Bacillus lysinoformis and application thereof in improving soil properties of gley soil

Abstract

The application belongs to the technical field of agricultural microbiology, and discloses a lysine bacillus and application of the lysine bacillus in improving the properties of gleyed paddy field soil, and particularly relates to application of lysine bacillus (Bacillus lysinoformis) in improving gleying of paddy soil and reducing total amount of reducing substances. Lysinibacillus sp. The application finds that the lysine bacillus can effectively increase the oxidation-reduction potential of gleyed paddy soil, reduce the total amount of soil reducing substances, the content of active reducing substances, and the content of ferrous ions and manganous ions, which provides selectivity for developing a new type of gleyed paddy soil improver.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology technology, specifically relating to a strain of lysinibacillus and its application in improving the properties of gleyed paddy soil. The lysinibacillus sp. L001 provided by this invention can increase the redox potential of gleyed paddy soil, reduce the total amount of reducing substances, reduce the content of active reducing substances, and reduce the content of ferrous and manganese. Background Technology

[0002] Gleyed soils are formed under conditions of prolonged flooding, leading to oxygen deficiency and reduced Fe content in the soil. 2+ Mn 2+ The accumulation of reducing substances and the decrease in redox potential lead to poor growth and development in rice. Soil gleying presents five major obstacles: closure, toxicity, infertility, rot, and coldness. Specifically, these can be manifested in the following four aspects: 1) Poor aeration. Due to waterlogging and high groundwater levels, poor drainage results in a redox potential Eh < 200mv, leading to a decrease in Fe in the soil. 2+ Mn 2+ Increased levels of reducing substances such as H2S have a toxic effect on rice plants and roots, resulting in slow growth, fewer tillers, and more black roots. 2) Cold soil conditions. Because the water and soil temperatures in gleyed paddy fields are 3–8℃ and 2–3℃ lower than in non-gleyed paddy fields, respectively, it may lead to stunted seedlings, delayed maturity, and low yields. 3) Low nutrient availability. Soil biological activity weakens, the availability of phosphorus and potassium in the soil is low, organic matter mineralization is inhibited, anaerobic conditions dominate, and reducing activity intensifies. 4) Poor structure. The topsoil layer is generally deep and heavy. Except in granite areas where the soil texture is coarser, most areas have dispersed soil particles and poor structure.

[0003] Currently, the main treatment measures for gley paddy soil are agricultural engineering methods, namely, ditching and drainage, lowering the groundwater level, and using sunlight to increase aeration, thereby reducing reducing substances in the soil. However, since gley paddy fields are mostly located in low-lying lake areas with high groundwater levels and difficult drainage, and the engineering costs are high, large-scale promotion is difficult. (2) Chemical method: This method involves adding chemical substances such as calcium peroxide to the soil to eliminate reducing substances. However, the chemical method has a short effective period, high cost, and poor economic feasibility.

[0004] Lysine Bacillus is a common environmental microorganism distributed in soil, rivers, and oceans. It has been reported that this bacterium can degrade pollutants such as cresol and sodium sulfide. Some strains can degrade heavy metals such as Cd and Cu. There are also reports that bacteria of this genus can control plant nematode diseases.

[0005] This invention has screened out Lysinibacillus sp. L001, which can effectively reduce the total amount of reducing substances, the content of active reducing substances, and the content of ferrous and manganese in the soil, and significantly increase the soil redox potential. While improving soil properties, it has no effect on rice growth and even improves the quality of rice seedlings, providing an effective technical measure for the improvement of low- and medium-yield fields in my country. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Lysinibacillus sp. L001, the preservation number of which is: CCTCC NO: M20221738.

[0007] Another object of the present invention is to provide the application of Lysinibacillus sp. L001 in improving the soil properties of gleyed paddy fields.

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] A strain of Lysinibacillus sp. L001 was obtained from gleyed rice soil in Yajiao Village, Guanyintang Town, Shashi District, Jingzhou City, Hubei Province using a dilution culture method. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on November 8, 2022, and is classified as Lysinibacillus sp. L001, with accession number CCTCC NO: M20221738, located at Wuhan University, Wuhan, China.

[0010] This bacterium is Gram-positive, rod-shaped, and produces spores. Its colonies are round, milky white, and smooth. It can grow at temperatures ranging from 10 to 37°C, with an optimal growth stability of 28°C and an optimal pH of 7.0. It can hydrolyze gelatin, is catalase-positive, and possesses peroxidase, alkaline phosphatase, β-galactosidase, and β-glucosidase activities.

[0011] The scope of protection of this invention also includes the application of Lysinibacillus sp. L001 in improving the properties of gleyed paddy soil, wherein improving the properties of gleyed paddy soil includes increasing the redox potential of gleyed paddy soil, reducing the total amount of reducing substances, reducing the content of active reducing substances, and reducing the content of ferrous and manganese.

[0012] Compared with the prior art, the present invention has the following advantages:

[0013] This invention is the first to discover that Lysinibacillus sp. L001 can increase the redox potential of gled paddy soil, reduce the total amount of reducing substances, reduce the content of active reducing substances, and reduce the content of ferrous and manganese, without affecting the growth of rice seedlings. Attached Figure Description

[0014] Figure 1 Graph of FeS oxidation capacity test of Bacillus lysine. Detailed Implementation

[0015] The technical solution of the present invention will be described in detail below with reference to preferred embodiments. These embodiments are for illustrative and explanatory purposes only and do not constitute a limitation on the technical solution of the present invention.

[0016] Example 1:

[0017] Screening of Lysine Bacillus L001 and determination of its oxidation effect on FeS

[0018] A strain of *Lysinibacillus* sp. L001 was obtained from gleyed rice soil in Yajiao Village, Guanyintang Town, Shashi District, Jingzhou City, Hubei Province, using a dilution culture method. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on November 8, 2022, and is classified as *Lysinibacillus* sp. L001, with accession number CCTCC NO: M 20221738, located at Wuhan University, Wuhan, China.

[0019] This bacterium is Gram-positive, rod-shaped, and produces spores. Colonies are round, milky white, and smooth. It can grow at temperatures ranging from 10-37°C, with optimal growth stability at 28°C and an optimal pH of 7.0. It can hydrolyze gelatin, is catalase-positive, and exhibits peroxidase, alkaline phosphatase, β-galactosidase, and β-glucosidase activities. A strain of *Lysinibacillus* sp. was ultimately obtained and deposited at the China Center for Type Culture Collection (CCTCC) on November 8, 2022. Its classification name is *Lysinibacillus* sp. L001, accession number is CCTCCNO: M 20221738, and its address is Wuhan University, Wuhan, China.

[0020] Preparation of Lysine Bacillus: The strain stored at -80℃ was activated on LB medium, and single colonies were picked and cultured on LB liquid medium for 24h to 48h for later use.

[0021] FeS oxidizing capacity test: Add 2.0g of ferrous sulfide powder to the bottom of a 10mL stoppered brown glass bottle, then add 10.0mL of sterile MWMW semi-solid culture medium (Aili Biotechnology (Shanghai) Co., Ltd.). After the culture medium solidifies, add 100μL (OD) 620 After adding 1.2% (Lysine-containing Bacillus) to MWMM medium, the glass bottles were placed in an incubator at 28°C and cultured in the dark for 5-14 days. A distinct brown Fe oxide layer appeared after 5 days of culture, and the color gradually deepened, reaching its deepest point at 14 days.

[0022] Example 2:

[0023] Determination of the effect of Bacillus lysine on gleying soil amendment

[0024] The experimental soil was taken from the gleyed paddy fields of Yajiao Village, Guanyintang Town, Shashi District, Jingzhou City. After being brought back to the laboratory, the soil was divided into flowerpots measuring 25cm (diameter) × 40cm (height), and a water layer of about 2cm was placed on the surface. The soil was allowed to stand for 3-5 days until the redox potential stabilized before use. 100, 50, and 20mL of 10% sodium chloride solution were then poured into the soil using 2cm diameter plastic tubes. 7 CFU / mL Bacillus lysine L001 was injected into rice soil in pots, with five injection points per pot, 10-15 cm below the surface soil. Ten pots were treated with each concentration, and each treatment was repeated three times. A water control was also included. After treatment, approximately 15 pre-germinated rice seeds (Quanyou 822) were sown in each pot and placed in a greenhouse for cultivation (28℃, 12h light, 12h dark). One week after rice sowing, 100, 50, and 20 mL of 10% CFU / mL Bacillus lysine L001 were injected into the soil. 7 CFU / mL Bacillus lysine was injected into pots using the same method. Soil redox potential (Eh) was measured using a TR-901 soil ORP meter (Shanghai Instrument & Electronics Scientific Instrument Co., Ltd.) one day before treatment and at 10, 20, and 30 days after treatment. Soil samples were collected from 10–15 cm below the surface for soil physicochemical property determination. According to the methods described in *Soil Agricultural Chemical Analysis Methods* (China Agricultural Science and Technology Press), edited by Lu Rukun, the total amount of soil reducing substances was determined using the aluminum sulfate extraction-potassium dichromate oxidation method; the determination of active reducing substances was performed using the 0.1 mol / L aluminum sulfate extraction-potassium periodate titration method; the soil reducing ferrous content was determined using the distilled water extraction-o-phenanthroline colorimetric method; and the reducing manganese content was determined using the distilled water extraction-potassium periodate colorimetric method. The obtained data were analyzed for variance using IBM SPSS 22.0. The calculation formula is as follows:

[0025] Increase value = Increase value of soil after treatment (1) - Increase value of oxygen in control (2)

[0026] (1) = Soil measurement value of treatment group - Measurement value of treatment group before treatment;

[0027] (2) = Soil measurement value of control group - measurement value of control group before treatment.

[0028] Using 1×10 7 Treatment with 100, 50, and 20 mL of CFU / ml Bacillus lysine significantly increased soil redox potential (Table 1). After 10 days of treatment, the soil redox potential in each treatment group was significantly higher than the control, reaching 30.33, 17.07, and -7.5 mV, respectively, representing increases of 70.2–28.38 mV compared to the control. After 20 days of treatment, the soil redox potential of the bacteria in each treatment group was significantly higher than the control, increasing by 81.34–34.1 mV. After 30 days of treatment, the soil redox potential of the bacteria in each treatment group was significantly higher than the control, increasing by 76.55–26.64 mV compared to the control.

[0029] Using 1×10 7 Treatment with 100, 50, and 20 mL of CFU / ml Bacillus lysine all reduced the total amount of reducing substances in the soil (Table 2). Ten days after treatment, the total amount of reducing substances in the soil for each treatment ranged from 5.74 to 10.74 cmol / kg, all showing a reduction compared to the control. The 100 mL treatment showed the largest reduction (6.68 cmol / kg), followed by the 50 and 20 mL treatments. Twenty days after treatment, the total amount of reducing substances in the soil for each treatment ranged from 4.02 to 8.15 cmol / kg, all showing a reduction compared to the control. The 100 mL treatment showed the largest reduction (7.38 cmol / kg), followed by the 50 and 20 mL treatments. Thirty days after treatment, the total amount of reducing substances in the soil for each treatment ranged from 5.45 to 8.78 cmol / kg, all showing a reduction compared to the control. The 100 mL treatment showed the largest reduction (6.84 cmol / kg), followed by the 50 and 20 mL treatments.

[0030] Using 1×10 7Treatment with 100, 50, and 20 mL of CFU / ml Bacillus lysine all reduced the total amount of active reducing substances in the soil (Table 3). Ten days after treatment, the total amount of active reducing substances in the soil for each treatment ranged from 2.25 to 6.44 cmol / kg, all showing a reduction compared to the control. The 100 mL treatment showed the largest reduction (3.79 cmol / kg), followed by the 50 and 20 mL treatments. Twenty days after treatment, the total amount of active reducing substances in the soil for each treatment ranged from 2.11 to 5.26 cmol / kg, all showing a reduction compared to the control. The 100 mL treatment showed the largest reduction (3.74 cmol / kg), followed by the 50 and 20 mL treatments. Thirty days after treatment, the total amount of active reducing substances in the soil for each treatment ranged from 2.64 to 5.33 cmol / kg, all showing a reduction compared to the control. The 100 mL treatment showed the largest reduction (3.2 cmol / kg), followed by the 50 and 20 mL treatments.

[0031] Using 1×10 7 Treatment with 100, 50, and 20 mL of CFU / ml Bacillus lysine all reduced soil ferrous content (Table 4). Ten days after treatment, the soil ferrous content in each treatment ranged from 3.11 to 5.08 cmol / kg, all showing a decrease compared to the control. The 100 mL treatment showed the largest reduction (2.03 cmol / kg), followed by the 50 and 20 mL treatments. Twenty days after treatment, the soil ferrous content in each treatment ranged from 2.79 to 4.55 cmol / kg, all showing a decrease compared to the control. The 100 mL treatment showed the largest reduction (2.34 cmol / kg), followed by the 50 and 20 mL treatments. Thirty days after treatment, the soil ferrous content in each treatment ranged from 2.44 to 4.07 cmol / kg, all showing a decrease compared to the control. The 100 mL treatment showed the largest reduction (2.72 cmol / kg), followed by the 50 and 20 mL treatments.

[0032] Using 1×10 7Treatment with 100, 50, and 20 mL of CFU / ml Bacillus lysine all reduced soil manganese content (Table 5). Ten days after treatment, the soil manganese content in each treatment ranged from 0.07 to 0.12 cmol / kg, all showing a reduction compared to the control. The 100 mL treatment showed the largest reduction (0.05 cmol / kg), followed by the 50 and 20 mL treatments. Twenty days after treatment, the soil manganese content in each treatment ranged from 0.06 to 0.12 cmol / kg, all showing a reduction compared to the control. The 50 mL treatment showed the largest reduction (0.05 cmol / kg), followed by the 50 and 20 mL treatments. Thirty days after treatment, the soil manganese content in each treatment ranged from 0.09 to 0.14 cmol / kg, all showing a reduction compared to the control. The 100 mL treatment showed the largest reduction (0.05 cmol / kg), followed by the 50 and 20 mL treatments.

[0033] Using 1×10 7 Treatments with 100, 50, and 20 mL of CFU / ml Bacillus lysine had no effect on seedling growth (Table 6). Thirty days after treatment, the rice plant height in each treatment ranged from 13.99 to 10.51 cm, all of which were higher than the control; the leaf age in each treatment ranged from 4.16 to 3.36, also higher than the control; and the fresh weight of rice ranged from 1.54 to 1.07 g / 10 plants, also higher than the control. This indicates that improved soil conditions were more suitable for rice growth.

[0034] Table 1. Effects of Bacillus lysine treatment on soil redox potential*

[0035]

[0036] *: The same letter in the table indicates no significant difference at the 0.05 level, while different letters indicate significant differences.

[0037] Table 2. Effects of Bacillus lysine treatment on total soil reducing substances*

[0038]

[0039] *: The same letter in the table indicates no significant difference at the 0.05 level, while different letters indicate significant differences.

[0040] Table 3. Effects of Bacillus lysine treatment on soil active reducing agent content*

[0041]

[0042] *: The same letter in the table indicates no significant difference at the 0.05 level, while different letters indicate significant differences.

[0043] Table 4. Effects of Bacillus lysine treatment on soil active ferrous content.

[0044]

[0045] *: The same letter in the table indicates no significant difference at the 0.05 level, while different letters indicate significant differences.

[0046] Table 5. Effects of Bacillus lysine treatment on soil active manganese content

[0047]

[0048] *: The same letter in the table indicates no significant difference at the 0.05 level, while different letters indicate significant differences.

[0049] Table 6. Effects of Bacillus lysine treatment on rice seedling quality 30 days after treatment*

[0050]

[0051] *: The same letter in the table indicates no significant difference at the 0.05 level, while different letters indicate significant differences.

Claims

1. An isolated strain of lysine-containing Bacillus ( Lysinibacillus sp. The preservation number of the lysine-containing Bacillus L001 is: CCTCC NO: M20221738.

2. The application of the Lysine Bacillus L001 as described in claim 1 in improving the properties of gleyed paddy field soil.

3. The application according to claim 2, wherein the improvement of the properties of gleyed paddy soil is an application of one or more of the following: increasing the redox potential of gleyed paddy soil, reducing the total amount of reducing substances, and reducing the content of active reducing substances.

4. In the application according to claim 3, the reduction of the content of active reducing substances is to reduce the content of ferrous and / or manganese.

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