A strain of Stenotrophomonas maltophilia and its application

Abstract

This invention discloses a strain of Stenotrophomonas maltophilia and its applications. It belongs to the technical field of agricultural microbiology and heavy metal pollution control. The strain SM28-3 of this invention is a strain of Stenotrophomonas maltophilia (… Stenotrophomonas maltophilia 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 some growth-promoting potential. Applying this strain allows for simultaneous rice production and remediation in soils with moderate to mild cadmium and arsenic co-contamination.

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 Stenotrophomonas maltophilia and its applications. 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, leading to rice contamination.

[0003] 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.

[0004] Currently, technologies for controlling cadmium and arsenic pollution in rice mainly include agronomic measures (paddy field water management and application of heavy metal passivating agents, etc.), soil pH adjustment, phytoremediation, and microbial remediation. 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 technology is receiving increasing attention due to its high efficiency, low cost, and lack of damage to the soil's structure and fertility.

[0005] However, there are few reports on strains that can simultaneously reduce the accumulation of cadmium and arsenic in rice.

[0006] Therefore, how to provide a highly efficient microbial strain for simultaneous cadmium and arsenic reduction is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a strain of Stenotrophomonas maltophilia 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 of rice grains, thus achieving safe rice production.

[0008] This invention utilizes Stenotrophomonas maltophilia, a siderotrophic carrier isolated from the rice rhizosphere, to effectively colonize rice roots. It chelates manganese and iron ions in the soil, enhancing the formation of an iron-manganese colloid film on the rice root surface. This effectively blocks harmful elements such as cadmium and arsenic from entering the root system and transporting to the aboveground parts, simultaneously reducing the cadmium and arsenic content in rice. This reduces the cadmium and arsenic content 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-1 This method, which achieves simultaneous production of cadmium and arsenic in rice in paddy fields with pH ≤ 6.5, has great application potential.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] A strain of Stenotrophomonas maltophilia, named SM28-3, is classified as ( Stenotrophomonas maltophilia It was deposited at the China Center for Type Culture Collection on June 23, 2025, with accession number CCTCC NO: M 20251437, and the deposit address is Wuhan University, Wuhan, China.

[0011] The above-mentioned Stenotrophomonas maltophilia was used to reduce cadmium and arsenic content.

[0012] Furthermore, the reduction of cadmium and arsenic content refers to reducing the cadmium and arsenic content in rice roots and rice grains.

[0013] The above-mentioned application of Stenotrophomonas maltophilia in increasing rice yield.

[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 SM28-3 of this invention is a maltophilic stenotrophomonad ( Stenotrophomonas maltophilia 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 image shows the morphology of strain SM28-3 in LB medium in Example 1 of this invention.

[0018] Figure 2 This is the phylogenetic tree of strain SM28-3 in Example 1 of the present invention.

[0019] Figure 3This is the morphology of Stenotrophomonas maltophilia SM28-3 in CAS medium in Example 3 of the present invention.

[0020] Figure 4 The morphology of Stenotrophomonas maltophilia SM28-3 in Monkina organophosphate medium in Example 3 of the present invention.

[0021] Figure 5 This is the morphology of Stenotrophomonas maltophilia SM28-3 in potassium medium in Example 3 of the present invention.

[0022] Figure 6 This is a graph showing the acid-base tolerance test results of Stenotrophomonas maltophilia SM28-3 in Example 4 of the present invention. Figure 6 A shows the trend of OD600 values ​​of Stenotrophomonas maltophilia SM28-3 over time in liquid LB medium with pH values ​​ranging from 4 to 10. Figure 6 B is a graph showing the trend of pH change of Stenotrophomonas maltophilia SM28-3 in liquid LB medium with pH values ​​of 4-10 over time.

[0023] Figure 7 The results of the changes in cadmium, iron, and manganese content in hydroponically grown rice after treatment with Stenotrophomonas maltophilia SM28-3 in Example 6 of this invention are shown. Figure 7 A represents the cadmium content in the root system. Figure 7 B represents the cadmium content in the aboveground parts. Figure 7 C represents the manganese content in the aboveground parts. Figure 7 D represents the iron content in the aboveground parts.

[0024] Figure 8 The figure shows the change in arsenic content in hydroponic rice after treatment with Stenotrophomonas maltophilia SM28-3 in Example 7 of this invention. Figure 8 A represents the arsenic content in the roots. Figure 8 B represents the arsenic content in the aboveground parts.

[0025] Figure 9 The changes in cadmium and arsenic content in rice rhizosphere after inoculation of Stenotrophomonas maltophilia SM28-3 into rice in mixed-contaminated soil in Example 8 of this invention.

[0026] Figure 10 The change in rice yield after inoculating cadmium- and arsenic-contaminated soil with Stenotrophomonas maltophilia SM28-3 in the rhizosphere of soil in Example 9 of this invention. Detailed Implementation

[0027] 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.

[0028] Example 1

[0029] Isolation, purification and identification of strain SM28-3

[0030] 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:

[0031] A 1g sample of rhizosphere soil from rice grown in cadmium-contaminated farmland was added to 10 mL of sterile physiological saline. The mixture was shaken for 15 minutes to obtain a microbial suspension. 0.5 mL of the suspension was then diluted to a final concentration of 10 mL. -1 Add 9% agar powder to chroma azuril (CAS) medium, sterilize at 115 °C for 30 minutes, and pour into plates. After cooling and solidification, transfer the diluted microbial suspension onto solid CAS medium and incubate at 30 °C for 168 h. Select colonies with a yellow halo and streak them multiple times on LB agar plates to isolate pure bacteria. After incubating all isolated single bacteria for 2 days, collect the bacterial cells from the plates and store them in 30% sterile glycerol at -80 °C.

[0032] The bacterial strain isolated from solid culture medium was inoculated into LB liquid medium and cultured at 30 ℃ and 180 rpm for 2 days to obtain bacterial suspension. 5 mL of the bacterial suspension was added to 1000 mL of hydroponic rice nutrient solution (Kimura B rice nutrient solution (QM4003, Beijing Coollab Technology Co., Ltd.)) containing 0.1 mg cadmium and arsenic respectively, and cultured for 2 weeks. The effects of different strains in reducing cadmium and arsenic in hydroponic rice were compared, and the strain with the highest simultaneous reduction effect on cadmium and arsenic was selected and named SM28-3. Strain SM28-3 can simultaneously reduce arsenic by 78.7% and cadmium by 78.6% in the aboveground parts of rice, reducing the aboveground arsenic content from 10.59 mg / kg in the control. -1 Reduced to 2.25 mg / kg -1 The cadmium content was 29.77 mg / kg, compared to the control. -1 Decreased to 6.66 mg kg -1 .

[0033] Strain SM28-3 appears as a smooth, milky-white substance in LB medium, with a smooth and moist surface. Figure 1 ).

[0034] Strains of strain SM28-3 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 and then uploaded to the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). BLAST sequence alignment and homology analysis were performed, and a phylogenetic tree was constructed using MEGA 7.0 software using the neighbor-joining method. Figure 2 The results showed that strain SM28-3 was related to Stenotrophomonas maltophilia (…). Stenotrophomonas maltophilia The sequences of ) have 97% homology.

[0035] The 16S rDNA sequencing results are as follows:

[0036]

[0037] Example 2

[0038] Preservation of strain SM28-3

[0039] Stenotrophomonas maltophilia SM28-3, its taxonomic name is Stenotrophomonas maltophilia It was deposited on June 23, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251437, and the deposit address is Wuhan University, Wuhan, China.

[0040] Example 3

[0041] Identification of siderophore production and phosphorus and potassium solubilization capabilities of Stenotrophomonas maltophilia SM28-3

[0042] 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 Stenotrophomonas maltophilia SM28-3 was streaked onto the CAS plate and incubated at 30 ℃ for 48 hours. After 48 hours, a significant yellow halo was observed on the CAS plate. Figure 3 This indicates that Stenotrophomonas maltophilia SM28-3 has a good siderophore production capacity.

[0043] Monkina Organophosphate 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 plates, and allow to cool naturally. Streak a single colony of Stenotrophomonas maltophilia SM28-3 onto Monkina Organophosphate Medium and incubate at 30 ℃ for 2-3 days. The colony will show good growth with a distinct transparent halo around it. Figure 4 This indicates that Stenotrophomonas maltophilia SM28-3 has a strong phosphate-solubilizing ability.

[0044] Potassium broth: 5 g sucrose, 2 g disodium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.005 g ferric chloride, 0.1 g calcium carbonate, 1 g potassium feldspar, 18 g agar, 1 L distilled water, pH adjusted to 7-7.5. A single colony of Stenotrophomonas maltophilia SM28-3 was streaked onto the potassium broth and incubated at 30°C for 2-3 days. The colony showed good growth with a distinct milky-white halo around it. Figure 5This indicates that Stenotrophomonas maltophilia SM28-3 has a strong potassium-solubilizing ability.

[0045] Example 4

[0046] Acid and alkali resistance test of Stenotrophomonas maltophilia SM28-3

[0047] Liquid LB medium (5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 1 L 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 Stenotrophomonas maltophilia SM28-3 were inoculated into liquid LB medium with different initial pH values. Samples were taken at 0 h, 12 h, 24 h, and 48 h of culture, and the OD600 and pH values ​​were measured.

[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 6 A) indicates that Stenotrophomonas maltophilia SM28-3 possesses extremely strong alkali resistance and relatively strong acid resistance. When cultured for 24 hours or more, Stenotrophomonas maltophilia SM28-3 maintains a bacterial culture pH of approximately 8.5 to adapt to the environment. Figure 6 B), which helps to improve acidic and alkaline environments.

[0049] Example 5

[0050] Adsorption experiment of Stenotrophomonas maltophilia SM28-3

[0051] Take 1 μL of Stenotrophomonas maltophilia SM28-3 bacterial suspension (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 1000μg L -1 5000 μg L -1 10000 μg L -1 50000 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] Stenotrophomonas maltophilia SM28-3 was cultured in LB medium containing cadmium for 60 hours, except for an initial cadmium concentration of 50,000 μg / L. -1 Except for the culture medium containing cadmium, the cadmium content in other culture media was reduced to a minimum, with cadmium removal rates ranging from 32.4% to 99.9%. This was achieved when the initial cadmium concentration in the solution was less than or equal to 10,000 μg / L. -1 At the same time, the cadmium removal rate was higher than 97%, indicating that Stenotrophomonas maltophilia SM28-3 can effectively adsorb and remove cadmium from contaminated solutions.

[0055] Example 6

[0056] Stenotrophomonas maltophilia SM28-3 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, four treatments were set up, as follows:

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

[0059] ② Inoculate with Stenotrophomonas maltophilia SM28-3;

[0060] ③ Add an initial concentration of 1 mg / L -1 Cadmium;

[0061] ④ Add an initial concentration of 1 mg / L -1 Cadmium was removed by inoculation with Stenotrophomonas maltophilia SM28-3.

[0062] The above inoculation with Stenotrophomonas maltophilia SM28-3 involved adding 5 mL of bacterial culture to the hydroponic container at a concentration of 8 × 10⁻⁶. 8 mL -1 Stenotrophomonas maltophilia SM28-3 bacterial suspension.

[0063] Rice samples were collected after two weeks of cultivation, and the metal content and dry weight of the roots and aboveground parts were measured.

[0064]

[0065] The results showed that Stenotrophomonas maltophilia SM28-3 significantly reduced the cadmium content in rice roots and aboveground parts. p <0.01) (Table 2, Figure 7 A, Figure 7(B) The aboveground parts decreased by 57.4%. It also promoted the increase of iron and manganese content in rice, especially under cadmium stress, significantly promoting the absorption of iron and manganese in rice, while reducing the absorption of cadmium (Table 2, Figure 7 C Figure 7 D).

[0066]

[0067] Meanwhile, Stenotrophomonas maltophilia SM28-3 helped alleviate cadmium stress on rice seedling growth, significantly promoted rice growth under cadmium stress, and increased rice aboveground growth and dry weight (Table 3).

[0068] Example 7

[0069] Stenotrophomonas maltophilia SM28-3 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, four treatments were set up, as follows:

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

[0072] ② Inoculate with Stenotrophomonas maltophilia SM28-3;

[0073] ③ Add an initial concentration of 0.1 mg / L -1 Arsenic;

[0074] ④ Add an initial concentration of 0.1 mg / L -1 Arsenic was inoculated with Stenotrophomonas maltophilia SM28-3.

[0075] The above inoculation with Stenotrophomonas maltophilia SM28-3 involved adding 5 mL of bacterial culture to the hydroponic container at a concentration of 8 × 10⁻⁶. 8 mL -1 Stenotrophomonas maltophilia SM28-3 bacterial suspension.

[0076] Rice samples were collected after two weeks of cultivation, and the metal content in the roots and aboveground parts was measured.

[0077]

[0078] The results showed that the arsenic content in the aboveground parts of rice was significantly reduced after inoculation. p <0.05) (Table 4, Figure 8 (), a decrease of 78.75%.

[0079] Example 8

[0080] Stenotrophomonas maltophilia SM28-3 is effective at reducing cadmium and arsenic in rice from contaminated soil.

[0081] Soil samples were collected from Heishan District, Yiyang City, Hunan Province, which was contaminated with cadmium and arsenic (total cadmium content was 0.46 mg / kg). -1 The total arsenic content was 30.02 mg / kg. -1 A rice pot experiment was conducted. Each bottomless plastic pot was filled with 3.5 kg of dry soil and 2.5 g of compound fertilizer, thoroughly mixed, and then submerged in water to a depth of 2 cm for two weeks to allow equilibration. One rice seedling was transplanted into each pot. Twenty days after transplanting, the rhizosphere was inoculated with Stenotrophomonas maltophilia SM28-3, with 50 mL of bacterial solution per seedling at a concentration of 8 × 10⁻⁶. 8 mL -1 Bacterial solution. 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.

[0082]

[0083] The results showed that inoculation with Stenotrophomonas maltophilia SM28-3 effectively reduced the total cadmium and arsenic content in rice, with the cadmium content in rice decreasing from 0.27 mg / kg. -1 Reduced to 0.14 mg / kg -1 This represents a reduction of approximately 48%, which is below the national safety standard (GB2762-2022, cadmium 0.2 mg / kg). -1 The total arsenic content in rice ranged from 0.47 mg / kg. -1 Reduced to 0.25 mg / kg -1 This reduced it by approximately 47% (Table 5). Figure 9 ).

[0084] Example 9

[0085] Stenotrophomonas maltophilia SM28-3 increases rice yield in contaminated soil

[0086] In the experiment of Case 8, the rice yield per pot was measured. The results showed that after inoculation with Stenotrophomonas maltophilia SM28-3, the rice yield increased from 7.82 g per pot to 13 g per pot, an increase of nearly 66%. This indicates that Stenotrophomonas maltophilia SM28-3 has a significant potential to increase rice yield. Figure 10 ).

[0087] 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.

[0088] 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 Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ), characterized in that, The Stenotrophomonas maltophiliae was named SM28-3 and its accession number is CCTCC NO: M 20251437.

2. The application of Stenotrophomonas maltophilia as described in claim 1 in reducing cadmium content in rice roots under cadmium stress.

3. The application of Stenotrophomonas maltophilia as described in claim 1 in reducing the cadmium and arsenic content in rice from soils contaminated with both cadmium and arsenic.

4. The application of Stenotrophomonas maltophilia as described in claim 1 in increasing rice yield in soils contaminated with cadmium and arsenic.

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