Serratia marcescens and application thereof

Abstract

The application discloses a strain of Serratia marcescans and application thereof, and belongs to the technical field of agricultural microorganisms and heavy metal pollution prevention and treatment. The Serratia marcescans isolated from the rhizosphere of rice is inoculated into the rhizosphere of rice for in-situ remediation of contaminated soil and rice planting, can effectively colonize the root of rice, and has high safety. The Serratia marcescans can adsorb and chelate manganese and iron ions in the soil, strengthen the formation of iron and manganese gel film on the root surface of rice, thereby blocking and controlling Cd elements from entering the root and the aboveground part of the plant; the Serratia marcescans can also increase the absorption of manganese and iron in the contaminated soil, promote the growth of rice and improve the yield of rice. The Serratia marcescans can make the Cd in rice in the medium and light contaminated soil reach the standard for safe production, and has a very good application prospect.

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 Serratia marcescens and its application. Background Technology

[0002] In recent years, heavy metal pollution in my country's soil has become a prominent issue, especially cadmium (Cd), with a soil sampling rate exceeding the standard reaching 7.0%, ranking first among all pollutants. Rice, due to the aerobic environment of paddy fields and the acid secretion from its roots, readily accumulates Cd, producing cadmium-contaminated rice, which seriously threatens the health of humans whose staple food is rice.

[0003] Currently, the main technology for controlling Cd content in rice is "VIP+n", which includes planting low-Cd rice varieties, managing farmland flooding, increasing soil pH, and other remediation techniques. Planting low-Cd rice varieties currently faces challenges in terms of agronomic traits and yield stability. Farmland flooding management presents difficulties for farmers. While increasing soil pH through the application of heavy metal passivating agents such as lime has unstable long-term effects and can easily lead to soil compaction, microbial remediation technology has gained attention due to its high efficiency, low cost, and lack of damage to the soil's structure and fertility.

[0004] Soil acidification is a serious problem in my country, with approximately 919 million mu (about 64.9 million hectares) of acidified arable land nationwide having a pH value less than 6.5. Soil acidification is widespread across the country, with a large distribution area, particularly prominent in the southern economic crop and grain-producing regions, as well as the Northeast, the "black soil granary." Soil acidification not only leads to deficiencies in nutrients such as potassium, calcium, and magnesium in farmland soil, exacerbating the toxicity of aluminum and manganese to plants, resulting in reduced yields or even crop failure, but also seriously affects the quality of agricultural products. Furthermore, soil acidification often exacerbates the leaching of heavy metal ions such as cadmium from the soil, increasing the risk of cadmium contamination in agricultural products. Measures to mitigate soil acidification mainly include rational fertilization, application of soil conditioners, and planting green manure. Among these, preparations rich in beneficial microorganisms can effectively regulate the soil microbial community structure and implement comprehensive measures for the improvement of the soil ecological environment.

[0005] However, there are currently very few strains that can simultaneously alleviate soil acidification and reduce cadmium accumulation in rice.

[0006] In summary, how to provide a microbial strain resource that can effectively alleviate soil acidification and reduce cadmium in paddy fields is a 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 Serratia marcescens and its application.

[0008] Serratia marcescens ZF11-1 can significantly reduce the Cd content in rice grains, ensuring safe rice production. It can also alleviate soil acidification, increase soil pH, and improve soil quality.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A strain of Serratia marcescens, named ZF11-1 and taxonomically named (Serratia marcescens), was deposited on June 23, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20251435, at Wuhan University, Wuhan, China.

[0011] The aforementioned application of Serratia marcescens in mitigating soil acidification and reducing cadmium levels.

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

[0013] The above-mentioned application of Serratia marcescens in chelating manganese and iron in soil.

[0014] The above-mentioned application of Serratia marcescens in the formation of iron film in rice roots.

[0015] The above-mentioned application of Serratia marcescens in increasing rice yield and rice plant biomass.

[0016] A microbial agent for mitigating soil acidification and reducing cadmium includes the aforementioned Serratia marcescens.

[0017] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows: The strain ZF11-1 of the present invention is a *Serratia marcescens* strain isolated from the rhizosphere of rice, and it exhibits higher safety when applied to in-situ removal of Cd pollution from rice. Compared with existing reports, this strain significantly improves the ability to reduce Cd in rice and also has strong growth-promoting potential. Applying this strain enables simultaneous production and remediation of rice in moderately to mildly Cd-contaminated soil, providing a safe, effective, and environmentally friendly bioremediation method. Attached Figure Description

[0018] 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.

[0019] Figure 1 This is a morphological diagram of strain ZF11-1 in Example 1 of the present invention, wherein, Figure 1 A represents the morphology of strain ZF11-1 in LB solid medium. Figure 1B represents the morphology of strain ZF11-1 in solid CAS medium. Figure 1 C represents the morphology of strain ZF11-1 in LB liquid medium.

[0020] Figure 2 This is the phylogenetic tree of strain ZF11-1 in Example 1 of the present invention.

[0021] Figure 3 This invention illustrates the changes in heavy metal content in hydroponic rice after inoculation with *Serratia marcescens* ZF11-1 in Example 4 of this invention. Figure 3 A represents the Cd concentration in the root system. Figure 3 B represents the Cd concentration in the aboveground parts of the plant. Figure 3 C represents the root Mn concentration. Figure 3 D represents the root Fe concentration.

[0022] Figure 4 The change in Mn / Fe content in the iron film of hydroponic rice after inoculation with Serratia marcescens ZF11-1 in Example 4 of the present invention.

[0023] Figure 5 This shows the changes in the growth of hydroponic rice after inoculation with Serratia marcescens ZF11-1 in Example 4 of the present invention.

[0024] Figure 6 The change in Cd content in rice after inoculating contaminated soil with Serratia marcescens ZF11-1 in Example 5 of the present invention was shown.

[0025] Figure 7 This invention illustrates the changes in rice yield and plant biomass after inoculating contaminated soil with *Serratia marcescens* ZF11-1 in Example 5 of this invention. Figure 7 A represents the rice yield result. Figure 7 B represents the plant biomass result.

[0026] Figure 8 The changes in pH of the culture medium before and after the growth of *Serratia marcescens* ZF11-1 in Example 6 of this invention, and the changes in soil pH after its addition to acidified soil, are described below. Figure 8 A represents the pH change in the culture medium before and after the growth of Serratia marcescens ZF11-1. Figure 8 B represents the change in soil pH after Serratia marcescens ZF11-1 was added to acidified soil. 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] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.

[0029] Example 1

[0030] Isolation, purification and identification of strain ZF11-1

[0031] Strain ZF11-1 was isolated and purified from rhizosphere soil samples of rice grown in Cd-contaminated farmland in Changsha County, Hunan Province. Details are as follows:

[0032] Add 1g of Cd-contaminated rice rhizosphere soil sample to 10mL of sterile physiological saline, shake on a shaker for 15 minutes to obtain a microbial suspension, and take 0.5mL of the microbial suspension to dilute to 10. -1 The concentration of the diluted microbial suspension was increased by 10 times. 9% agar powder was added to chromium azuril (CAS) medium, sterilized at 115 °C for 30 minutes, poured into plates, and allowed to cool and solidify. The resulting suspension was then transferred to solid CAS medium and incubated at 30 °C for 120 h. Colonies with a yellow halo were selected and streaked multiple times to isolate pure cultures. After incubating all isolated single bacteria for 2 days, the bacterial cells on the plates were collected and stored in 30% sterile glycerol at -80 °C.

[0033] The bacterial strains isolated from solid culture medium were 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 containing 1 mg Cd (Kimura B rice nutrient solution (QM4003, Beijing Coolplay Technology Co., Ltd.)), and cultured for 2 weeks. The effects of different strains on reducing Cd in hydroponic rice were compared, and the strain with the best Cd-reducing effect was selected and named ZF11-1, which reduced the Cd content in the aboveground parts of hydroponic rice by 41.36%.

[0034] In CAS medium, a yellow halo is visible around the colony, indicating that strain ZF11-1 can secrete siderophores (…). Figure 1 B). In LB liquid medium, the bacterial culture initially appears milky white or pale yellow, gradually turning rose-red with increasing culture time. Figure 1 C).

[0035] When strain ZF11-1 was inoculated into LB solid medium, it grew rapidly, appearing as a rose-red color on LB solid plates with a smooth and moist surface. Figure 1 A).

[0036] Strains ZF11-1 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 sequencing results are shown below. The data was then assembled using SeqMan Pro software and 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 ZF11-1 had 97% sequence homology with Serratia marcescens.

[0037] The 16S rDNA sequencing results are as follows:

[0038]

[0039] Example 2

[0040] Preservation of strain ZF11-1

[0041] Serratia marcescens ZF11-1 was deposited on June 23, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251435, at Wuhan University, Wuhan, China.

[0042] Example 3

[0043] Adsorption experiment of Serratia marcescens ZF11-1

[0044] Take 1 μL of Serratia marcescens ZF11-1 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 1000 μg 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.

[0045] The results are shown in the table below.

[0046] Table 1 Adsorption experimental data (unit: μg L) -1 )

[0047]

[0048] 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 60 h, the Cd concentration decreased to its lowest level, and the Cd removal rate reached 97.43~99.86%. When the shaking time was greater than 60 h, the Cd concentration decreased to its lowest level, except for an initial Cd concentration of 50000 μg / L. -1 In addition, the Cd concentration increased slightly in the other treatments, indicating that Serratia marcescens ZF11-1 may have a small amount of desorption of Cd in the later stage, and the adsorption time should be controlled within 60 h.

[0049] Example 4

[0050] Serratia marcescens ZF11-1 reduces the efficiency of Cd reduction in hydroponic rice.

[0051] 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, two treatments were set up, as detailed below:

[0052] ①CK group: 1 mg / L of Cd was added, but no bacterial culture was inoculated;

[0053] ②ZF11-1 group: Add an initial concentration of 1 mg / L of Cd and inoculate with Serratia marcescens ZF11-1.

[0054] The above inoculation with Serratia marcescens ZF11-1 involved adding 5 mL of bacteria at a concentration of 1×10⁻⁶ to the hydroponic container. 8 Serratia marcescens ZF11-1 bacterial suspension per mL.

[0055] Rice samples were collected after two weeks of cultivation. The heavy metal content and dry weight of the roots and aboveground parts were measured, and the growth of the rice was observed. The results are shown in Table 2. Figures 3-5 As shown.

[0056] Table 2. Effects of Serratia marcescens ZF11-1 treatment on rice seedling growth.

[0057]

[0058] The results showed that *Serratia marcescens* ZF11-1 significantly reduced Cd content in rice roots and aboveground parts (p < 0.01). Figure 3 A, Figure 3 B). It promoted the increase of manganese and iron content in rice roots, especially under Cd stress, significantly promoting manganese absorption while reducing Cd absorption. Figure 3 C Figure 3 D). It enhanced the formation of the iron film on the root surface, increasing the iron film's retention of Cd in the solution. Figure 4 Meanwhile, *Serratia marcescens* ZF11-1 helped alleviate the stress of Cd on rice seedling growth and significantly promoted the growth of rice under Cd stress. Figure 5 This increased the dry weight and aboveground length of rice (Table 2).

[0059] In summary, *Serratia marcescens* ZF11-1 possesses siderophore secretion capabilities, enabling it to chelate manganese and iron in the soil. It forms a rich iron film on the root surface of rice in contaminated soil, increasing the adsorption of heavy metal Cd and preventing its entry into the root system and translocation to the aboveground parts. Furthermore, this invention utilizes *Serratia marcescens* ZF11-1 to chelate manganese and iron in contaminated soil, promoting the absorption of manganese and iron by rice, thereby antagonizing Cd and reducing Cd absorption by rice.

[0060] Example 5

[0061] The efficiency of Serratia marcescens ZF11-1 in reducing Cd in rice from contaminated soil

[0062] Cd-contaminated soil samples were collected from an area in Yiyang City, Hunan Province (total Cd content: 0.46 mg / kg). -1 The effective Cd concentration is 0.21 mg / kg. -1 A rice pot experiment was conducted. Each bottomless plastic pot was filled with 1 kg of dry soil and 0.5 g of compound fertilizer, thoroughly mixed, and then submerged in water to a depth of 2 cm for two weeks. One rice seedling was transplanted into each pot. Twenty days after transplanting, the control group (CK) received no treatment, while the experimental group (ZF11-1) was inoculated with *Serratia marcescens* ZF11-1 in the rhizosphere, with each seedling inoculated with 50 mL of the bacterial solution at a concentration of 1×10⁻⁶. 8 Samples / mL. After the rice matures, collect rice plant samples, clean them, and dry them.

[0063] Cd in rice was determined according to the method in GB 5009.15-2014, and the results are as follows: Figure 6 As shown.

[0064] The results showed that inoculation with *Serratia marcescens* ZF11-1 effectively reduced the Cd content in rice, decreasing it from 0.068 mg / kg. -1 Reduced to 0.029 mg / kg -1 This represents a 57% reduction.

[0065] The dry weight of rice grains and the dry weight of stems and leaves were measured in each pot of rice, and the results are as follows: Figure 7 As shown.

[0066] The results showed that after inoculation with Serratia marcescens ZF11-1, rice yield increased from an average of 2.17 g per plant to 3.72 g per plant, an increase of nearly 71%. Figure 7 A). Simultaneously, inoculation with *Serratia marcescens* ZF11-1 also significantly increased the dry weight of rice plants (A). Figure 7 B). This indicates that the strain can effectively promote rice growth in polluted soil and has significant potential to increase rice yield.

[0067] Example 6

[0068] The ability of Serratia marcescens ZF11-1 to produce alkali and its efficiency in mitigating soil acidification

[0069] Add 1 mL (approximately 10) 8 Serratia marcescens ZF11-1 bacterial suspension was inoculated into 150 mL of LB medium and cultured for 48 hours. A blank control medium without inoculation was set up. The pH value of the solution was measured after 48 hours. The results showed that Serratia marcescens ZF11-1 had a strong alkali-producing ability, effectively increasing the pH value of LB medium by 1.74 units. Figure 8 A).

[0070] Add 1 mL (approximately 10) 8 Serratia marcescens ZF11-1 (1 cell) was inoculated into 150 mL of LB medium and cultured for 48 hours to prepare ZF11-1 bacterial suspension. The ZF11-1 suspension was then added to two soils with different acidification levels at a solid-liquid ratio of 1:2.5, stirred for 48 hours, and allowed to stand for 7 days. The initial pH of soil 1 was 6.63, and the initial pH of soil 2 was 5.97. Treatment with ZF11-1 bacterial suspension significantly increased the pH of both soils: soil 1 increased by 1.83 units to 8.46, and soil 2 increased by 2.42 units to 8.39. The pH of both soils changed from acidic to alkaline (…). Figure 8 B).

[0071] 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.

[0072] 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 Serratia marcescens, characterized in that, The described *Serratia marcescens* strain, named ZF11-1 and classified as *Serratia marcescens*, was deposited on June 23, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251435, located at Wuhan University, Wuhan, China.

2. The application of Serratia marcescens as described in claim 1 in alleviating soil acidification and reducing cadmium.

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

4. The application of Serratia marcescens as described in claim 1 in chelating manganese and iron in soil.

5. The application of Serratia marcescens as described in claim 1 in the formation of iron film in rice roots.

6. The application of Serratia marcescens as described in claim 1 in increasing rice yield and rice plant biomass.

7. A microbial agent for mitigating soil acidification and reducing cadmium, characterized in that, Includes *Serratia marcescens* as described in claim 1.

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