of Cu and Cd, and change their chemical speciation, but also play a role in phosphate solubilization and increase the available phosphate content in the soil.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13020333/s1, Table S1. The primers, reaction system and procedure of PCR amplification used in Illumina high−throughput sequencing; Table S2: Media and their formulas used in this study; Table S3. The primers, reaction system and procedure of PCR amplification used in molecular identification of the isolates; Table S4: Analysis of similarities endophytic microbial community in castor based on Bray–Curtis distance; Figure S1: Phylogenetic tree based on 16S rDNA sequence obtained by the Maximum likelihood (ML) method with 1000 replicates; Figure S2: Effect of inoculated endophytic bacteria on soil pH value.

Author Contributions

Conceptualization, Q.L. and H.H. (Hongqing Hu); validation, Q.L.; formal analysis, Q.L.; investigation, Q.L. and Y.S.; resources, H.H. (Huan He); data curation, Q.L.; writing—original draft preparation, Q.L.; writing—review and editing, H.H. (Hongqing Hu) and Q.F.; visualization, Q.L., H.H. (Hongqing Hu), Q.F. and J.Z.; supervision, H.H. (Hongqing Hu), Q.F. and J.Z.; project administration, Q.L. and H.H. (Hongqing Hu); funding acquisition, H.H. (Hongqing Hu). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Joint Key Funds of the National Natural Science Foundation of China (U21A20237) and Chinese Postdoctoral Science Foundation (2021M691165).

Data Availability Statement

Data available from the author.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Afzal, M.; Khan, Q.M.; Sessitsch, A. Endophytic bacteria: Prospects and applications for the phytoremediation of organic pollutants. Chemosphere 2014, 117, 232–242. [Google Scholar] [CrossRef] [PubMed]
  2. Ma, Y.; Rajkumar, M.; Zhang, C.; Freitas, H. Beneficial role of bacterial endophytes in heavy metal phytoremediation. J. Environ. Manag. 2016, 174, 14–25. [Google Scholar] [CrossRef] [PubMed]
  3. Yuan, M.; He, H.D.; Xiao, L.; Zhong, T.; Liu, H.; Li, S.B.; Deng, P.Y.; Ye, Z.H.; Jing, Y.X. Enhancement of Cd phytoextraction by two Amaranthus species with endophytic Rahnella sp. JN27. Chemosphere 2014, 103, 99–104. [Google Scholar] [CrossRef]
  4. Han, Y.L.; Wang, R.; Yang, Z.R.; Zhan, Y.H.; Ma, Y.; Ping, S.Z.; Zhang, L.W.; Lin, M.; Yan, Y.L. 1−Aminocyclopropane−1−Carboxylate deaminase from Pseudomonas stutzeri A1501 facilitates the growth of rice in the presence of salt or heavy metals. J. Microbiol. Biotechnol. 2015, 25, 1119–1128. [Google Scholar] [CrossRef] [PubMed]
  5. Wang, Q.; Ge, C.F.; Xu, S.A.; Wu, Y.J.; Sahito, Z.A.; Ma, L.Y.; Pan, F.S.; Zhou, Q.Y.; Huang, L.K.; Feng, Y.; et al. The endophytic bacterium Sphingomonas SaMR12 alleviates Cd stress in oilseed rape through regulation of the GSH−AsA cycle and antioxidative enzymes. BMC Plant Biol. 2020, 20, 1–14. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Masmoudi, F.; Tounsi, S.; Dunlap, C.A.; Trigui, M. Endophytic halotolerant Bacillus velezensis FMH2 alleviates salt stress on tomato plants by improving plant growth and altering physiological and antioxidant responses. Plant Physiol. Biochem. 2021, 165, 217–227. [Google Scholar] [CrossRef]
  7. Robinson, R.J.; Fraaije, B.A.; Clark, I.M.; Jackson, R.W.; Hirsch, P.R.; Mauchline, T.H. Endophytic bacterial community composition in wheat (Triticum aestivum) is determined by plant tissue type, developmental stage and soil nutrient availability. Plant Soil 2016, 405, 381–396. [Google Scholar] [CrossRef]
  8. Ma, Y.; Weisenhorn, P.; Guo, X.; Wang, D.; Yang, T.; Shi, Y.; Zhang, H.; Chu, H. Effect of long−term fertilization on bacterial communities in wheat endosphere. Pedosphere 2021, 31, 538–548. [Google Scholar] [CrossRef]
  9. Wang, Q.; Pan, F.S.; Xu, X.M.; Rafiq, M.T.; Yang, X.E.; Chen, B.; Feng, Y. Cadmium level and soil type played a selective role in the endophytic bacterial community of hyperaccumulator Sedum alfredii Hance. Chemosphere 2021, 263, 127986. [Google Scholar]
  10. Ruiz Olivares, A.; Carrillo−Gonzalez, R.; Gonzalez−Chavez, M.D.C.A.; Soto Hernandez, R.M. Potential of castor bean (Ricinus communis L.) for phytoremediation of mine tailings and oil production. J. Environ. Manag. 2013, 114, 316–323. [Google Scholar] [CrossRef]
  11. Andreazza, R.; Bortolon, L.; Pieniz, S.; Camargo, F.A.O. Use of high−yielding bioenergy plant castor bean (Ricinus communis L.) as a potential phytoremediator for copper−contaminated soils. Pedosphere 2013, 23, 651–661. [Google Scholar] [CrossRef]
  12. Kang, W.; Bao, J.G.; Zheng, J.; Hu, H.Q.; Du, J.K. Distribution and chemical forms of copper in the root cells of castor seedlings and their tolerance to copper phytotoxicity in hydroponic culture. Environ. Sci. Pollut. Res. Int. 2015, 22, 7726–7734. [Google Scholar] [CrossRef] [PubMed]
  13. Khan, M.M.; Islam, E.; Irem, S.; Akhtar, K.; Ashraf, M.Y.; Iqbal, J.; Liu, D. Pb−induced phytotoxicity in para grass (Brachiaria mutica) and Castorbean (Ricinus communis L.): Antioxidant and ultrastructural studies. Chemosphere 2018, 200, 257–265. [Google Scholar] [CrossRef] [PubMed]
  14. Xiong, P.P.; He, C.Q.; Oh, K.; Chen, X.P.; Liang, X.; Liu, X.Y.; Cheng, X.; Wu, C.L.; Shi, Z.C. Medicago sativa L. enhances the phytoextraction of cadmium and zinc by Ricinus communis L. on contaminated land in situ. Ecol. Eng. 2018, 116, 61–66. [Google Scholar] [CrossRef]
  15. He, C.Q.; Zhao, Y.P.; Wang, F.F.; Oh, K.; Zhao, Z.Z.; Wu, C.L.; Zhang, X.Y.; Chen, X.P.; Liu, X.Y. Phytoremediation of soil heavy metals (Cd and Zn) by castor seedlings: Tolerance, accumulation and subcellular distribution. Chemosphere 2020, 252, 126471. [Google Scholar] [CrossRef] [PubMed]
  16. Nie, F.H. New comprehensions of Hyperaccumulator. Ecol. Environ. 2005, 14, 136–138. (In Chinese) [Google Scholar]
  17. Bhargava, A.; Carmona, F.F.; Bhargava, M.; Srivastava, S. Approaches for enhanced phytoextraction of heavy metals. J. Environ. Manag. 2012, 105, 103–120. [Google Scholar] [CrossRef]
  18. Bauddh, K.; Singh, R.P. Cadmium tolerance and its phytoremediation by two oil yielding plants Ricinus communis (L.) and Brassica juncea (L.) from the contaminated soil. Int. J. Phytoremediat. 2012, 14, 772–785. [Google Scholar] [CrossRef]
  19. Wood, J.L.; Tang, C.; Franks, A.E. Microbial associated plant growth and heavy metal accumulation to improve phytoextraction of contaminated soils. Soil Biol. Biochem. 2016, 103, 131–137. [Google Scholar] [CrossRef] [Green Version]
  20. Ashraf, M.A.; Hussain, I.; Rasheed, R.; Iqbal, M.; Riaz, M.; Arif, M.S. Advances in microbe−assisted reclamation of heavy metal contaminated soils over the last decade: A review. J. Environ. Manag. 2017, 198, 132–143. [Google Scholar] [CrossRef]
  21. Franco−Franklin, V.; Moreno−Riascos, S.; Ghneim−Herrera, T. Are endophytic bacteria an option for increasing heavy metal tolerance of plants? a meta−analysis of the effect size. Front. Environ. Sci. 2021, 8, 603668. [Google Scholar] [CrossRef]
  22. Bulgarelli, D.; Rott, M.; Schlaeppi, K.; van Themaat, E.V.L.; Ahmadinejad, N.; Assenza, F.; Rauf, P.; Huettel, B.; Reinhardt, R.; Schmelzer, E.; et al. Revealing structure and assembly cues for Arabidopsis root−inhabiting bacterial microbiota. Nature 2012, 488, 91–95. [Google Scholar] [CrossRef]
  23. Lundberg, D.S.; Lebeis, S.L.; Paredes, S.H.; Yourstone, S.; Gehring, J.; Malfatti, S.; Tremblay, J.; Engelbrektson, A.; Kunin, V.; del Rio, T.G.; et al. Defining the core Arabidopsis thaliana root microbiome. Nature 2012, 488, 86–90. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Bulgarelli, D.; Garrido−Oter, R.; Muench, P.C.; Weiman, A.; Droege, J.; Pan, Y.; McHardy, A.C.; Schulze−Lefert, P. Structure and function of the bacterial root microbiota in wild and domesticated Barley. Cell Host Microbe 2015, 17, 392–403. [Google Scholar] [CrossRef] [Green Version]
  25. Sheng, X.F.; Xia, J.J.; Jiang, C.Y.; He, L.Y.; Qian, M. Characterization of heavy metal−resistant endophytic bacteria from rape (Brassica napus) roots and their potential in promoting the growth and lead accumulation of rape. Environ. Pollut. 2008, 156, 1164–1170. [Google Scholar] [CrossRef] [PubMed]
  26. Schwyn, B.; Neilands, J.B. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 1987, 160, 47–56. [Google Scholar] [CrossRef]
  27. Penrose, D.M.; Glick, B.R. Methods for isolating and characterizing ACC deaminase−containing plant growth−promoting rhizobacteria. Physiol. Plant 2003, 118, 10–15. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Behera, B.C.; Yadav, H.; Singh, S.K.; Sethi, B.K.; Mishra, R.R.; Kumari, S.; Thatoi, H. Alkaline phosphatase activity of a phosphate solubilizing Alcaligenes faecalis, isolated from Mangrove soil. Biotechnol. Res. Innov. 2017, 1, 101–111. [Google Scholar] [CrossRef]
  29. Yoon, S.H.; Ha, S.M.; Kwon, S.; Lim, J.; Chun, J. Introducing EzBioCloud: A taxonomically united database of 16S rRNA gene sequences and whole−genome assemblies. Int. J. Syst. Evol. Microbiol. 2017, 67, 1613–1617. [Google Scholar] [CrossRef]
  30. Rauret, G.; López−Sánchez, J.F.; Sahuquillo, A.; Rubio, R.; Davidson, C.; Ure, A.; Quevauviller, P. Improvement of the BCR three step sequential extraction procedure prior to the certification of new sediment and soil reference materials. J. Environ. Monit. 1999, 1, 57–61. [Google Scholar] [CrossRef]
  31. Compant, S.; Clément, C.; Sessitsch, A. Plant growth−promoting bacteria in the rhizo− and endosphere of plants: Their role, colonization, mechanisms involved and prospects for utilization. Soil Biol. Biochem. 2010, 42, 669–678. [Google Scholar] [CrossRef] [Green Version]
  32. Deng, S.Q.; Ke, T.; Li, L.T.; Cai, S.W.; Zhou, Y.Y.; Liu, Y.; Guo, L.M.; Chen, L.Z.; Zhang, D.Y. Impacts of environmental factors on the whole microbial communities in the rhizosphere of a metal−tolerant plant: Elsholtzia haichowensis Sun. Environ. Pollut. 2018, 237, 1088–1097. [Google Scholar] [CrossRef] [PubMed]
  33. Sun, X.L.; Xu, Z.H.; Xie, J.Y.; Hesselberg−Thomsen, V.; Tan, T.M.; Zheng, D.Y.; Strube, M.L.; Dragos, A.; Shen, Q.R.; Zhang, R.F.; et al. Bacillus velezensis stimulates resident rhizosphere Pseudomonas stutzeri for plant health through metabolic interactions. ISME J. 2022, 16, 774–787. [Google Scholar] [CrossRef] [PubMed]
  34. Durand, A.; Leglize, P.; Lopez, S.; Sterckeman, T.; Benizri, E. Noccaea caerulescens seed endosphere: A habitat for an endophytic bacterial community preserved through generations and protected from soil influence. Plant Soil 2022, 472, 257–278. [Google Scholar] [CrossRef]
  35. Zhou, Y.; Wei, Y.L.; Zhao, Z.J.; Li, J.S.; Li, H.M.; Yang, P.Z.; Tian, S.Z.; Ryder, M.; Toh, R.; Yang, H.T.; et al. Microbial communities along the soil−root continuum are determined by root anatomical boundaries, soil properties, and root exudation. Soil Biol. Biochem. 2022, 171, 108721. [Google Scholar] [CrossRef]
  36. Huang, J.; Zhu, X.Y.; Lu, J.; Sun, Y.; Zhao, X.Q. Effects of different land use types on microbial community diversity in the shizishan mining area. Environ. Sci. 2019, 40, 5551–5560. (In Chinese) [Google Scholar]
  37. Stefanowicz, A.M.; Kapusta, P.; Szarek−Łukaszewska, G.; Grodzińska, K.; Niklińska, M.; Vogt, R.D. Soil fertility and plant diversity enhance microbial performance in metal−polluted soils. Sci. Total Environ. 2012, 439, 211–219. [Google Scholar] [CrossRef]
  38. Sun, A.Q.; Jiao, X.Y.; Chen, Q.L.; Wu, A.L.; Zheng, Y.; Lin, Y.X.; He, J.Z.; Hu, H.W. Microbial communities in crop phyllosphere and root endosphere are more resistant than soil microbiota to fertilization. Soil Biol. Biochem. 2021, 153, 108113. [Google Scholar] [CrossRef]
  39. Sun, W.H.; Xiong, Z.L.; Li, W.; Soares, M.A.; White, J.F., Jr.; Li, H.Y. Bacterial communities of three plant species from Pb−Zn contaminated sites and plant−growth promotional benefits of endophytic Microbacterium sp. (strain BXGe71). J. Hazard. Mater. 2019, 370, 225–231. [Google Scholar] [CrossRef]
  40. Lv, X.; Wang, Q.K.; Zhang, X.Y.; Hao, J.J.; Li, L.; Chen, W.; Li, H.K.; Wang, Y.H.; Ma, C.P.; Wang, J.L.; et al. Community structure and associated networks of endophytic bacteria in pea roots throughout plant life cycle. Plant Soil 2021, 468, 225–238. [Google Scholar] [CrossRef]
  41. Glick, B.R.; Cheng, Z.Y.; Czarny, J.; Jin, D. New perspectives and approaches in plant growth−promoting rhizobacteria research. Eur. J. Plant Pathol. 2007, 119, 329–339. [Google Scholar] [CrossRef]
  42. Grobelak, A.; Kokot, P.; Hutchison, D.; Grosser, A.; Grosser, A. Plant growth−promoting rhizobacteria as an alternative to mineral fertilizers in assisted bioremediation−sustainable land and waste management. J. Environ. Manag. 2018, 227, 1–9. [Google Scholar] [CrossRef] [PubMed]
  43. Sharma, P. Efficiency of bacteria and bacterial assisted phytoremediation of heavy metals: An update. Bioresour. Technol. 2021, 328, 124835. [Google Scholar] [CrossRef]
  44. Sarwar, N.; Imran, M.; Shaheen, M.R.; Ishaque, W.; Kamran, M.A.; Matloob, A.; Rehim, A.; Hussain, S. Phytoremediation strategies for soils contaminated with heavy metals: Modifications and future perspectives. Chemosphere 2017, 171, 710–721. [Google Scholar] [CrossRef]
  45. Tessier, A.; Campbell, P.G.C.; Bisson, M. Sequential extraction procedure for the speciation of particulate trace metals. Anal. Chem. 1979, 51, 844–851. [Google Scholar] [CrossRef]
  46. Yang, P.; Zhou, X.F.; Wang, L.L.; Li, Q.S.; Zhou, T.; Chen, Y.K.; Zhao, Z.Y.; He, B.Y. Effect of phosphate−solubilizing bacteria on the mobility of insoluble cadmium and metabolic analysis. Int. J. Environ. Res. Public Health 2018, 15, 1330. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Li, W.L.; Wang, J.F.; Lv, Y.; Dong, H.J.; Wang, L.L.; He, T.; Li, Q.S. Improving cadmium mobilization by phosphate−solubilizing bacteria via regulating organic acids metabolism with potassium. Chemosphere 2020, 244, 125475. [Google Scholar] [CrossRef] [PubMed]
  48. Chen, L.; Luo, S.L.; Li, X.J.; Wan, Y.; Chen, J.L.; Liu, C.B. Interaction of Cd−hyperaccumulator Solanum nigrum L. and functional endophyte Pseudomonas sp. Lk9 on soil heavy metals uptake. Soil Biol. Biochem. 2014, 68, 300–308. [Google Scholar] [CrossRef]
  49. Wang, Q.; Ma, L.Y.; Zhou, Q.Y.; Chen, B.; Zhang, X.C.; Wu, Y.J.; Pan, F.S.; Huang, L.K.; Yang, X.E.; Feng, Y. Inoculation of plant growth promoting bacteria from hyperaccumulator facilitated non−host root development and provided promising agents for elevated phytoremediation efficiency. Chemosphere 2019, 234, 769–776. [Google Scholar] [CrossRef]
  50. Wu, B.; He, T.; Wang, Z.; Qiao, S.; Wang, Y.; Xu, F.; Xu, H. Insight into the mechanisms of plant growth promoting strain SNB6 on enhancing the phytoextraction in cadmium contaminated soil. J. Hazard. Mater. 2020, 385, 121587. [Google Scholar] [CrossRef]
  51. Liu, C.J.; Lin, H.; Dong, Y.B.; Li, B. Increase of P and Cd bioavailability in the rhizosphere by endophytes promoted phytoremediation efficiency of Phytolacca acinosa. J. Hazard. Mater. 2022, 431, 128546. [Google Scholar] [CrossRef] [PubMed]
  52. Wang, L.; Lin, H.; Dong, Y.B.; Li, B.; He, Y.H. Effects of endophytes inoculation on rhizosphere and endosphere microecology of Indian mustard (Brassica juncea) grown in vanadium−contaminated soil and its enhancement on phytoremediation. Chemosphere 2020, 240, 124891. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. The composition of endophytic bacterial community in the castor. (A) The NMDS analysis of endophytic bacterial community; (B) Relative abundance of the endophytic bacterial community at phylum level; (C) Comparison of the relative abundance of endophytic bacteria between the same tissues at phylum level (* 0.01 < p ≤ 0.05, ** 0.001 < p ≤ 0.01, *** p ≤ 0.001); (D) Relative abundance of the endophytic bacterial community at genus level.
Figure 1. The composition of endophytic bacterial community in the castor. (A) The NMDS analysis of endophytic bacterial community; (B) Relative abundance of the endophytic bacterial community at phylum level; (C) Comparison of the relative abundance of endophytic bacteria between the same tissues at phylum level (* 0.01 < p ≤ 0.05, ** 0.001 < p ≤ 0.01, *** p ≤ 0.001); (D) Relative abundance of the endophytic bacterial community at genus level.
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Figure 2. Cu mobilization and variances of pH values in supernatant. (A) Cu2+ concentration in supernatant; (B) pH of supernatant.
Figure 2. Cu mobilization and variances of pH values in supernatant. (A) Cu2+ concentration in supernatant; (B) pH of supernatant.
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Figure 3. DTPA extractable Cu (A) and Cd (B) in soil. The different lowercase letters in the figures indicate significant differences (p < 0.05).
Figure 3. DTPA extractable Cu (A) and Cd (B) in soil. The different lowercase letters in the figures indicate significant differences (p < 0.05).
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Figure 4. The chemical speciation of Cu (A) and Cd (B) in soil. The different lowercase letters in the figures indicate significant differences (p < 0.05).
Figure 4. The chemical speciation of Cu (A) and Cd (B) in soil. The different lowercase letters in the figures indicate significant differences (p < 0.05).
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Figure 5. Available P concentration in soil with different inoculation of endophytic bacteria. The different lowercase letters in the figure indicate significant differences (p < 0.05).
Figure 5. Available P concentration in soil with different inoculation of endophytic bacteria. The different lowercase letters in the figure indicate significant differences (p < 0.05).
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Table
Table 1. The basic characteristics of soils in two sampling sites.
Table 1. The basic characteristics of soils in two sampling sites.
Sampling SitesCu
mg/kg		Cd
mg/kg		pHAvailable P mg/kgOM
(g/kg)		NH4+−N
mg/kg		NO3−−N
mg/kg
Tonglushan693.84.38.104.47.03.83.4
Gangxia833.09.57.7622.439.55.859.2
Table
Table 2. Observed OTUs and Alpha diversity indices in Ricinus communis L.
Table 2. Observed OTUs and Alpha diversity indices in Ricinus communis L.
SourcesSamplesSequencesOTUsSimpsonChao1Coverage (%)
Tonglushan castorTR36,1167830.14361.099.9
TS45,6625050.23264.799.8
TL48,5791100.7171.999.8
Gangxia castorYR52,46811240.09654.899.6
YS55,58410610.19678.299.4
YL63,4099020.20443.699.5
Table
Table 3. Plant growth−promoting traits and heavy metal tolerance for isolated endophytic bacteria strains.
Table 3. Plant growth−promoting traits and heavy metal tolerance for isolated endophytic bacteria strains.
StrainPGP TraitsHeavy Metal
Tolerance
IAA
(mg/L)		Siderophores Production
((λ0 − λ)/λ0)		ACCD
μmol α− KA/(h·mg)		Phosphate SolubilizationNitrogen FixationCu2+
(mg/L)		Cd2+
(mg/L)
Soluble P
Concentration
(mg/L)		pH in
Supernatant
TR151.8 ± 1.50.159−−−+12010
TR249.4 ± 0.20.26473.7 ± 2.9−−+60025
TR3119.1 ± 15.40.245138.1 ± 42.2141.5 ± 8.05.0 ± 0.1+600150
TR493.4 ± 19.40.232107.4 ± 43.8314.7 ± 71.24.7 ± 0.4+600150
TR546.6 ± 12.30.14138.6 ± 1.8492.9 ± 14.44.3 ± 0.1+50010
TR6140.1 ± 9.50.09827.6 ± 2.5−−+40050
TR74.0 ± 0.00.161−416.2 ± 10.04.6 ± 0.1+50010
TR863.5 ± 7.80.10971.4 ± 7.6190.7 ± 5.14.9 ± 0.0+600100
TR1073.1 ± 11.90.215−479.4 ± 26.24.5 ± 0.0+600100
TR1143.4 ± 0.1−35.9 ± 0.1483.1 ± 23.54.4 ± 0.1+60025
TR127.1 ± 0.00.289109.6 ± 41.7115.5 ± 29.05.0 ± 0.1+600150
TR1340.0 ± 1.20.130−−−+50025
TR1432.6 ± 1.30.22322.6 ± 2.5−−+30010
TR1513.0 ± 1.00.309143.8 ± 6.1130.1 ± 5.05.1 ± 0.0+600150
TR166.6 ± 1.30.35822.0 ± 2.0495.5 ± 10.34.5 ± 0.0+1100100
TR1825.7 ± 1.60.29147.4 ± 1.4127.4 ± 7.35.0 ± 0.0+600150
TR2029.6 ± 2.60.24137.9 ± 1.6−−+50010
TR2120.9 ± 5.2−233.9 ± 30.687.2 ± 4.15.0 ± 0.0+500100
TR2353.5 ± 9.00.33950.2 ± 2.8450.6 ± 7.44.4 ± 0.0+50010
TS136.4 ± 1.40.23623.8 ± 1.3−−+120−
TS210.8 ± 0.70.092−−−+12025
TS811.9 ± 0.10.58228.7 ± 0.4−−+4025
TL16.8 ± 0.00.11133.8 ± 0.2−−+12010
TL25.6 ± 0.00.282−−−−12050
TR2413.4 ± 0.50.250−467.9 ± 40.94.5 ± 0.1+12050
YR150.3 ± 6.80.30218.6 ± 0.4−−+120−
YR228.5 ± 6.60.32043.9 ± 3.6−−+60010
YL18.1 ± 0.00.268−445.3 ± 2.14.5 ± 0.1+12050
YL26.3 ± 0.00.241−85.6 ± 30.95.1 ± 0.2+8050
YL34.9 ± 0.40.22020.1 ± 3−−+120−
YL438.3 ± 2.1−22.5 ± 0.4−−+12010
YL522.0 ± 1.2−−−−+120−
YL613.8 ± 0.00.130−−−+12050
YL76.2 ± 0.0−29.2 ± 0.0−−+12025
YS133.1 ± 1.90.344−183.3 ± 12.24.9 ± 0.0+40200
YS254.0 ± 3.10.34719.3 ± 2.2−−+12010
YS3124.3 ± 3.40.30621.5 ± 4.7−−+500200
YS426.9 ± 6.40.34441.5 ± 0.4−−+20010
YS510.9 ±0.00.232−469.8 ± 9.24.7 ± 0.0+12025
YS64.0 ± 0.0−−−−−12025
YS715.3 ± 6.00.32732.9 ± 2.9−−+120−
YS818.1 ± 1.20.23211.6 ± 3.7−−+30010
YS99.0 ± 0.00.26118.8 ± 0.5−−+12025
YS1042.3 ± 1.40.30032.5 ± 11.1−−+12010
“(λ0 − λ)/λ0)” represents the ability to produce siderophores, and the higher the value is, the higher the capacity for siderophore production will be. “ACCD” and “α− KA” mean ACC deaminase and α−ketobutyrate, respectively. “−” means the strain does not possess the corresponding growth−promoting ability. “+” indicates the strain has the ability to perform nitrogen fixation.
Table
Table 4. The 16S rDNA gene sequence similarity analysis of the eight isolates.
Table 4. The 16S rDNA gene sequence similarity analysis of the eight isolates.
StrainsAccession NumberClosest Relative StrainSimilarity (%)Genus
TR4OP310027Enterobacter kobei (CP017181)99.8Enterobacter
TR8OP310028Buttiauxella agrestis (JMPI01000079)99.3Buttiauxella
TR16OP310029Acinetobacter pittii (APQP01000001)99.0Acinetobacter
TR18OP310030Enterobacter kobei (CP017181)99.8Enterobacter
TR21OP310031Enterobacter huaxiensis (FYBI01000003)99.3Enterobacter
YL1OP310032Bacillus altitudinis (ASJC01000029)99.2Bacillus
YS3OP310033Proteus myxofaciens (LXEN01000172)98.5Proteus
YS5OP310034Metabacillus idriensis (AY904033)99.8Metabacillus
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Li, Q.; Fu, Q.; Zhu, J.; Sun, Y.; He, H.; Hu, H. Endophytic Bacteria in Ricinus communis L.: Diversity of Bacterial Community, Plant−Growth Promoting Traits of the Isolates and Its Effect on Cu and Cd Speciation in Soil. Agronomy 2023, 13, 333. https://doi.org/10.3390/agronomy13020333

AMA Style

Li Q, Fu Q, Zhu J, Sun Y, He H, Hu H. Endophytic Bacteria in Ricinus communis L.: Diversity of Bacterial Community, Plant−Growth Promoting Traits of the Isolates and Its Effect on Cu and Cd Speciation in Soil. Agronomy. 2023; 13(2):333. https://doi.org/10.3390/agronomy13020333

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Li, Qian, Qingling Fu, Jun Zhu, Yuxin Sun, Huan He, and Hongqing Hu. 2023. "Endophytic Bacteria in Ricinus communis L.: Diversity of Bacterial Community, Plant−Growth Promoting Traits of the Isolates and Its Effect on Cu and Cd Speciation in Soil" Agronomy 13, no. 2: 333. https://doi.org/10.3390/agronomy13020333

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