Prussella sp. Capable of reducing antibiotic resistance genes and promoting growth and application of Prussella sp.

Abstract

The invention discloses a Plusteria monocytogenes strain capable of reducing antibiotic resistance genes and promoting growth and application of the Plusteria monocytogenes strain. The strain is separated from rice soil, and is identified as Plusteria based on physiological and biochemical characteristics and 16S rRNA (ribosomal Ribonucleic Acid) analysis. The Plusteria bacteria provided by the invention have a remarkable reduction effect on antibiotic resistance genes in the organic fertilizer; pot experiment results show that the strain can effectively reduce abundance of antibiotic resistance genes at the upper part and the root part of the lettuce and can significantly promote growth of the lettuce.

Description

Technical Field

[0001] This invention relates to the field of biocontrol technology, specifically to a strain of Priestella that combines the functions of reducing antibiotic resistance genes and promoting plant growth, and its applications. Background Technology

[0002] With the widespread use of antibiotics in medicine, agriculture, and animal husbandry, the accumulation and spread of antibiotic resistance genes (ARGs) in the environment has become a global public health problem. ARGs can spread in microbial communities through horizontal gene transfer (HGT), leading to the emergence of multidrug-resistant strains and seriously threatening human health and ecological security. ARGs are divided into intracellular ARGs and extracellular ARGs. Extracellular ARGs are produced through active bacterial secretion or bacterial death and lysis. Intracellular ARGs mainly spread horizontally through conjugation and transduction; extracellular ARGs spread horizontally through transformation. Reducing extracellular ARGs can achieve the goal of decreasing ARG abundance and effectively controlling the horizontal transfer of ARGs.

[0003] Organic fertilizers are a significant source of soil ARGs, containing abundant extracellular ARGs. Adding ARG-degrading strains is crucial for reducing the environmental risks posed by ARGs in organic fertilizers. On the other hand, in the field of sustainable agricultural development, some bacteria, due to their nitrogen-fixing, phosphorus-solubilizing, and plant hormone-secreting properties, possess the ability to promote plant growth. Although some Bacillus and Pseudomonas strains have been reported to possess both growth-promoting and organic pollutant-degrading functions, there are currently no reports of strains that simultaneously promote growth and reduce ARGs.

[0004] In summary, developing a microbial strain that combines highly efficient ARGs reduction with significant plant growth promotion can simultaneously address the needs of environmental pollution and agricultural production, while avoiding the limitations of single-function strains in practical applications. This has significant scientific and practical value. Based on this, this invention isolated and screened a functional bacterium from paddy soil that can effectively reduce ARGs and promote plant growth. This bacterium was added to well-rotted organic fertilizer and potted soil, and its effects on reducing ARGs in organic fertilizer, ARGs in the aboveground and root parts of lettuce, and increasing yield were measured, with the aim of creating a novel bio-organic fertilizer product for ARGs reduction. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Priestella that has both antibiotic resistance gene reduction and growth promotion functions, and its applications, in order to overcome the shortcomings of the prior art.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] In the first aspect, the present invention isolated and screened a strain of Priestella HY-3, classified and named Priestiasp., with accession number CCTCC NO: M 2025787.

[0008] Research has shown that HY-3 can efficiently degrade organic fertilizers and antibiotic resistance genes in plants, and significantly promote plant growth.

[0009] Secondly, this invention protects the use of the aforementioned Priestella HY-3 bacteria in reducing antibiotic resistance genes and / or promoting plant growth.

[0010] Thirdly, this invention protects the use of the aforementioned Priestella HY-3 bacteria in the preparation of microbial agents that reduce antibiotic resistance genes and / or promote plant growth.

[0011] Fourthly, this invention protects the use of the aforementioned Priestella HY-3 bacteria in the preparation of microbial organic fertilizers that reduce antibiotic resistance genes and / or promote plant growth.

[0012] Fifthly, the present invention protects a microbial agent, characterized in that the agent contains the aforementioned Priestella HY-3 bacteria or a culture containing the Priestella HY-3 bacteria as described in claim 1.

[0013] Sixthly, the present invention protects the use of the aforementioned microbial agents in reducing antibiotic resistance genes and / or promoting plant growth.

[0014] Seventhly, the present invention protects the use of the aforementioned microbial agents in the preparation of microbial organic fertilizers that reduce antibiotic resistance genes and / or promote plant growth.

[0015] Eighthly, the present invention protects a microbial organic fertilizer containing the Priestella bacteria HY-3 described above or the microbial agent described in claim 5.

[0016] Preferably, the content of the microbial organic fertilizer is not less than 1.0 × 10⁻⁶. 3 CFU / g.

[0017] Ninthly, the present invention protects the use of the microbial organic fertilizer described above in reducing antibiotic resistance genes and / or promoting plant growth.

[0018] In a tenth aspect, the present invention protects a method for reducing antibiotic resistance genes and / or promoting plant growth, said method being achieved by applying the aforementioned Priestella bacteria HY-3, the aforementioned microbial inoculant, or the aforementioned microbial organic fertilizer to the target plant.

[0019] In the specific implementation plan, the plant is lettuce.

[0020] In the specific implementation plan, reducing antibiotic resistance genes refers to reducing the content of antibiotic resistance genes in organic fertilizers and the above-ground parts and roots of plants.

[0021] The beneficial effects of this invention are:

[0022] This invention targets the effective reduction of the abundant antibiotic resistance gene content in organic fertilizers and the curbing of antibiotic resistance gene transfer caused by fertilization. A strain of Priestella HY-3 was screened that can significantly reduce the abundance of antibiotic resistance genes in organic fertilizers and lettuce, and has significant growth-promoting function. This provides technical support for reducing the content of antibiotic resistance genes in organic fertilizers and lettuce and increasing crop biomass, and has broad application prospects.

[0023] Biological Preservation Information:

[0024] A bacterium of the genus *Priestia*, HY-3, classified as *Priestia sp.*, was deposited on April 15, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025787. Attached Figure Description

[0025] Figure 1 The effect of strain HY-3 on the ability to degrade extracellular DNA.

[0026] Figure 2 A phylogenetic tree was constructed based on the 16S rRNA gene sequence of strain HY-3.

[0027] Figure 3 The effect of inoculating strain HY-3 on the content of antibiotic resistance genes in organic fertilizer.

[0028] Figure 4 To investigate the effect of inoculating strain HY-3 on the content of antibiotic resistance genes in lettuce. Detailed Implementation

[0029] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0030] Example 1: Isolation and Identification of Functional Strains

[0031] (1) Sample collection:

[0032] The soil was collected and separated from the topsoil of rice paddies in Hengyang City, Hunan Province.

[0033] (2) Test culture medium:

[0034] YN medium (g / L): 10 g sucrose, 2 g dipotassium hydrogen phosphate, 0.1 g peptone, 1 g ammonium sulfate, 0.2 g sodium chloride, 0.6 g yeast extract, 0.5 g calcium chloride, 0.5 g magnesium sulfate heptahydrate, pH 7.0. For solid medium, add 1.6-1.8 g agar powder.

[0035] (3) Preparation of extracellular DNA:

[0036] E. coli carrying the plasmid containing the ampicillin resistance gene (Amp) was transferred into LB medium containing kanamycin (50 μg / mL) and cultured with shaking at 37°C for 24 h. The bacterial culture was then centrifuged at 10,000 rpm, and the plasmid was extracted using a plasmid extraction kit. The extracted plasmid is the extracellular DNA containing the antibiotic resistance gene. The LB medium formula (1 L) is as follows: 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride.

[0037] (4) Isolation and screening of strains:

[0038] Take 1 g of soil and place it into a 15 mL centrifuge tube containing 10 mL of sterile water. Shake at 30°C and 200 rpm for 24 h to completely disperse the soil. After standing for 5 min, take 100 μL of the soil supernatant in a clean bench and place it into a 2 mL sterile centrifuge tube. Add 900 μL of sterile water and mix thoroughly to obtain 10 g of soil. -1 The diluent, obtained using the same method, yielded 10. -2 10 -3 10 -4 10 -5 10 -6 10 -7 A series of diluents are provided for plate coating. Use a dilution of 10... -4 10 -5 10 -6 10 -7 100 μL of soil dilution was spread onto YN medium plates, which were then inverted and incubated at 30°C for 24 h. Colony morphology, color, moisture content, and gloss were observed. Different colonies were picked and placed in LB liquid test tubes, which were then placed in a shaker for incubation. The bacterial solution was then streaked onto a plate for purification to obtain single colonies.

[0039] (5) Screening of strains that degrade extracellular antibiotic resistance genes:

[0040] The monoclonal strain obtained in step (4) was transferred to YN medium and cultured in a shaker at 30°C for 24 h. The bacterial culture was centrifuged at 10000 rpm, and 9 μL of the supernatant was collected and mixed with 1 μL of 200 ng / μL extracellular DNA. The mixture was then incubated at 30°C for 2 h. After the culture was complete, 2 μL of loading buffer was added to the above system, mixed well, and 5 μL of the solution was subjected to agarose gel electrophoresis. The disappearance of the DNA band was observed; strains with no DNA band were those capable of degrading extracellular DNA. The strain screened in this invention was named HY-3, and the results of its extracellular DNA degradation ability are as follows: Figure 1 As shown.

[0041] (6) Classification and identification of strain HY-3

[0042] After incubation at 30°C for 24 hours on YN solid medium, strain HY-3 produced round, disc-shaped colonies of a pale yellowish-white color. The colonies had smooth, regular edges, a moist surface, and were easily picked from the medium. Gram staining, glucose fermentation, and gelatin liquefaction were positive. Indole reaction, starch hydrolysis, phenylalanine dehydrogenase test, nitrate reduction test, citrate utilization, oxidase, H2S production, and lactose fermentation were negative. The phylogenetic tree analysis based on the 16S sequence showed that strain HY-3 had the highest homology with *Priestia aryabhattai* B8W22 and was less homologous to *Bacillus sp.*. Based on the phylogenetic tree analysis and physiological and biochemical characteristics, strain HY-3 was identified as a member of the genus *Priestia*. Figure 2 The phylogenetic tree results showed that this fungus is harmless to crops and non-pathogenic to humans and animals. Strain HY-3 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2025787.

[0043] Example 2: Effect of Priestia sp. HY-3 on the abundance of antibiotic resistance genes in well-rotted organic fertilizer

[0044] Weigh 20 g of well-rotted chicken manure organic fertilizer into a 50 mL centrifuge tube, and centrifuge at 1×10⁻⁶ ppm. 7 Add CFU / g of inoculum to strain HY-3, mix well, and incubate statically at 28℃ for 28 days. On day 0 and day 28, 0.5 g of organic fertilizer samples were collected to extract DNA. The abundance changes of antibiotic resistance genes in the organic fertilizer were analyzed using quantitative real-time PCR. The PCR reaction program was: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 10 s, annealing extension at 60℃ for 30 s, 95℃ for 15 s, for 40 cycles. The primer sequences used are shown in Table 1 below.

[0045] Table 1 Primers used for antibiotic resistance gene detection

[0046]

[0047] The results are as follows Figure 3 As shown, the total absolute abundance of antibiotic resistance genes in organic fertilizer without the addition of strain HY-3 did not change significantly after 28 days of cultivation. However, the total absolute abundance of antibiotic resistance genes in organic fertilizer treated with strain HY-3 decreased significantly by about 55% after 28 days of cultivation, indicating that strain HY-3 has a good degradation effect on antibiotic resistance genes in organic fertilizer.

[0048] Example 3: Investigating the effects of strain HY-3 combined with organic fertilizer on lettuce biomass and the content of antibiotic resistance genes in lettuce using a pot experiment.

[0049] The pot experiment was conducted in the greenhouse of the College of Resources and Environment, Nanjing Agricultural University, Nanjing City, Jiangsu Province. Soil for the pot experiment was taken from vegetable soil in Jiangning District, Nanjing City. After natural air drying, it was sieved through an 8 mm sieve, and 3 kg of soil sample was placed in each pot (specifications: 20 cm in diameter and 14 cm in height). The experiment was divided into three treatment groups: Treatment Group 1: pure soil treatment; Treatment Group 2: soil treated with 4% (by weight) chicken manure organic fertilizer; Treatment Group 3: soil treated with 4% (by weight) chicken manure organic fertilizer, with a final concentration of 5 × 10⁻⁶. 8 The bacterial suspension was administered at CFU / g; each group had three replicates. The vegetable variety used in the experiment was Italian lettuce (Lactuca sativa L. var. ramosa Hort.). One week after transplanting, treatment group 3 was inoculated with HY-3 bacterial suspension, which was applied directly to the rhizosphere of the lettuce. After 50 days of regrowth following the inoculation, the biomass of the lettuce was measured.

[0050] The biomass of each treatment group is shown in Table 2. In terms of aboveground and root fresh weight, the lettuce biomass of the organic fertilizer treatment group was superior to that of the soil group. Furthermore, the treatment group receiving both organic fertilizer and HY-3 showed a yield increase of approximately 58.8% compared to the group receiving only organic fertilizer. In conclusion, adding HY-3 bacterial suspension during organic fertilizer application has a good growth-promoting effect on lettuce plants.

[0051] Table 2 Biomass of lettuce in growth promotion experiment

[0052]

[0053] Note: Data in the table represents mean ± standard deviation.

[0054] Endophytic bacteria were extracted from lettuce leaves and roots from treatment groups 1, 2, and 3. The extraction process was as follows: The second-to-last and third-to-last leaves were used for extracting microbial DNA from the lettuce leaves, and all lettuce roots were used for extracting microbial DNA from the lettuce roots. First, the lettuce leaves and roots were rinsed with sterile water for 2 min, then soaked in 70% ethanol for 1 min, then soaked in 2% sodium hypochlorite for 3 min, then soaked again in 70% ethanol for 1 min, and finally rinsed three times with sterile water. The lettuce leaves and roots were ground into a fine powder using liquid nitrogen. 0.5 g of the plant sample was weighed and endophytic DNA was extracted using a soil DNA extraction kit (Qiagen). Finally, quantitative real-time PCR was used to analyze the abundance changes of antibiotic resistance genes in the lettuce endophytes. The PCR reaction program was: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 10 s, annealing extension at 60℃ for 30 s, 95℃ for 15 s, for 40 cycles. The primer sequences used are shown in Table 3 below:

[0055] Table 3 Primers used for antibiotic resistance gene detection

[0056]

[0057] Results of antibiotic resistance gene content determination in lettuce endophytes: Figure 4 As shown, regarding the content of antibiotic resistance genes in lettuce roots, the abundance of antibiotic resistance genes in the organic fertilizer treatment increased approximately 10-fold compared to the untreated group. However, the addition of organic fertilizer and HY-3 treatment restored the abundance of antibiotic resistance genes in roots to the level of the untreated group. Regarding the content of antibiotic resistance genes in lettuce leaves, the organic fertilizer treatment increased the abundance of antibiotic resistance genes approximately 27-fold compared to the untreated group. The addition of organic fertilizer and HY-3 treatment resulted in a slightly lower abundance of antibiotic resistance genes in leaves compared to the untreated group. These results indicate that the addition of HY-3 bacterial suspension can significantly mitigate the environmental risks posed by antibiotic resistance genes in organic fertilizers, which is of great significance for food security.

[0058] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A strain of Priestella HY-3, classified and named. Priestia sp., accession number CCTCC NO: M2025787.

2. The use of the Priestella HY-3 bacteria as described in claim 1 in reducing antibiotic resistance genes and / or promoting plant growth.

3. The use of the Priestella HY-3 bacteria as described in claim 1 in the preparation of microbial agents that reduce antibiotic resistance genes and / or promote plant growth.

4. The use of the Priestella HY-3 bacteria as described in claim 1 in the preparation of microbial organic fertilizers that reduce antibiotic resistance genes and / or promote plant growth.

5. A microbial inoculant, characterized in that, The bacterial agent contains Priester bacteria HY-3 as described in claim 1 or a culture containing Priester bacteria HY-3 as described in claim 1.

6. The use of the microbial agent according to claim 5 in reducing antibiotic resistance genes and / or promoting plant growth.

7. The use of the microbial agent according to claim 5 in the preparation of microbial organic fertilizers that reduce antibiotic resistance genes and / or promote plant growth.

8. A microbial organic fertilizer, characterized in that, The fertilizer contains the Priestella bacteria HY-3 as described in claim 1 or the microbial agent as described in claim 5; Preferably, the content of the microbial organic fertilizer is not less than 1.0 × 10⁻⁶. 3 CFU / g.

9. The use of the microbial organic fertilizer according to claim 8 in reducing antibiotic resistance genes and / or promoting plant growth.

10. A method for reducing antibiotic resistance genes and / or promoting plant growth, characterized in that, The method is achieved by applying the Priestella bacteria HY-3 as described in claim 1, the microbial agent as described in claim 5, or the microbial organic fertilizer as described in claim 8 to the target plant.

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