Pseudomonas canavaninivorans RB14 and its application

Abstract

The application discloses a Pseudomonas canavaninivorans RB14 endophytic strain of Sasa veitchii and application thereof, belongs to the technical field of microorganisms and heavy metal contaminated soil remediation, and the Pseudomonas canavaninivorans RB14 endophytic strain of Sasa veitchii is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 38454. The Sasa veitchii endophytic bacteria RB14 disclosed by the application can significantly promote the growth of Sasa veitchii in common soil and heavy metal lead-zinc stress environment, especially in the lead-zinc stress environment, and has a significant promoting effect on lead-zinc enrichment and transport of Sasa veitchii, growth, stress resistance and soil nutrients. The bacteria can be applied to production, have the effects of promoting lead-zinc enrichment, transport and growth of plants, and have important significance for remediation of heavy metal contaminated soil and protection of an ecological environment.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a bacterium RB14 isolated from the roots of *Indocalamus fragilis* and its application in enhancing the remediation potential of *Indocalamus fragilis* in lead-zinc contaminated soil, particularly the application of using endophytic bacteria to assist bamboo plants in remediating lead-zinc contaminated soil. Background Technology

[0002] my country is a major producer of lead and zinc mineral resources. While lead and zinc mining and smelting have driven national economic development, they have also caused serious soil lead pollution. Lead-zinc tailings, as typical mining waste sites, generally suffer from high heavy metal content, nutrient deficiency, and structural deterioration in their soil, severely inhibiting the natural restoration of vegetation and easily leading to the formation of large-scale ecological degradation zones. Furthermore, their tailings are rich in phosphorus (Pb). 2+ Zn 2+ Long-term exposure to heavy metal ions can easily cause ecological risks.

[0003] Phytoremediation technology has attracted much attention in the treatment of heavy metal pollution in soil due to its advantages such as environmental friendliness, low cost, and high efficiency. *Inula japonica*, a perennial evergreen plant belonging to the genus *Inula* of the Poaceae family, is an ideal soil remediation plant compared to hyperaccumulators, possessing characteristics such as a well-developed root system, rapid biomass accumulation, and strong stress resistance.

[0004] However, traditional phytoremediation technologies suffer from limitations such as lengthy remediation cycles and low heavy metal accumulation, restricting their large-scale application. Microbial-assisted phytoremediation (MAP) overcomes these limitations by leveraging the growth-promoting, detoxifying, and resistance-enhancing effects of microorganisms on plants, representing a novel and highly promising strategy for soil heavy metal remediation. In MAP systems, tolerant plants serve as the core carriers, not only fixing and absorbing heavy metals through their roots but also regulating the rhizosphere microenvironment via root exudates, recruiting beneficial microorganisms to build a mutually beneficial symbiotic system, thereby synergistically enhancing plant stress resistance and remediation efficacy.

[0005] Currently, research on microbial-assisted phytoremediation mainly focuses on directly isolating and screening microorganisms from contaminated soil. However, this method is inefficient, and the screened strains often lack specificity. Since lead-zinc tailings areas commonly suffer from heavy metal pollution and nutrient deficiencies, developing symbiotic microorganisms that simultaneously possess highly efficient growth-promoting functions, facilitate heavy metal remediation, and are easily colonized has been a long-standing but unresolved problem in this field. Summary of the Invention

[0006] One of the objectives of this invention is to provide an endophytic strain of *Pseudomonas canavaninivorans* RB14 to solve the aforementioned problems.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] An endophytic strain of *Pseudomonas canavaninivorans* RB14, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38454, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, on April 27, 2026.

[0009] The endophytic Pseudomonas canavaninivorans RB14 provided by this invention is a Gram-negative bacterium. The optimal growth temperature is 25-35℃, and the optimal pH is 4.0-10.0.

[0010] The second objective of this invention is to provide an application of the above-mentioned endophytic strain Pseudomonas canavaninivorans RB14 of *Phyllostachys edulis* in the remediation of lead-zinc contaminated soil.

[0011] As a preferred technical solution, the Pseudomonas canavaninivorans RB14 endophytic strain is used to enhance the remediation potential of Pseudomonas canavaninivorans for lead-zinc contaminated soil.

[0012] As a further preferred technical solution, the lead concentration in the lead-zinc contaminated soil is up to 1500 mg / kg. The bacterium RB14 is effective at 500 mg / L Pb. 2+ The lead enrichment level in the culture medium was as high as 7.00±1.36 mg / g.

[0013] As a further preferred technical solution, the bamboo is *Ruozhu* var. *mairei*.

[0014] The third objective of this invention is to provide the application of the aforementioned Pseudomonas canavaninivorans RB14 in promoting the growth of bamboo under high concentrations of lead and zinc stress.

[0015] As a preferred technical solution, the *Pseudomonas canavaninivorans* RB14 increases the available phosphorus content in the soil. This strain exhibits a high inorganic phosphorus solubility of 112.50 ± 6.93 mg / L.

[0016] Compared with the prior art, the advantages of the present invention are as follows:

[0017] Compared with the control group without fungal treatment, the application of Pseudomonas canavaninivorans RB14 significantly promoted the growth of *Indocalamus canavaninivorans*, improved its resistance, and enhanced soil nutrients in both ordinary soil and soil under high concentrations of lead and zinc stress. The improvement was even more pronounced under high concentrations of lead and zinc stress (p < 0.05). At 500 mg / L Pb... 2+ The lead accumulation in the culture medium reached as high as 7.00±1.36 mg / g. Under high-concentration lead-zinc stress, compared with the control, after fungicide application, the leaf area of ​​*Inula spp.* increased by 90.01%, and the areas of roots, rhizomes, stems, and leaves increased by 72.64%, 32.57%, 37.38%, and 62.25%, respectively, with a total biomass increase of 42.84%. Physiologically, after fungicide application, the free proline content in the roots of *Inula spp.* increased by 138.28%, and the MDA content decreased by 47.92%, effectively enhancing the plant's stress resistance (p<0.05). Soil nutrient formulation... After application of the fungus, the available nitrogen content in the soil increased by 157.28%, the available phosphorus content increased by 34.67%, and the activities of urease and phosphatase increased by 22.70% and 174.11%, respectively. It significantly promoted the accumulation of lead and zinc and their translocation to the aboveground parts, increasing the total accumulation of lead and zinc in *Pseudomonas canavaninivorans* by 52.27% and 24.27%, respectively. In particular, the lead content in the roots increased by 73.44%, and the underground-to-aboveground translocation coefficients of lead and zinc increased by 37.62% and 36.93%, respectively. *Pseudomonas canavaninivorans* RB14 can be applied in production, possessing the functions of alleviating lead and zinc stress, promoting plant growth, accumulating and translocating lead and zinc, and activating soil nutrients. This invention provides a new method and approach for the ecological restoration of lead-zinc tailings areas and the remediation of heavy metal-contaminated soil by bamboo plants assisted by endophytic bacteria, and also provides excellent microbial resources. Attached Figure Description

[0018] Figure 1 The results of morphological and molecular biological identification of the endophytic bacterium RB14 of this invention are as follows. Figure 1 In the middle: AB: Colony morphology; C: Neighbor-Joining phylogenetic tree;

[0019] Figure 2 The lead enrichment and phosphorus solubility capabilities of the endophytic bacterial strain RB14 of this invention; Figure 2 In Chinese: A: Morphology on microporous filter membranes; B: Lead enrichment and inorganic phosphorus dissolution capacity; C: Inorganic phosphorus dissolution rings;

[0020] Figure 3 Comparison of growth of *Ipomoea purpurea* treated with the endophytic bacteria RB14 of the present invention with that without treatment; Figure 3In the middle: CK0: ordinary soil; CK1: ordinary soil + RB14 bacterial solution; T0: lead-zinc soil; T1: lead-zinc soil + RB14 bacterial solution, and... Figure 4 same;

[0021] Figure 4 The restorative effect of applying the endophytic bacteria RB14 of the present invention on *Ipomoea purpurea*; Figure 4 In the figure: AB: comparison of leaf area and biomass; CD: comparison of free proline and MDA content; EH: comparison of available nitrogen, available phosphorus, urease activity and phosphatase activity in soil; IJ: comparison of Pb enrichment in whole plant and content in each organ; KL: comparison of Zn enrichment in whole plant and content in each organ; lowercase letters a, b, c, etc. in the figure indicate significant differences among bamboo species within different categories at the p<0.05 level. Detailed Implementation

[0022] To explain the technical content, objectives, and effects of the present invention in detail, the following specific embodiments further illustrate the content of the present invention. However, the content of the present invention is far more than the following embodiments.

[0023] The LB medium of the present invention has the following formula: 10.0 g / L tryptone + 5.0 g / L yeast extract + 10.0 g / L sodium chloride, with a pH of 6.8-7.2.

[0024] The LBA culture medium of the present invention has the following formula: LB + 20.0 g / L agar.

[0025] The IPB culture medium of the present invention has the following formula: 10 g / L glucose + 0.5 g / L ammonium sulfate + 0.3 g / L sodium chloride + 0.3 g / L potassium chloride + 0.3 g / L magnesium sulfate heptahydrate + 0.03 g / L ferrous sulfate heptahydrate + 1.0 g / L manganese sulfate + 5.0 g / L calcium phosphate.

[0026] The IPA culture medium of the present invention has the following formula: IPB + 20.00 g / L agar.

[0027] The OPA culture medium of the present invention has the following formula: 10 g / L glucose + 0.5 g / L ammonium sulfate + 0.3 g / L sodium chloride + 0.3 g / L potassium chloride + 0.3 g / L magnesium sulfate heptahydrate + 0.03 g / L ferrous sulfate heptahydrate + 1.0 g / L manganese sulfate + 2 g / L lecithin + 20.00 g / L agar.

[0028] Example 1

[0029] Isolation, screening and molecular biological identification of endophytic RB14 strains

[0030] (1) Strain strain isolation:

[0031] Root samples were collected from *Imperata cylindrica* (a type of bamboo) that had grown for 3 years in a lead-zinc tailings area in Sichuan Province. The samples were first rinsed thoroughly with running water, then dried with absorbent paper and cut into approximately 5 mm segments. The roots were then subjected to surface sterilization treatment following this procedure: rinsing with running water for 30 minutes, followed by rinsing with sterile water 3–5 times; immersion in 75% ethanol for 2 minutes, followed by rinsing with sterile water 3–5 times again; then immersion in 5% sodium hypochlorite solution for 5 minutes, and repeated rinsing with sterile water 3–5 times. 100 µL of the sterile water from the final rinse was spread onto LBA medium and incubated upside down at 30°C in the dark for 5 days to verify the surface sterilization effect. 1.0 g of the surface-sterilized roots were placed in a sterile mortar, 5 mL of sterile water was added, and the roots were ground to obtain a tissue homogenate. 100 μL of serially diluted tissue homogenate was spread onto LBA medium. After inoculation, solid plates were inverted and incubated in the dark at 30°C for 5–7 days. Once colonies had grown, single colonies were selected based on morphological differences and inoculated onto LBA medium for isolation and purification. The purified strains were stored in 25% (v / v) glycerol and kept at -80°C for long-term preservation.

[0032] (2) Strain screening:

[0033] Lead tolerance of isolated strains was initially screened by plating them onto LBA medium containing 500 mg / L Pb [Pb(NO3)2]. Pb-free LBA was used as a control. The cultures were incubated at 30 °C in the dark for 3 days to observe colony formation and measure the diameter of individual colonies. Subsequently, the lead tolerance of the initially screened strains was tested using LB medium containing gradient concentrations of Pb (0, 300 mg / L, 500 mg / L, 750 mg / L, and 1000 mg / L). After incubation at 30 °C with shaking at 180 rpm in the dark for 3 days, the OD of the bacterial culture was measured. 600 Absorbance (SpectraMax® ABS) was used to screen for strains with high tolerance to lead.

[0034] The selected strains were inoculated onto inorganic phosphorus agar (IPA) and organic phosphorus agar (OPA) media, respectively, and cultured at 28°C in the dark for 7 days. The ability to dissolve inorganic phosphorus (IP) and organic phosphorus (OP) was qualitatively evaluated by observing and measuring the diameter of the transparent circles appearing around the colonies. The nitrogen-fixing capacity of the strains was tested using Ashby solid medium; after 7 days of culture, normal growth was considered indicative of nitrogen-fixing ability. Siderophore production capacity was tested using CAS agar medium; after 7 days of culture, a change in the medium around the colonies from blue to orange indicated siderophore secretion activity, and the secretion capacity was assessed by the ratio of the secretion circle diameter (D) to the colony diameter (D0) (D / D0). Using 1-aminocyclopropane-1-carboxylic acid ester (ACC) as the sole nitrogen source, the growth of the strains on DF basal medium and ADF solid medium supplemented with ACC was compared to qualitatively evaluate their ACC deaminase production capacity. The strains were inoculated onto the two culture media mentioned above, and after incubation at 28°C in the dark for 7 days, the colony diameter was measured, and the DADF / DDF ratio was calculated. When D... ADF / D DF When the ratio is >1, the strain is considered to be able to produce ACC deaminase, and the higher the ratio, the stronger the enzyme production capacity. All evaluation experiments were performed with three biological replicates. Finally, cluster heatmaps and the rank-sum ratio (RSR) model were used to analyze the qualitative evaluation of the PGP functional traits of each strain, and the endophytic strain RB14 with excellent overall performance was selected.

[0035] (3) Morphological and molecular biological identification of endophytic strain RB14:

[0036] like Figure 1 A and Figure 1 As shown in Figure B, the endophytic strain RB14 forms round, pale yellow, loosely textured colonies with smooth surfaces and regular edges on LBA medium. The endophytic *Pseudomonas canavaninivorans* RB14 provided by this invention is a Gram-negative bacterium. The optimal growth temperature is 25-35℃, and the optimal pH is 4.0-10.0.

[0037] Genomic DNA was extracted from RB14 cells using the CTAB method, and PCR amplification was performed using universal primers 341F (SEQ ID NO:1: CCTAYGGGRBGCASCAG) and 806R (SEQ ID NO:2: GGACTACNNGGGTATCTAAT) for the hypervariable region of bacterial 16S rDNA V3-V4. The amplified products were sequenced by Beijing Qingke Biotechnology Co., Ltd., and the sequence of the obtained 16S rRNA amplified product is shown in SEQ ID NO:3 below.

[0038]

[0039] The aforementioned 16S rDNA sequence has been uploaded to GenBase, accession number: C_AA167429.1 (https: / / ngdc.cncb.ac.cn / genbase / ). The obtained sequence was subjected to BLASTn homology alignment in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / , accessed May 10, 2025). The ClustalW method was used to align the sequence with other 16S rDNA sequences of the most similar species. The alignment results showed that strain RB14 had a 99.93% similarity to the 16S rDNA sequence of *Pseudomonas canavaninivorans*, with 100% coverage. The 16S rDNA sequence of strain RB14 and its homologous sequences were integrated using MEGA 12 software, and a phylogenetic tree was constructed using the Neighbor-Joining method. The results preliminarily confirm strain RB14 as *Pseudomonas canavaninivorans*. (See...) Figure 1 C.

[0040] Example 2

[0041] This embodiment tests the lead accumulation capacity and growth-promoting properties of the endophytic bacterium RB14 strain.

[0042] (1) Test of lead accumulation capacity of Pseudomonas canavaninivorans RB14 strain

[0043] To accurately assess the lead accumulation capacity of the bacterial strains, a 0.22 μm sterile microporous membrane was used to cover the surface of LBA medium containing 500 mg / L Pb [Pb(NO3)2 as the lead source], effectively separating the bacteria from the medium without affecting their normal growth. The activated candidate strains were inoculated into the center of the membrane and incubated upside down in the dark at 28°C for 7 days. Clean colonies on the membrane were collected, dried at 50°C for 24 hours, and weighed. The bacteria were then digested using a nitric acid-perchloric acid system, and the lead content was determined using inductively coupled plasma optical emission spectrometry (ICP-OES) (PerkinElmer, Optima 8000, USA).

[0044] The results are as follows Figure 2 As shown, Pseudomonas canavaninivorans RB14 exhibited excellent lead enrichment capacity, with a lead enrichment capacity of up to 7.00±1.36 mg / g in 500 mg / L lead medium.

[0045] (2) Test of growth-promoting characteristics of Pseudomonas canavaninivorans RB14 strain

[0046] The inorganic phosphorus solubility of RB14 was quantitatively evaluated. The strain was inoculated into inorganic phosphorus broth (IPB) medium and cultured at 30°C and 180 rpm for 7 days. After centrifugation at 8000 rpm for 20 minutes, the supernatant was collected, and the soluble phosphorus content was determined using the molybdenum blue method to quantitatively evaluate its inorganic phosphorus solubility. All quantitative evaluation experiments were performed in triplicate.

[0047] The results are as follows Figure 2 As shown, the inorganic phosphorus solubility of Pseudomonas canavaninivorans RB14 strain is 112.50±6.93 mg / L, exhibiting excellent plant growth-promoting characteristics.

[0048] Example 3

[0049] Verification of the effect of Pseudomonas canavaninivorans RB14 in assisting the remediation of lead-zinc contaminated soil by *Indocalamus fragilis*.

[0050] (1) Experimental design

[0051] Soil containing 1500 mg / kg Pb and 1500 mg / kg Zn was prepared by exogenously adding Pb(NO3)2 and ZnSO4. After stabilization for 2-3 months, 4 kg of air-dried lead-contaminated soil (with ordinary soil without added lead and zinc as a negative control) was filled into 40 cm diameter circular plastic flowerpots according to the experimental design. *Imperata cylindrica* seedlings were then propagated by burying rhizomes, with 3 rhizome segments evenly buried in each pot, 3 pots per treatment. Each rhizome segment was 15 cm long and had a uniform diameter. The experimental treatments were as follows:

[0052] CK0: Ordinary soil (negative control);

[0053] CK1: Ordinary soil + RB14 bacterial solution (negative treatment);

[0054] T0: Lead-zinc soil (1500 mg / kg Pb + 1500 mg / kg Zn, positive control);

[0055] T1: Lead-zinc soil + RB14 bacterial solution (positive treatment).

[0056] After the above treatment, they are placed in a greenhouse for cultivation, where they are only given normal watering, weeding and other maintenance, without any additional fertilization.

[0057] (2) Preparation and application of bacterial solution

[0058] The bacterial strain was removed from -80℃ and inoculated into LBA. It was then incubated upside down in a dark environment at 30℃ for 5-7 days to activate the culture. Next, the activated pure colonies were transferred to 50mL of LB solution, sealed with a breathable membrane, and incubated at 30℃ and 180-200 rpm in a shaker for 5-7 days to obtain the stock solution. The OD of the stock solution was... 600 Centrifuge at approximately 1,4000 rpm for 10 minutes, discard the supernatant; then wash twice by centrifugation and resuspend in 50 mL of sterile water to prepare a stock solution; dilute 10 times with sterile water before application.

[0059] After 3-4 months of cultivation, apply 50 mL of bacterial solution to each pot by root irrigation, once every 20 days, for a total of 3 applications. 120 days after the completion of the inoculum application, harvest the samples and determine the relevant indicators.

[0060] (3) Effects of Pseudomonas canavaninivorans RB14

[0061] Three to four months after the application of the inoculant, growth, physiological parameters, soil nutrients, and heavy metal accumulation were measured in different treatment groups. The experimental results are as follows: Figure 3 and Figure 4 As shown in the figure. Compared with the control without fungal application, the application of Pseudomonas canavaninivorans RB14 significantly promoted the growth, improved the resistance, and enhanced soil nutrients of *Indocalamus 'Beautiful Beauty'* planted in both ordinary soil and lead-zinc stressed soil, with the improvement effect being more pronounced under lead-zinc stress (p < 0.05). Under lead-zinc stress, compared with the control, after fungal application, the leaf area of ​​*Indocalamus 'Beautiful Beauty' increased by 90.01%, and the areas of roots, rhizomes, stems, and leaves increased by 72.64%, 32.57%, 37.38%, and 62.25%, respectively, while the total biomass of the plant increased by 42.84%. Physiologically, after fungal application, the free proline content in the roots of *Indocalamus 'Beautiful Beauty' increased by 138.28%, and the MDA content decreased by 47.92%, effectively enhancing the plant's stress resistance (p < 0.05). Regarding soil nutrients, after the application of the fungicide, the available nitrogen content in the soil increased by 157.28%, the available phosphorus content increased by 34.67%, and the activities of urease and phosphatase increased by 22.70% and 174.11%, respectively. The high activity of phosphatase and the relatively low increase in available phosphorus content may be due to the precipitation of some phosphate ions by lead binding, which reduced the stress of lead on the plant roots. Pseudomonas canavaninivorans RB14 significantly promoted the accumulation of lead and zinc and their translocation to the aboveground parts, increasing the total accumulation of lead and zinc in Pseudomonas canavaninivorans by 52.27% and 24.27%, respectively. In particular, the lead content in the roots increased by 73.44%, and the underground to aboveground translocation coefficients of lead and zinc increased by 37.62% and 36.93%, respectively.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An endophytic strain of *Pseudomonas canavaninivorans* RB14, characterized in that, The endophytic strain of *Pseudomonas canavaninivorans* RB14, described above, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38454, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, on April 27, 2026.

2. The application of the Pseudomonas canavaninivorans RB14 endophytic strain of *Pseudomonas canavaninivorans* as described in claim 1 in the remediation of lead-zinc contaminated soil.

3. The application according to claim 2, characterized in that, The endophytic strain Pseudomonas canavaninivorans RB14 from *Indocalamus stenoptera* was used to enhance the remediation potential of *Indocalamus stenoptera* for lead-zinc contaminated soil.

4. The application according to claim 3, characterized in that, The lead concentration in the lead-zinc contaminated soil reached as high as 1500 mg / kg.

5. The application according to claim 2, characterized in that, The bamboo mentioned is the beautiful bamboo.

6. The application of Pseudomonas canavaninivorans RB14 as described in claim 1 in promoting the growth of bamboo under high concentration lead and zinc stress.

7. The application according to claim 6, characterized in that, The Pseudomonas canavaninivorans RB14 enhances the ability of bamboo to accumulate and translocate lead and zinc.

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