Corrosion-promoting and growth-promoting multiple-effect-in-one compound bacterium for straw returning and application of multiple-effect-in-one compound bacterium

Abstract

The invention discloses a multi-effect-in-one compound bacterium capable of promoting decay and growth during straw returning, which consists of lysinibacillus fusiformis H93 and bacillus rollei X96, the preservation numbers of the lysinibacillus fusiformis H93 and the bacillus rollei X96 are respectively CGMCC (China General Microbiological Culture Collection Center) NO.33633 and CGMCC NO.33632, and the compound bacterium is preserved in China General Microbiological Culture Collection Center on February 24, 2025. The invention further discloses a complex microbial inoculant prepared from the strain and application of the complex microbial inoculant in promoting degradation of wheat straw directly returned to the field and promoting growth of rice. In different rice and wheat crop rotation soil in Jiangsu 13, a bacterium of lysinibacillus fusiformis H93 is screened from Huaian soil with the best crop growth promoting effect, a bacterium of bacillus rolfsii X96 is screened from Yuzhou soil with the best straw degradation effect, and the formed compound bacteria have the effects of straw direct returning to the field to promote decay and crop growth promoting.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and in particular to a multi-functional composite bacteria having the functions of decomposing straw and promoting crop growth, and applications thereof. Background Art

[0002] Global crop straw production has increased rapidly over the years. According to the Food and Agriculture Organization (FAO), global crop residue production has tripled over the past 50 years, from 1961 (1.552 billion tons) to 2011 (4.635 billion tons), reaching 5.287 billion tons in 2020. Cereal crops account for nearly 75% of global crop straw production, with wheat, rice, and corn accounting for nearly 90% of cereal crop straw production, with their respective contributions being almost equal.

[0003] However, current straw return to fields still faces challenges such as low straw degradation efficiency in the soil and susceptibility to disease, significantly hindering the widespread adoption of straw return. The degradation rate of straw in soil and the survival of pathogens it carries depend on the soil's microbial community. Therefore, regulating the soil microbial community and increasing beneficial strains that promote straw degradation is an effective approach for efficient and healthy straw return. However, research on microorganisms that both enhance the degradation efficiency of direct straw return and promote crop growth remains limited. Summary of the Invention

[0004] The purpose of the present invention is to provide a composite bacteria with multiple effects of returning straw to the field, promoting decomposition and growth, and its application, so as to solve the shortcomings of the existing technology.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A straw return to field rotting and growth-promoting bacterial agent, characterized by comprising one or a combination of lysinibacillus fusiformis H93 or Bacillus roseaevich X96; wherein Lysinibacillus fusiformis H93 has a preservation number of CGMCC NO.33633; and Bacillus roseaevich X96 has a preservation number of CGMCC NO.33632.

[0007] In one embodiment, the straw return-to-field rot-promoting and growth-promoting bacterial agent of the present invention is a combination of Lysinibacillus fusiformis H93 and Bacillus roseaevich X96. The combination can be mixed in any proportion. In a preferred embodiment, the ratio of Lysinibacillus fusiformis H93 to Bacillus roseaevich X96 is 1:1.

[0008] The present invention also provides a method for preparing the straw return to field rot-promoting and growth-promoting bacterial agent, which can be cultured according to the bacterial culture method in the art. For example, Bacillus fusiformis H93 or Bacillus rosea X96 are inoculated into 1 / 10 TSB liquid culture medium and cultured at 25-28°C and 160-180 rpm / min for 2-3 days to obtain a fermentation liquid, centrifuge, discard the supernatant, and resuspend in sterile water to obtain a single bacterial agent of the two bacterial strains. The bacterial concentration of the bacterial agent can be (1-5)×10 8 CFU / mL, more specifically, can be 1×10 8 CFU / mL. When a composite bacterial agent is used, the single bacterial agents are mixed in proportion.

[0009] The present invention also provides a spindle-shaped lysinophilic Bacillus H93, which was deposited in the General Microbiology Center of the China Culture Collection Administration on February 24, 2025, with a deposit number of CGMCCNO.33633.

[0010] The present invention also provides a Bacillus rosea X96, which has been deposited in the General Microbiology Center of the China Culture Collection Administration on February 24, 2025, with a deposit number of CGMCC NO.33632.

[0011] The present invention also provides the use of the straw-returning-to-field decay-promoting and growth-promoting bacterial agent or the lysinophilic Bacillus H93 or the Bacillus rosea X96 in straw degradation.

[0012] The present invention also provides the use of the straw return to field decay-promoting and growth-promoting bacterial agent or the fusiform Lysinibacillus H93 or the Roseburia X96 in promoting straw degradation and / or crop growth during direct straw return to the field.

[0013] In a specific example, the application of the present invention is to mix the straw return to field rot-promoting and growth-promoting bacteria agent or spindle-shaped Lysinibacillus H93 or Roseburia X96 with straw and bury it in the soil. In a specific example, 1×10 8 CFU of microorganisms.

[0014] The straw of the present invention may be a common crop straw in the art, such as wheat straw.

[0015] In one embodiment, the crop of the present invention is wheat or rice.

[0016] Beneficial effects of the present invention:

[0017] The present invention screened out fusiform lysinophilic Bacillus H93 from Huai'an soil samples, and screened out bacterium Roseburia X96 from Xuzhou soil samples. The composed composite bacteria have the functions of promoting the decomposition of straw by directly returning it to the field and promoting the growth of crops, and have good promotion value for promoting the decomposition of straw by directly returning it to the field in rice-wheat rotation farmland in Jiangsu Province.

[0018] The data of the examples of the present invention show that in a potted plant verification test, the bacterial agent of the present invention can significantly increase the fresh weight and dry weight of rice, and significantly increase the straw degradation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the effect of different soils on the degradation rate of wheat straw in a wheat pot experiment.

[0020] Figure 2 This paper shows the effect of different soils on the degradation rate of wheat straw in a rice pot experiment.

[0021] Figure 3 This is the effect of different soils on the fresh weight of wheat plants in a wheat pot experiment.

[0022] Figure 4 This is the effect of different soils on the fresh weight of rice plants in a rice pot experiment.

[0023] Figure 5 Effects of different bacterial agent treatments on the aboveground fresh weight of 35-day-old rice plants.

[0024] Figure 6 Effects of different bacterial agent treatments on the aboveground dry weight of 35-day-old rice plants.

[0025] Figure 7 Effects of different bacterial agent treatments on the degradation rate of 35-day-old wheat straw in rice.

[0026] Figure 8 This is the 16s rRNA phylogenetic tree of strain H93.

[0027] Figure 9 This is the 16s rRNA phylogenetic tree of strain X96.

[0028] Figure 10 This is a picture of the growth of strain H93 on 1 / 10 TSB solid medium.

[0029] Figure 11 This is a picture of the growth of strain X96 on 1 / 10 TSB solid medium.

[0030] Different letters above the bars indicate significant (P < 0.05) differences defined by one-way ANOVA.

[0031] Biomaterial deposit information

[0032] Strain H93, classified as Lysinibacillus fusiformis, was deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 24, 2025, and the deposit number is CGMCCNO.33633.

[0033] Strain X96, classified as Bosea robiniae, was deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 24, 2025, and the deposit number is CGMCC NO.33632. DETAILED DESCRIPTION

[0034] The following examples and figures are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0035] Unless otherwise specified, the diameter of the plates (culture dishes) involved in the following examples is 90 mm.

[0036] Unless otherwise specified, the culture media involved in the following examples are shown in Table 1.

[0037] Unless otherwise specified, the reagents involved in the following examples are shown in Table 2.

[0038] Table 1 Culture medium involved in each embodiment

[0039]

[0040]

[0041] Table 2 Reagents involved in each embodiment

[0042]

[0043] Example 1: Experiment on burying straw in potted plants

[0044] Wheat seeds: Jimai No. 22;

[0045] Rice seeds: Y Liangyou 689

[0046] Wheat straw: taken from Lianyungang City, Jiangsu Province, cut into 5-6 cm length, with a moisture content of about 15wt%.

[0047] The main farming pattern in Jiangsu Province is rice-wheat rotation. Therefore, this study selected rice-wheat rotation soils and grassland soils from 13 cities in Jiangsu as research objects, and used wheat and rice as rotation test crops for research.

[0048] From each region's farmland soil, three replicates were collected using a nine-point sampling method. Field soil samples from the 0-20 cm depth were mixed to form one sample. The samples were then passed through a 10-mesh sieve to remove stones and visible plant debris. Sterilized soil from the Nanjing sampling site (Science and Technology Park, Nanjing Institute of Vegetable and Flower Sciences) was used as background soil. Sterilized soil from the 14 regions was sterilized by gamma irradiation. 10% of the soil samples collected from each region were inoculated into the sterilized soil and mixed evenly, resulting in a 1:9 ratio of farmland soil to sterilized soil. Three replicates were performed for each of the three samples from each region. One kilogram of soil was weighed from each replicate to obtain soils with identical physical and chemical properties but distinct microbial communities. The sterilized background soil was also used as a blank control. For the experiment, 5 g of wheat straw, cut into 5-6 cm segments, was placed in a 100-mesh nylon mesh bag. A 3-cm layer of soil was added to a pot, followed by three straw bags, totaling 15 g. The remaining soil was then added to the pot.

[0049] Three wheat seeds with uniform sprout length were sown in each pot. The temperature was controlled at 15-20°C. The lighting time during the planting period was 8:00-20:00, the relative humidity was 40%, and an equal amount of sterile water was added as needed.

[0050] After 30 days, sampling was performed to collect rhizosphere soil, soil surrounding straw, buried straw, and plant fresh weight. The plant was removed from the soil with its roots, minimizing root breakage. The soil adhering to the root surface was shaken off, representing the rhizosphere soil. The buried straw was removed from the pot, and the soil adhering to the surface of the mesh bag was shaken off, representing the soil surrounding the straw. Plant fresh weight was measured by repeatedly washing the roots of the removed plant with clean water until no soil remained, wiping them dry, and weighing them. The straw degradation rate was also measured. The straw mesh bag was removed and rinsed with water to remove any surface soil. The wheat straw inside was placed in an envelope and oven-dried at 60°C for 96 hours. The weight of the remaining wheat straw was then measured. The difference between the initial dry weight and the remaining weight, divided by the initial dry weight, was the straw degradation rate.

[0051] After the soil for planting wheat is air-dried, it is passed through a 10-mesh sieve and a season of rice is planted. Six replicates are set up for each treatment. As with wheat pot planting, wheat straw should also be buried. Select rice seeds with consistent bud length after germination and sow them in pots with a sowing depth of 1-2 cm. Sow 3 seeds per pot and add an equal amount of sterile water to soak the soil. During the planting period, the lighting time is 8:00-20:00, the temperature is controlled at 25℃-30℃, and the relative humidity is 60%. Add water in equal amounts as needed. Water with sterile water every day for the first week to keep the soil surface moist; then water with sterile water every 1 day, and keep the soil 2-3 cm flooded each time. After 30 days, take samples in the same way as the wheat pot.

[0052] The results are as follows Figure 1-Figure 4 As shown, in both pot experiments, the straw degradation rate of the treatment group containing 10% non-sterile soil was higher than that of the sterile soil control group. In the wheat sampling, the straw degradation rate of the ck treatment group was 27.40%, and the fresh weight of each plant was 1.60g. The straw degradation rate of the Xuzhou treatment group was 40.91% ( Figure 1 ), the fresh weight of each plant in the Huai'an treatment group was 2.1g ( Figure 3 ); in rice samples, the straw degradation rate of the ck treatment group was 29.93%, with a fresh weight of 5.30 g per pot of plant; the straw degradation rate of the Xuzhou treatment group was 36.09% ( Figure 2 ), the fresh weight of each plant in the Huai'an treatment group was 6.82g ( Figure 4 ); Comprehensive comparison showed that the straw degradation rate of the Xuzhou treatment group was higher, and the plant fresh weight data of the Huai'an treatment group was the best.

[0053] Example 2 Broad-spectrum screening

[0054] The soil attached to the straw of the Xuzhou treatment group obtained from the last rice sampling in Example 1 was selected to screen indigenous functional strains with the ability to degrade lignin and cellulose, and the rhizosphere soil of the Huai'an treatment group was selected to screen indigenous functional strains with the ability to degrade lignin and cellulose and produce growth hormone.

[0055] The specific method is as follows: 10g of soil sample was weighed and added to 90mL of sterile water, and three replicates were set up and recorded as XZ1; XZ2; XZ3; HA1; HA2; HA3. The sample was shaken at 170rpm / min at 25℃ for about 30min, and then allowed to stand for 10min. The upper layer of soil suspension was taken as 10 -1 This concentration gradient. And so on, make 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Different concentration gradient soil suspensions. Use different culture media to isolate and culture fungi and bacteria. Bengal red culture medium is used for fungi. 10-2 , 10 -3 , 10 -4 0.1 ml of soil suspension of three concentration gradients was applied and cultured in a constant temperature incubator at 28°C for 2-3 days; 1 / 10 TSB solid culture medium was used for bacteria, and 10 -3 , 10 -4 , 10 -5 , 10 -6 Four concentration gradients of soil suspension (0.1 mL) were applied and incubated in a 28°C incubator for 2-3 days. During this time, the growth of the strains on the plates was observed, as were the characteristics of the colonies (color, shape, and texture) on the culture medium. Single colonies with distinct morphological characteristics were selected from the appropriate concentration gradient (high biodiversity). The fungus was inoculated onto PDA medium, and the bacteria were streaked onto a new 1 / 10 TSB solid medium. The plates were then incubated in an inverted culture at 28°C, and purification was continued until only one strain was observed on the plates, with single bacterial colonies present.

[0056] Example 3 Functional strain screening

[0057] 1) Determination of cellulose degradation ability:

[0058] The purified strains (fungi and bacteria) obtained from the Xuzhou and Huai'an treatment groups in Example 2 were inoculated into CMC-Na culture medium, inverted and cultured in a 28°C constant temperature incubator for 72 hours, and their ability to degrade cellulose was measured. -1 1 mL of Congo red solution was added to a CMC-Na culture medium plate, and the dye solution was discarded after standing for 30 min. 1 mol·L -1 The strain was then washed with a 0.5% NaCl solution and allowed to stand for 1 hour, after which any excess color was removed. The appearance of a transparent ring around the colony indicates that the strain has the ability to degrade cellulose. The diameter of the transparent ring (c (mm)) and the diameter of the colony (H (mm)) were measured, and the Hc value was calculated using the following formula. The identification data for the strains screened in the Xuzhou treatment group are shown in Table 3, and the identification results for the strains screened in the Huai'an treatment group are shown in Table 4.

[0059] Hc = diameter of transparent zone / diameter of colony

[0060] 2) Lignin degradation ability:

[0061] The purified strains (fungi, bacteria) obtained from the Xuzhou and Huai'an treatment groups in Example 2 were inoculated on PDA-aniline blue culture medium, inverted and cultured in a 28°C constant temperature incubator for 72 hours to determine their ability to degrade lignin. If a transparent circle appears around the colony, it indicates that the strain has the ability to degrade lignin. Measure the diameter c (mm) of the transparent circle and the diameter of the colony H (mm), and calculate the Hc value according to the following formula. The identification data of the strains screened in the Xuzhou treatment group are shown in Table 3, and the identification results of the strains screened in the Huai'an treatment group are shown in Table 4.

[0062] Hc = diameter of transparent zone / diameter of colony

[0063] 3) Quantitative determination of IAA production capacity:

[0064] The strains with degradation ability in the Huai'an treatment group were rescreened for functional strains capable of producing growth hormone.

[0065] a. Bacteria

[0066] First, prepare TSB liquid culture medium containing L-tryptophan, that is, add 100 μL of L-tryptophan solution to every 100 mL of culture medium (filter with a 0.22 μm water filter membrane and then add to the culture medium), mix well, and then draw 3 mL into a 10 mL shaking tube. Pick the strain with the ability to degrade cellulose and lignin after purification from the Huai'an treatment group and inoculate it into the shaking tube. Culture on a shaking table (30°C, 170 rpm / min) for 48 hours. Then, 1 mL of bacterial solution was transferred to a 2 mL centrifuge tube and centrifuged at 1000 rpm / min and 4°C for 10 min. 100 μL of the supernatant was transferred to a 96-well plate, and an equal volume of 100 μL of color development solution Fecl3·6H2O was added. The plate was kept in the dark for 30 min. The blank sample was 100 μL of TSB liquid medium (without L-tryptophan solution) and 100 μL of color development solution Fecl3·6H2O was added. Red was positive, and the darker the color, the higher the hormone concentration. The OD value was measured using a microplate reader. 530 The IAA content per unit volume of fermentation broth was calculated using a standard curve, with the control group at 0 μg / mL. The standard curve was constructed using a gradient dilution of analytically pure IAA. The IAA production capacity of the screened strains in the Huai'an treatment group is shown in Table 4.

[0067] b. Fungi

[0068] First, prepare PDB liquid culture medium containing L-tryptophan, that is, add 100 μL of L-tryptophan solution to every 100 ml of culture medium (filtered with a 0.22 μm water filter membrane before adding to the culture medium), mix well, and then draw 10 mL into a 25 μL conical flask. Pick the purified fungi with the ability to degrade cellulose and lignin, press with the tail of the yellow gun tip, take a 5 mm diameter bacterial cake and inoculate it into the conical flask, culture on a shaking table (30 ° C, 170 rpm / min) for 96 hours, then draw 1 mL of bacterial solution into a 2 mL centrifuge tube and centrifuge at 1000 rpm / min for 10 minutes, draw 100 μL of the supernatant into a 96-well plate, add an equal volume of color development solution Fecl3·6H2O100 μL, and let it stand in the dark for 30 minutes. The blank sample is 100 μL. PDB liquid culture medium (without L-tryptophan solution) was added with 100 μL of color developing solution FeCl3·6H2O. Red was positive. The darker the color, the higher the hormone concentration. The OD 530The IAA content per unit volume of fermentation broth was calculated using a standard curve, with the control group at 0 μg / mL. The standard curve was constructed using a gradient dilution of analytically pure IAA. The IAA production capacity of the screened strains in the Huai'an treatment group is shown in Table 4.

[0069] Table 3 Degradation ability of Xuzhou strains on cellulose and lignin

[0070]

[0071]

[0072] Table 4 Huai'an strains have the ability to degrade and produce IAA

[0073]

[0074]

[0075] Example 4: Verification test of straw buried potted plants

[0076] The microorganisms screened in Example 3 were classified according to different functional dimensions, namely, cellulose degradation ability, lignin degradation ability, and IAA production ability. Two strains with the best function were selected for each dimension. Since the fungus Z0 was particularly prominent in the fungal degradation effect, Z0 was selected as the test strain, that is, four degradation strains Z0, X96, X66, and X33 (numbered 1, 2, 3, and 4, respectively) and two growth-promoting strains H93 and A62 (numbered A and B, respectively) were composed of single bacteria (1, 2, 3, 4, A, and B), double bacteria combinations (12, 34, 1A, 2A, 3B, and 4B), three bacteria combinations (12A, 14A, 23B, and 34B), and six bacteria combinations (1234AB). The non-inoculated microorganisms were used as the control, for a total of 18 treatments, with 6 replicates for each treatment, to carry out potted verification tests.

[0077] The soil used in the potted plant experiment was sterile soil, which was collected from Liuhe District, Nanjing City, Jiangsu Province. It was dried in the sun, passed through a 10-mesh net, and sterilized by γ-rays.

[0078] Wheat straw: taken from Lianyungang City, Jiangsu Province, with a moisture content of about 15wt%, cut into 5-6cm length.

[0079] Rice seeds: Y Liangyou 689

[0080] Preparation of microbial agent:

[0081] Single bacterial agent: Activated bacterial strains (strain X96, strain X66, strain X33, and strain H93) were inoculated into 1 / 10 TSB liquid medium and cultured at 25°C and 170 rpm / min for 2 days to obtain fermentation broth. The supernatant was removed by centrifugation at 7000 rpm / min for 10 minutes, and sterile water was added to adjust the bacterial broth concentration to 1×10 8 CFU / mL, and the single bacterial agent of the bacteria was obtained.

[0082] Preparation of single fungal agent: The activated fungal strains (strain Z0, strain A62) were inoculated on PDA culture plates and cultured at 28°C for 10 days to obtain spore-forming fungal plates. Spores and hyphae on the surface of the fungal strain plates were scraped with sterile water, and the fungal spores in the agent were determined by hemocytometer method at 1×10 8 To facilitate the subsequent preparation of fungal agents, all spores counted on the hemocytometer were recorded as viable bacteria, and the fungal spore concentration was 1×10 8 CFU / mL.

[0083] Compounding of compound bacterial agents: The corresponding single bacterial agents were mixed at a volume ratio of 1:1 (i.e., a bacterial ratio of 1:1) to obtain the compound bacterial agent. The total concentration of the compound bacterial agent was also 1×10 8 CFU / mL.

[0084] In specific application, for a single bacterial agent: the concentration of microorganisms inoculated per pot is 5×10 7 CFU / mL; for the dual-bacteria combination, the inoculum size of each microorganism was 2.5×10 7 CFU / mL; for the three-microorganism combination: the inoculum size of each microorganism is 1.67×10 7 CFU / mL; for the six-microorganism combination: the inoculum size of each microorganism is 8.3×10 6 CFU / mL.

[0085] Each pot was first filled with soil about 3 cm deep, and then 3 bags of wheat straw were added, each bag of straw was 5 g, and 30 ml of sterile water or 30 ml of fungicide was poured on the surface of the wheat straw according to the treatment. Each gram of wheat straw required 1×10 8 CFU of microorganisms, that is, each pot contains 1.5×10 9 CFU of microorganisms.

[0086] Continue filling the pots with soil to a total depth of approximately 15 cm, with approximately 1 kg of soil per pot. Add an equal amount of water to each pot until completely soaked. Select rice seeds with consistent bud length after germination and sow them in the pots at a depth of 1-2 cm, sowing 3 seeds per pot. Add an equal amount of sterile water to soak the soil thoroughly. During the planting period, light exposure was maintained from 8:00 AM to 8:00 PM, the temperature was controlled between 25°C and 30°C, and the relative humidity was 60%. Water was added as needed. For the first week, water the soil daily with sterile water to keep the soil surface moist. Thereafter, water the soil with sterile water every other day, keeping the soil submerged by 2-3 cm. Samples were harvested 35 days after sowing. The aboveground portion of the rice was removed with scissors and the fresh weight of the aboveground portion was determined. After the measurement, the aboveground portion of the rice was individually placed in an envelope and placed in an oven at 60°C for 96 hours. The aboveground dry weight of the rice was then determined. The net bag containing wheat straw buried in the soil was dug out, the soil on the surface was washed off with water, the wheat straw inside was taken out and placed in an envelope, and then placed in an oven at 60°C for 96 hours to dry. The weight of the remaining wheat straw was measured and the straw degradation rate was calculated.

[0087] The biomass of each treatment Figure 5-6 As shown in the data, in terms of aboveground fresh weight and aboveground dry weight, the aboveground fresh weight and aboveground dry weight of the CK treatment without inoculation of microorganisms were the lowest, only 8.48g and 1.42g respectively. The treatments inoculated with microorganisms (single microorganisms and compound microorganisms) were better than the CK treatment. Among them, the aboveground dry weight of the treatment group (2A) inoculated with the compound microorganism composed of H93 and X96 reached 1.94g, which was significantly increased by 36.62% compared with the sterile control group.

[0088] The results of wheat straw degradation rate under each treatment are as follows Figure 7 As shown in the figure, the wheat straw degradation rate of the CK treatment without inoculation was the lowest, at 37.21%. The straw degradation rate of the treatment group (2A) inoculated with a composite inoculum composed of H93 and X96 reached 43.83%, an increase of 17.79% over the sterile control group, significantly better than the other treatment groups.

[0089] In summary, the composite bacterial agent prepared by strains H93 and X96 has the effects of promoting the decomposition of straw directly returned to the field and promoting the growth of crops.

[0090] Example 5 Identification of strains H93 and X96

[0091] The colonies of strain H93 cultured on 1 / 10 TSB solid medium at 25℃ for 3 days were round, with irregular edges, white, and opaque. Figure 10 ). By comparing the 16s rRNA gene sequence of strain H93 with similar sequences and constructing a phylogenetic tree, the results are as follows Figure 8As shown, it has a high degree of homology with Lysinibacillus fusiformis. Combined with the colony morphology and 16s rRNA phylogenetic tree of strain H93, it was identified as Lysinibacillus fusiformis. Strain H93 was deposited with the General Microbiology Center of the China General Culture Collection Administration on February 24, 2025, with the deposit number CGMCC No. 33633.

[0092] The colonies of strain X96 after culturing on 1 / 10 TSB medium at 25℃ for 3 days were round, white, with moist, smooth, transparent and neat edges. Figure 11 ). By comparing the 16s rRNA gene sequence of strain X96 with similar sequences and constructing a phylogenetic tree, the results are as follows Figure 9 As shown, it has a high degree of homology with Bosea robiniae. Combined with the colony morphology and 16s rRNA phylogenetic tree of strain X96, it was identified as Bosea robiniae. Strain X96 was deposited with the General Microbiology Center of the China Culture Collection Administration on February 24, 2025, with the deposit number CGMCC NO. 33632.

Claims

1. A straw return to field rot-promoting and growth-promoting bacterial agent, characterized in that: It is one or a combination of Bacillus fusiformis H93 and Bacillus reuteri X96; wherein the preservation number of Lysinibacillus fusiformis H93 is CGMCC NO.33633; the preservation number of Bacillus reuteri X96 is CGMCC NO.33632.

2. The straw return to field decomposition and growth-promoting microbial agent according to claim 1, characterized in that: It is a combination of Lysinibacillus fusiformis H93 and Bacillus rosea X96.

3. The straw return to field rot-promoting and growth-promoting bacterial agent according to claim 2, characterized in that: The ratio of Lysinibacillus fusiformis H93 and Bacillus roseauii X96 bacteria was 1:

1.

4. The method for preparing the straw-returning-to-field-decomposition-promoting bacterial agent according to any one of claims 1 to 3, characterized in that: Lysinibacillus fusiformis H93 or Bacillus rhodesii X96 were inoculated into 1 / 10 TSB liquid culture medium and cultured at 25-28°C and 160-180 rpm / min for 2-3 days to obtain fermentation broth, which was centrifuged, the supernatant was discarded, and the mixture was resuspended in sterile water to obtain a single bacterial agent of the two bacterial strains; preferably, the bacterial concentration of the bacterial agent is (1-5)×10 8 CFU / mL; preferably 1×10 8 CFU / mL; when it is a combined composite bacterial agent, the single bacterial agents are mixed in proportion.

5. A spindle-shaped lysinophilic Bacillus H93, with a deposit number of CGMCC No. 33633.

6. A Bacillus rosea X96, with a deposit number of CGMCC No. 33632.

7. Use of the straw-returning agent for promoting decay and growth according to any one of claims 1 to 3, or the spindle-shaped Lysinibacillus H93 according to claim 5, or the Bacillus rosea X96 according to claim 6 in straw degradation; the application specifically comprises mixing the straw-returning agent for promoting decay and growth according to any one of claims 1 to 3, or the spindle-shaped Lysinibacillus H93 according to claim 5, or the Bacillus rosea X96 according to claim 6 with straw and burying the mixture in the soil; preferably, 1×10 8 CFU of microorganisms.

8. Use of the straw return-to-field decay-promoting and growth-promoting agent according to any one of claims 1 to 3, or the spindle-shaped Lysinibacillus H93 according to claim 5, or the Bacillus rosea X96 according to claim 6 in promoting straw degradation and / or crop growth during direct straw return to the field; the application specifically comprises mixing the straw return-to-field decay-promoting and growth-promoting agent according to any one of claims 1 to 3, or the spindle-shaped Lysinibacillus H93 according to claim 5, or the Bacillus rosea X96 according to claim 6 with straw and burying the mixture in the soil; preferably, 1×10 8 CFU of microorganisms.

9. The use according to claim 7 or 8, characterized in that The straw is wheat straw.

10. The use according to claim 8, characterized in that The crop is wheat or rice.

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