Pseudomonas moorei and application thereof

Abstract

The application discloses a Pseudomonas molinii strain and application thereof, and belongs to the technical field of agricultural microorganisms and heavy metal pollution prevention and control. Pseudomonas mosselii The strain PMZ1 is a Pseudomonas molinii strain isolated from the rhizosphere of rice and has higher safety when applied to in-situ removal of cadmium and arsenic pollution in the rice. Compared with the existing reports, the strain has a significant improvement in the ability of reducing cadmium and arsenic in the rice, can realize synchronous reduction of cadmium and arsenic, and has a certain growth promotion potential. The strain can be applied to realize production and repair of the rice in the soil with medium and light cadmium and arsenic composite pollution.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology and heavy metal pollution control technology, and more specifically to a strain of Pseudomonas molluscum and its application. Background Technology

[0002] In recent years, heavy metal pollution in my country's soil has become a prominent issue, particularly cadmium (Cd) and arsenic (As), with exceedance rates reaching 7.0% and 2.7% respectively, ranking first and third among all pollutants. Rice, due to its unique anatomical structure, root acid and oxygen secretion, and the alternating wet and dry environment of paddy fields during cultivation, is particularly prone to accumulating cadmium and arsenic.

[0003] Currently, technologies for controlling cadmium and arsenic pollution in rice mainly include agronomic measures (paddy field water management, application of heavy metal passivating agents, etc.), phytoremediation, and microbial remediation. However, all of these have some drawbacks. Water management is difficult for farmers to implement, and the effects of applying heavy metal passivating agents are unstable and their remediation efficiency gradually decreases over time. Phytoremediation has a long cycle and slow efficiency. Microbial remediation technologies mainly include microbial leaching, in-situ passivation with microbial fertilizers, and microbial-assisted phytoremediation. Due to its high remediation efficiency, low cost, and lack of damage to the soil's structure and fertility, it is receiving increasing attention.

[0004] Cadmium and arsenic in paddy fields often exhibit opposite chemical properties. When farmland is flooded and the soil Eh level decreases, rice accumulates less cadmium but more arsenic. Conversely, when paddy fields are in an aerobic environment and the soil Eh level increases, rice accumulates less arsenic but more cadmium. Therefore, it is difficult to control cadmium and arsenic pollution in paddy fields simultaneously.

[0005] Therefore, how to provide a microbial strain that can simultaneously reduce the cadmium and arsenic content in rice is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a strain of *Pseudomonas molluscum* and its applications. This strain can not only promote the growth of rice in polluted paddy fields, but also significantly reduce the cadmium and arsenic content in rice grains, thus achieving safe rice production.

[0007] Inoculating rice rhizosphere with *Pseudomonas molluscum*, a siderogenic carrier isolated from the rice rhizosphere, effectively colonizes the rice roots, chelates manganese and iron ions in the soil, and enhances the formation of an iron-manganese colloid film on the rice root surface. This simultaneously blocks harmful elements such as cadmium and arsenic from entering the root system and transporting to the aboveground parts, thus reducing the cadmium and arsenic content in rice. This reduces the concentration of cadmium and arsenic in rice with moderate to mild pollution (total soil Cd ≤ 1.5 mg / kg). -1 pH ≤ 5.5 or total soil Cd ≤ 2.0 mg / kg -1 pH ≤ 6.5; Total soil arsenic ≤ 150 mg / kg -1This method, which allows rice to be produced in paddy fields with pH ≤ 6.5 while simultaneously meeting cadmium and arsenic standards, has great application potential.

[0008] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0009] A strain of *Pseudomonas molluscum*, named PMZ1, is classified as (…). Pseudomonas mosselii It was deposited on September 1, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20251942, and the deposit address is Wuhan University, Wuhan, China.

[0010] The aforementioned application of Pseudomonas molluscum in reducing cadmium and arsenic levels.

[0011] Furthermore, the reduction of cadmium and arsenic content refers to reducing the cadmium and arsenic content in rice roots and rice grains.

[0012] The above-mentioned application of Pseudomonas molluscum in increasing rice yield.

[0013] A microbial agent for reducing cadmium and arsenic content, comprising the aforementioned *Pseudomonas molluscum*.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0015] The strain PMZ1 of this invention is a strain of Pseudomonas molluscum (… Pseudomonas mosselii This strain, isolated from the rhizosphere of rice, exhibits enhanced safety when applied to in-situ removal of cadmium and arsenic pollution in rice. Compared to existing reports, this strain significantly improves the ability to reduce cadmium and arsenic in rice, achieving simultaneous reduction of both, and also possesses certain growth-promoting potential. Applying this strain enables simultaneous production and remediation of rice in mildly to moderately contaminated soils with combined cadmium and arsenic pollution, providing a safe, effective, and environmentally friendly bioremediation method. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 The colony morphology of strain PMZ1 in Example 2 of this invention is shown below. Figure 1 A represents the morphology of strain PMZ1 in LB medium. Figure 1 B represents the morphology of strain PMZ1 in CAS medium. Figure 1C represents the morphology of strain PMZ1 in Monkina organophosphate medium.

[0018] Figure 2 This is the phylogenetic tree of strain PMZ1 in Example 2 of the present invention.

[0019] Figure 3 This is a diagram showing the results of the acid-base tolerance experiment of *Pseudomonas molluscum* PMZ1 in Example 4 of the present invention. Figure 3 A is a graph showing the trend of OD600 value of Pseudomonas molluscum PMZ1 over time in liquid LB medium with pH values ​​ranging from 4 to 10. Figure 3 B is a graph showing the trend of pH change over time for Pseudomonas molluscum PMZ1 in liquid LB medium with a pH range of 4 to 10.

[0020] Figure 4 The cadmium content in the rice roots and aboveground parts, and the cadmium and arsenic content in the iron film on the root surface, in Example 6 of the present invention, are as follows: Figure 4 A represents the cadmium content in rice roots. Figure 4 B represents the cadmium content in the aboveground parts of rice. Figure 4 C represents the metal content of the root surface iron film.

[0021] Figure 5 The arsenic content in the rice roots and aboveground parts in Example 7 of the present invention is shown below. Figure 5 A represents the arsenic content in rice roots. Figure 5 B represents the arsenic content in the aboveground parts of rice.

[0022] Figure 6 The content of cadmium, total arsenic, and inorganic arsenic in rice grains in Example 8 of the present invention, wherein, Figure 6 A represents the cadmium content of rice grains. Figure 6 B represents the total arsenic and inorganic arsenic content of rice grains.

[0023] Figure 7 This refers to the rice yield in Example 9 of the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1

[0026] Isolation and purification of strain PMZ1

[0027] This strain was isolated and purified from the rhizosphere soil sample of rice grown in cadmium-contaminated farmland in Changsha County, Hunan Province. Details are as follows:

[0028] Add 1g of cadmium-contaminated rice rhizosphere soil sample to 10mL of sterile physiological saline, shake for 15 minutes to obtain a microbial suspension, and then dilute 0.5mL of the microbial suspension to a final concentration of 10 mL. -1 Double, applied to a solution containing 300 mg L -1 LB agar medium containing cadmium. Colonies were selected and further cultured on medium containing 300 mg L... -1 Streaking cadmium onto LB agar medium and repeating this process until a single colony is obtained.

[0029] The bacterial strain isolated from LB agar was inoculated into LB liquid medium (LB liquid medium composition: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride, deionized water as solvent, pH 7.0±0.1), and cultured at 30 ℃ and 180 rpm for 2 days to obtain the bacterial suspension. 5 mL of the bacterial suspension was added to 1000 mL of medium containing 1 mg / L of [a specific solution / concentration]. -1 In hydroponic rice nutrient solution (Kimura B rice nutrient solution (QM4003, Beijing Coolplay Technology Co., Ltd.)), after culturing for 2 weeks, the effects of different strains on reducing cadmium and arsenic in hydroponic rice were compared. The strain with the best simultaneous reduction of cadmium and arsenic was screened and named PMZ1. The results showed that the cadmium and arsenic content in the aboveground parts of the control group rice was 29.78 mg kg. -1 and 10.59 mgkg -1 The PMZI strain simultaneously reduced cadmium by 82% and arsenic by 80% in the aboveground parts of rice.

[0030] Example 2

[0031] Identification of strain PMZ1

[0032] (1) Morphological identification

[0033] When strain PMZ1 was inoculated into LB solid medium, it grew rapidly, appearing milky white on LB plates with a smooth and moist surface. It grew rapidly within a pH range of 6-8 and a temperature range of 25-35°C. Figure 1 A).

[0034] (2) Physiological and biochemical identification

[0035] Chromium azuril (CAS) medium was prepared. The CAS detection medium was purchased from Beijing Coollab Technology Co., Ltd., catalog number PM0821-1L. 9% agar powder was added, and the mixture was thoroughly mixed before sterilization at 115 ℃ for 30 minutes and then poured into plates. After cooling and solidification, a single colony of strain PMZ1 was streaked onto a CAS plate and incubated at 30 ℃ for 48 h. After 48 h, a significant yellow halo was observed on the CAS plate, indicating that the strain has good siderophore production ability. Figure 1 B).

[0036] Monkina Organic Phosphorus Medium: 10 g glucose, 0.5 g ammonium sulfate, 0.3 g potassium chloride, 0.3 g sodium chloride, 0.03 g ferrous sulfate heptahydrate, 0.3 g magnesium sulfate heptahydrate, 0.03 g manganese sulfate tetrahydrate, 5 g calcium carbonate, 0.2 g lecithin, 15-18 g agar powder, 1 L distilled water, pH adjusted to 7-7.5. Sterilize at 115 ℃ for 30 minutes, pour into plates, and allow to cool naturally.

[0037] Single colonies of strain PMZ1 were streaked onto Monkina organophosphate medium and incubated at 30°C for 48 h. A distinct transparent halo was observed around the colonies. Figure 1 C) indicates that this strain has a strong phosphorus solubilizing ability.

[0038] (3) Molecular biological identification

[0039] Strains of strain PMZ1 were picked from liquid culture medium and transferred to centrifuge tubes. DNA was extracted using a bacterial genomic DNA extraction kit, and PCR amplification was performed using 16S universal primers: 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID No. 1) and 1492R (5'-GGCTACCTTGTTACGACTT-3', SEQ ID No. 2). The PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the data was assembled using SeqMan Pro software. The data was then uploaded to the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) for BLAST sequence alignment and homology analysis. A phylogenetic tree was constructed using MEGA 7.0 software using the neighbor-joining method. Figure 2 The results showed that strain PMZ1 was related to *Pseudomonas molluscum* (…). Pseudomonas mosselii The sequences of ) have 97% homology.

[0040] The 16S rDNA sequencing results are as follows:

[0041]

[0042] Example 3

[0043] Preservation of strain PMZ1

[0044] Pseudomonas molluscum PMZ1, its classification name is Pseudomonas mosselii It was deposited on September 1, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251942, and the deposit address is Wuhan University, Wuhan, China.

[0045] Example 4

[0046] Acid and alkali resistance test of Pseudomonas molluscum PMZ1

[0047] Liquid LB medium (5g yeast extract, 10g tryptone, 10g sodium chloride, 1L distilled water) was prepared and the pH was adjusted to 4 (4.10), 5 (5.09), 6 (6.07), 7 (6.88), 8 (8.01), 9 (9.04), and 10 (9.94) with HCl and NaOH, respectively. Single colonies of *Pseudomonas morganii* PMZ1 were inoculated into LB medium with different initial pH values, and samples were taken at 0 h, 12 h, 24 h, and 48 h of culture to determine the OD600 and pH.

[0048] The results showed that, except for the culture medium with an initial pH of 4.1, the strain could grow rapidly in all other culture media after 24 hours, with OD600 values ​​reaching above 2.5. Figure 3 A) indicates that *Pseudomonas molluscum* PMZ1 possesses extremely strong alkali resistance and relatively strong acid resistance. When cultured for 24 hours or more, *Pseudomonas molluscum* PMZ1 maintains a bacterial culture pH of approximately 8.5 to adapt to the environment. Figure 3 B), which helps to improve acidic and alkaline environments.

[0049] Example 5

[0050] The efficiency of Pseudomonas morganii PMZ1 in adsorbing cadmium from aqueous solution

[0051] Take 1 μL of Pseudomonas molluscum PMZ1 bacterial culture (CFU = 10). 8 (1 cell per ml) was added to 150 ml of LB medium containing different Cd concentrations, with the initial Cd concentration of the medium being 0 μg / L. -1 100 μg L -1 1000 μg L -1 5000 μg L -1 10000 μg L -150000 μg L -1 80000 μg L -1 The oscillation speed was 150 rpm, and the oscillation sampling times were 0 h, 4 h, 8 h, 12 h, 24 h, 32 h, 38 h, 48 h, 60 h, 72 h, 84 h, and 108 h.

[0052] The results are shown in Table 1.

[0053]

[0054] With increasing oscillation time, the Cd content at different initial concentrations decreased significantly, especially when the initial Cd concentration was below 10000 μg / L. -1 When the shaking time was 72 h, the Cd concentration decreased to its lowest level, and the Cd removal rate reached 70-97.7%. When the shaking time was longer than 72 h, the Cd concentration decreased to its lowest level, except when the initial Cd concentration was greater than 50,000 μg / L. -1 In addition, the Cd concentration increased slightly in other treatments, indicating that *Pseudomonas molluscum* PMZ1 may have a small amount of Cd desorption potential in the later stages, and the adsorption time should be controlled within 72 h. Furthermore, when the initial Cd concentration is greater than 50,000 μg / L... -1 As the adsorption time increases, the cadmium concentration in the solution gradually decreases.

[0055] Example 6

[0056] Pseudomonas morganii PMZ1 reduces the efficiency of cadmium production in hydroponic rice.

[0057] Germinated rice seeds were transplanted into 1000 mL 96-well black hydroponic containers and pre-cultured for two weeks. Kimura B rice nutrient solution was used. After two weeks, five treatments were set up, as follows:

[0058] ①CK (no heavy metals added, no bacterial inoculation);

[0059] ② Add an initial concentration of 0.1 mg / L -1 Cadmium;

[0060] ③ Add an initial concentration of 0.1 mg / L -1 Cadmium was inoculated with Pseudomonas morganii PMZ1.

[0061] ④ Add an initial concentration of 1 mg / L -1 Cadmium;

[0062] ⑤ Add the initial concentration of 1 mg / L -1 Cadmium was inoculated with Pseudomonas morganii PMZ1.

[0063] The above inoculation with *Pseudomonas morganii* PMZ1 involved adding 5 mL of bacteria to the hydroponic container at a concentration of 8 × 10⁻⁶. 8 mL -1 Fluid of Pseudomonas molluscum PMZ1.

[0064] Rice samples were collected after two weeks of cultivation, and the metal content in the roots and aboveground parts was measured.

[0065]

[0066] The results showed that *Pseudomonas molluscum* PMZ1 significantly reduced cadmium content in rice roots and aboveground parts (p < 0.01), with the highest reduction of cadmium in aboveground parts reaching 82% (Table 2). Figure 4 A and Figure 4 B).

[0067] Meanwhile, *Pseudomonas molluscum* PMZ1 can also significantly increase the iron and manganese content in the root surface iron film. Figure 4 C). In both cadmium-containing and cadmium-free conditions, *Pseudomonas molluscum* PMZ1 inoculation significantly increased the amount of Fe and Mn in the root iron film. Compared to the control at the same cadmium level, the PMZ1 treatment increased root surface Fe content by up to 161% and root surface Mn content by up to 155%. The increase in the amount of root surface iron film is beneficial for controlling the entry of heavy metals cadmium and arsenic into the roots and their translocation to the aboveground parts.

[0068] Example 7

[0069] Pseudomonas morganii PMZ1 reduces the efficiency of arsenic reduction in hydroponic rice.

[0070] Germinated rice seeds were transplanted into 1000 mL 96-well black hydroponic containers and pre-cultured for two weeks. Kimura B rice nutrient solution was used. After two weeks, five treatments were set up, as follows:

[0071] ①CK (no heavy metals added, no bacterial inoculation);

[0072] ② Add an initial concentration of 0.1 mg / L -1 Arsenic;

[0073] ③ Add an initial concentration of 0.1 mg / L -1 Arsenic was inoculated with Pseudomonas möbius PMZ1.

[0074] ④ Add an initial concentration of 1 mg / L -1 Arsenic;

[0075] ⑤ Add the initial concentration of 1 mg / L -1 Arsenic was inoculated with Pseudomonas morganii PMZ1.

[0076] The above inoculation with *Pseudomonas morganii* PMZ1 involved adding 5 mL of bacteria to the hydroponic container at a concentration of 8 × 10⁻⁶.8 mL -1 Fluid of Pseudomonas molluscum PMZ1.

[0077] Rice samples were collected after two weeks of cultivation, and the metal content in the roots and aboveground parts was measured.

[0078]

[0079] The results showed that the arsenic content in both rice roots and aboveground parts was significantly reduced after inoculation (p < 0.05), especially in the aboveground parts where the arsenic content decreased by about 80% (Table 3). Figure 5 ).

[0080] Example 8

[0081] The efficiency of Pseudomonas morganii PMZ1 in reducing cadmium and arsenic in contaminated rice in soil.

[0082] Soil samples were collected from Heishan District, Yiyang City, Hunan Province, which was contaminated with cadmium and arsenic (total Cd 0.45 mg / kg). -1 Total As 30.6 mg / kg -1 The experiment involved planting control and potted rice plants inoculated with PMZ1 bacterial solution in the rhizosphere during the tillering stage. The specific procedure was as follows: Each bottomless plastic pot was filled with 3.5 kg of dry soil and 2.5 g of compound fertilizer. After thorough mixing, the soil was submerged to a depth of 2 cm for two weeks to achieve equilibration. One rice seedling was transplanted into each pot. Twenty days after transplanting, the control group (CK) received no treatment, while the experimental group (PMZ1) received rhizosphere inoculation with *Pseudomonas molybditidis* PMZ1, with each seedling inoculated with 50 mL of the bacterial solution at a concentration of 8 × 10⁻⁶. 8 mL -1 After the rice matures, rice plant samples are collected, cleaned, and dried. Cadmium and arsenic in the rice are determined according to the methods in GB / T 5009.15-2014 and GB / T 5009.11-2014, respectively.

[0083]

[0084] The results showed that inoculation with *Pseudomonas morganii* PMZ1 effectively reduced the total cadmium and arsenic content in rice, with the cadmium content decreasing from 0.27 mg / kg. -1 Reduced to 0.16 mg / kg -1 ( p <0.01), a reduction of approximately 41%, which is lower than the national safety standard (National Standard GB2762-2022, 0.2 mg / kg cadmium). -1 The total arsenic content in rice ranged from 1.23 mg / kg. -1 Reduced to 0.67 mg / kg -1 ( p <0.01), a decrease of approximately 46%; the inorganic arsenic content in rice decreased from 0.43 mg / kg.-1 Reduced to 0.18 mg / kg -1 ( p <0.01), below the national safety standard (National Standard GB 2762-2022, Inorganic Arsenic 0.35 mg kg). -1 (Table 4, Figure 6 ).

[0085] Example 9

[0086] Pseudomonas molluscum PMZ1 increases rice yield in contaminated soil

[0087] The yield of rice per pot was measured in Implementation Case 6.

[0088]

[0089] The results showed that after inoculation with *Pseudomonas morganii* PMZ1, rice yield increased from 4.17 g per pot to 6.27 g per pot, an increase of nearly 50%. This indicates that this strain has significant potential to improve rice yield (Table 5). Figure 7 ).

[0090] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0091] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of *Pseudomonas molluscum*, characterized in that, The *Pseudomonas molluscum* strain was named PMZ1, and its classification was named ( Pseudomonas mosselii It was deposited on September 1, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251942, and the deposit address is Wuhan University, Wuhan, China.

2. The application of Pseudomonas molluscum as described in claim 1 in reducing cadmium and arsenic content.

3. The application of *Pseudomonas molluscum* as described in claim 2 in reducing cadmium and arsenic content, characterized in that... The reduction of cadmium and arsenic content refers to reducing the cadmium and arsenic content in rice roots and rice grains.

4. The application of Pseudomonas molluscum as described in claim 1 in increasing rice yield.

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