A compound microbial agent for selenium-enriched rice and a preparation method and application thereof

Abstract

The present application relates to the technical field of microbial preparation, in particular to a compound microbial inoculant for selenium-enriched rice, a preparation method and application thereof.The compound microbial inoculant comprises selenium-reducing LYB-S bacterial liquid, bacillus subtilis bacterial liquid, xanthan gum, nano zinc oxide, nano iron oxide and water; wherein, in terms of mass percentage, the addition amount of the selenium-reducing LYB-S bacterial liquid is 50%, the addition amount of the bacillus subtilis bacterial liquid is 25%, the addition amount of the xanthan gum is 0.3%, the addition amount of the nano zinc oxide is 2%, the addition amount of the nano iron oxide is 1%, and the balance is water, and the total mass percentage is 100%. The compound microbial inoculant can be used to increase the selenium content of rice. In addition, the compound microbial inoculant can also promote the growth of rice simultaneously, increase the number of grains per panicle and panicle weight, and realize yield increase.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, specifically to a compound microbial agent for selenium enrichment of rice, its preparation method, and its application. Background Technology

[0002] Selenium is an essential micronutrient for the human body, participating in the regulation of the activity of various enzymes and playing an important role in enhancing immunity, preventing chronic diseases, anti-oxidation, and anti-cancer properties. Selenium deficiency may lead to various diseases, such as thyroid disease, diabetes, reproductive disorders, and obesity. Because the human body's own selenium intake is limited, and the ingested selenium cannot be effectively absorbed and utilized to perform its important physiological functions, it is necessary to obtain it from the daily diet.

[0003] As rice is the staple food of more than half of the world's population, increasing its selenium content is an effective way to improve the selenium nutritional status of the population. Currently, rice selenium enrichment technology mainly falls into two categories: soil application of selenium fertilizer and foliar spraying of selenium fertilizer. Commonly used selenium sources are inorganic selenium such as sodium selenite or sodium selenate, but direct application presents several problems: inorganic selenium is easily fixed or leached by the soil, resulting in low utilization; excessive application can easily cause soil pollution and plant toxicity; and the inorganic selenium absorbed by rice needs to be converted within the plant, leading to unstable organic conversion efficiency.

[0004] Nano-selenium is a red, zero-valent elemental selenium with a diameter of approximately 20 to 500 nanometers. It is readily soluble in water and does not easily transform into a blackish-gray elemental selenium. It features small particle size, large specific surface area, and high bioactivity. Compared to traditional selenium supplements, nano-selenium exhibits higher bioavailability and bioactivity, and its toxicity is significantly lower than that of inorganic selenium. While nanoparticle synthesis can be achieved through physical, chemical, or biological methods, the use of large quantities of toxic chemicals, high temperatures, and expensive equipment, as well as the potential adverse effects of surface adsorption of toxic chemicals in physical and chemical methods, presents challenges. Biosynthesis is favored over traditional chemical and physical methods due to its use of biological organisms such as plants, microalgae, and other microorganisms, which have lower environmental toxicity. Microbial synthesis of nano-selenium typically involves adding microorganisms to a sodium selenite solution, where their metabolism reduces high-valent selenium to nano-sized selenium. Simultaneously, organic substances such as proteins, polysaccharides, and lipids are generated on the surface of the nano-selenium, contributing to its structural stability. However, single nano-selenium formulations are prone to aggregation and loss in field applications, and their effects on plant growth and selenium absorption and transport are limited. Therefore, developing a compound microbial agent that integrates nano-selenium biosynthesis, stabilization, and synergistic promotion functions is of great significance for the safe, efficient, and sustainable production of selenium-enriched rice. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a compound microbial agent for selenium enrichment of rice and its preparation method, and to use the compound microbial agent to increase the selenium content of rice. Furthermore, this compound microbial agent, while enriching selenium, can simultaneously promote rice growth, increase the number of grains per panicle and panicle weight, thereby increasing yield.

[0006] To achieve the above-mentioned objectives, the specific technical solution of this invention is as follows:

[0007] A compound microbial agent for selenium enrichment of rice comprises selenium-reducing LYB-S bacterial solution, Bacillus subtilis bacterial solution, xanthan gum, nano zinc oxide, nano iron oxide, and water. The addition amounts, by mass percentage, are: selenium-reducing LYB-S bacterial solution 40%-60%, Bacillus subtilis bacterial solution 20%-30%, xanthan gum 0.1%-0.5%, nano zinc oxide 1%-3%, nano iron oxide 0.5%-2%, with the remainder being water, and the total mass percentage is 100%.

[0008] Furthermore, in the aforementioned compound microbial agent for selenium enrichment of rice, the selenium-reducing LYB-S bacterial solution is *Bacillus belyssae* (…). Bacillus velezensis LYB-S bacterial culture. Bacillus belyss ( Bacillus velezensis LYB-S is a strain capable of reducing sodium selenite to red elemental selenium nanoparticles. The reduced red selenium nanoparticles are spherical with a diameter ranging from 100 to 200 nanometers, and the reduction efficiency is 50.11 ± 6.03%. Bacillus subtilis also possesses properties that improve soil structure, promote crop growth, and enhance disease resistance. Therefore, this application also protects Bacillus belyssioides (LYB-S). Bacillus velezensis Application of LYB-S in the reduction of sodium selenite to red elemental nano-selenium.

[0009] Furthermore, in the compound microbial agent for selenium enrichment of rice, the particle size of the nano zinc oxide and nano iron oxide is 30-50 nm.

[0010] Furthermore, in the aforementioned compound microbial agent for selenium enrichment of rice, the preparation method of the selenium-reducing LYB-S bacterial solution includes the following steps:

[0011] Bacillus berberis ( Bacillus velezensisThe LYB-S strain (hereinafter referred to as LYB-S strain) was transferred to YD medium (LYB-S special medium) for activation. The activated strain was then cultured to the logarithmic growth phase. The strain cultured to the logarithmic growth phase was then inoculated into YD medium containing sodium selenite. At this time, the LYB-S strain reduced sodium selenite to obtain nano-selenium. The cultured LYB-S strain and the nano-selenium produced were centrifuged and the precipitate was collected. The precipitate was resuspended in sterile water to obtain the LYB-S bacterial solution (selenium-reduced LYB-S bacterial solution).

[0012] Preferably, the LYB-S bacterial culture contains 10 viable LYB-S bacteria. 8 -10 10 CFU / g, the concentration of nano-selenium is 5–20 g / L.

[0013] As a preferred embodiment of this application, the formulation of the LYB-S special culture medium is as follows: sucrose 10 g / L, K2HPO4 2 g / L, (NH4)2SO4 1 g / L, MgSO4·7H2O 0.5 g / L, yeast extract 0.5 g / L, and NaCl 0.1 g / L. The inoculation ratio of the strain cultured to the logarithmic phase is 2% (by volume percentage); the concentration of sodium selenite in the YD culture medium is 5 mmol / L; and the centrifugation conditions are 8000 r / min for 15 min.

[0014] Furthermore, the preparation method of Bacillus subtilis bacterial solution in the aforementioned compound microbial agent for selenium enrichment of rice includes the following steps:

[0015] Bacillus subtilis was inoculated into LB medium for activation, and then the activated strain was cultured to the logarithmic phase. The strain cultured to the logarithmic phase was then inoculated into LB liquid medium for shaking culture, and the precipitate was collected by centrifugation. Finally, the precipitate was resuspended in sterile water to obtain Bacillus subtilis bacterial suspension.

[0016] In a preferred embodiment of this application, the inoculation ratio of the strain cultured to the logarithmic growth phase is 2% (by volume percentage); the shaking culture conditions are 30 °C, 180 r / min, and 36 h; the centrifugation conditions are 8000 r / min and 15 min; and the viable count in the Bacillus subtilis culture is 10-1. 8 -10 10 CFU / g.

[0017] As a preferred embodiment of this application, the preparation method of the compound microbial agent for selenium enrichment of rice described above includes the following steps: first, xanthan gum is dissolved in sterile water to prepare a colloidal solution; then, the obtained colloidal solution, selenium-reducing LYB-S bacterial solution and Bacillus subtilis bacterial solution are stirred and mixed evenly in proportion to obtain a mixed solution; finally, nano zinc oxide and nano iron oxide are added to the mixed solution in sequence, stirred and mixed evenly again, and the pH is adjusted.

[0018] In a preferred embodiment of this application, in the preparation method of the compound microbial agent for selenium enrichment of rice, the stirring conditions are 500 r / min and the stirring time is 15 min; the pH is adjusted to 7.2-7.5.

[0019] This application also protects the use of the compound microbial agent for selenium enrichment of rice described above in increasing the selenium content of rice.

[0020] Furthermore, while enriching with selenium, it can simultaneously promote rice growth, increase the number of grains per panicle and panicle weight, thereby increasing yield; it can also be used to improve soil nutrients, etc.

[0021] In this scheme, the role of each component is:

[0022] 1. The core functional bacteria (selenium-reducing LYB-S bacteria, as nano-selenium generating bacteria) can reduce inorganic selenium (sodium selenite) in situ to bio-nano-selenium. Nano-selenium is not only far less toxic than sodium selenite, but is also more easily absorbed and converted by plant roots. Bacillus subtilis, as a powerful growth-promoting bacterium, promotes rice root development and enhances plant health by secreting growth hormones, producing antibacterial substances, and activating plant systemic resistance. When combined with selenium-reducing LYB-S bacteria, it can jointly improve the root system's ability to absorb and transport selenium.

[0023] 2. Xanthan gum, as a biopolysaccharide colloid in this application, acts as a binder and protectant. It can encapsulate microbial cells and nanomaterials, improving the survival rate of strains in the compound microbial agent during the granulation process, and forming a local protective film after application to the soil, thus slowing down the death rate of functional bacteria.

[0024] 3. Nano-zinc oxide and nano-iron oxide: Both are trace elements required by plants and can correct soil nutrient deficiencies. More importantly, nanomaterials have unique surface and small-size effects, which may adsorb and enrich selenium in the soil, providing selenium accumulation sites for functional bacteria; at the same time, they may stimulate the physiological activity of plant roots, creating positive interactions with microorganisms and jointly enhancing the microenvironment for selenium absorption.

[0025] 4. Safety and Efficiency: The entire system is based on biosynthesis and bio-promoting, avoiding the direct use of high-concentration inorganic selenium, making it environmentally friendly. The combination of multiple components creates a three-dimensional selenium enrichment model of "microbial reduction + root promotion + microenvironment regulation," which improves the conversion rate and enrichment of organic selenium in rice.

[0026] Compared with existing technologies, the present invention has the following advantages:

[0027] (1) Selenium-enriched, efficient and safe: It can stably increase the selenium content of rice to a selenium-enriched level of 155 μg / kg, and through biotransformation, it generates nano-selenium with higher activity, which is safer than direct application of inorganic selenium fertilizer.

[0028] (2) Significant yield increase: While enriching with selenium, it can simultaneously promote rice growth, increase the number of grains per panicle and the weight of panicle, thereby increasing yield and solving the problem that traditional selenium enrichment technology may affect yield.

[0029] (3) Improve soil nutrients: The multifunctional microorganisms in the inoculant can effectively increase the content of soil organic matter and available phosphorus, enrich the soil with selenium, and ensure the sustainability of agricultural production.

[0030] (4) Synergistic effect of multiple technologies: Through the combination of nano-selenium-producing bacteria, Bacillus subtilis and nanomaterials, the synergistic effect of multiple technologies such as selenium form transformation, root growth promotion and microenvironment regulation is achieved. Attached Figure Description

[0031] Figure 1 This is a transmission scanning electron microscope image of nano-selenium;

[0032] Figure 2 A graph showing the available phosphorus content in rice paddy soil;

[0033] Figure 3 This is a graph showing the organic matter content of rice paddy soil.

[0034] Figure 4 This is a diagram showing the influence of the compound microbial inoculants in this application on rice growth and yield.

[0035] Among them, (a) plant height of rice; (b) panicle length of rice; (c) number of panicles per rice plant; and (d) panicle weight of rice.

[0036] Figure 5 This is a graph showing the relationship between the selenium content in rice treated with compound microbial agents and rice in the control group. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and examples. It should be understood that the specific examples described herein are merely illustrative and not intended to limit the invention. Furthermore, it should be understood that after reading this invention, those skilled in the art can make various modifications and alterations to it, but these equivalent forms also fall within the scope defined by the appended claims.

[0038] Unless otherwise specified, "%" in the following examples refers to percentage content by mass.

[0039] The Bacillus berleis in this application ( Bacillus velezensis LYB-S is an existing strain with accession number CGMCC NO.34429, which has been disclosed in the patent application number 2025110533227. This application will not provide accession certificate.

[0040] The Bacillus subtilis strain used in this application is a commercially available strain purchased from Mingzhou Biotechnology, product code: B12039.

[0041] Example 1:

[0042] Selenium-reducing LYB-S bacteria (Bacillus belysinus) Bacillus velezensis Application of LYB-S in the reduction of sodium selenite to red elemental nano-selenium.

[0043] To investigate the reduction efficiency of this bacterium in reducing sodium selenite to nano-selenium, the following experiment was conducted:

[0044] YD culture medium formula: sucrose 10 g / L, K2HPO4 2 g / L, (NH4)2SO4 1 g / L, MgSO4·7H2O 0.5 g / L, yeast extract 0.5 g / L, NaCl 0.1 g / L, pH 7.0.

[0045] The LYB-S strain was inoculated into YD medium and activated at 30 °C and 180 r / min for 12 h. The activated bacterial culture was then transferred to YD medium containing 5 mmol / L sodium selenite at an inoculation rate of 2% (by volume percentage). A medium without sodium selenite was used as a control. The cultures were incubated at 30 °C and 180 r / min for 60 h.

[0046] After cultivation, the bacterial culture was centrifuged at 5000×g and 4℃ for 20 min, and the supernatant was discarded. The precipitate was washed three times with 1 mmol / L NaCl solution. The final collected red precipitate was fully resuspended in 30 mL of 1 mol / L Na2S·9H2O solution, reacted for 30 min, and then centrifuged at 10000×g for 30 min. The absorbance of the supernatant was measured at a wavelength of 500 nm.

[0047] Preparation of standard curve: Accurately weigh black selenium powder and prepare standard solutions of 0, 20, 40, 60, 80, and 100 mg / L with Na2S solution. Measure the absorbance at 500 nm using the same method and plot the standard curve.

[0048] Calculate the elemental selenium content in the supernatant based on the standard curve, and calculate the reduction rate:

[0049] Reduction rate (%) = (mass of elemental selenium / initial mass of sodium selenite) × 100%.

[0050] The experiment was repeated three times, and the average reduction efficiency of sodium selenite by LYB-S bacteria was measured to be 50.11% ± 6.03%.

[0051] 2. Characterization of nano-selenium

[0052] Purification of nano-selenium: The cultured bacterial solution was centrifuged at 8000 rpm for 15 min, and the precipitate was collected. The precipitate obtained by centrifugation with 150 mL of the cultured bacterial solution was then resuspended in 2 mL of sterile water to obtain LYB-S bacterial solution; the viable count of LYB-S in this bacterial solution was 10-1. 9 The concentration of nano-selenium was 15 g / L. The resuspended material (i.e. the obtained LYB-S bacterial solution) was transferred to pre-made sucrose density gradient tubes, which were prepared with sucrose of 50%, 60%, and 70% by mass. After centrifugation at 8000 r / min for 10 min, the red precipitate at the bottom of the centrifuge tube was collected, which is the nano-selenium reduced by LYB-S.

[0053] TEM scanning electron microscopy of nano-selenium: The collected red precipitate was dried in a vacuum freeze dryer for 48 h. Conductive adhesive was then applied to a copper stage of a scanning electron microscope (SEM). Small amounts of the lyophilized nano-selenium powder were placed on the conductive adhesive using a toothpick. After gold sputtering, the surface morphology of the nano-selenium particles was observed using SEM. Specific results are shown in [link to SEM document]. Figure 1 , Figure 1 This is a transmission scanning electron microscope image of nano-selenium.

[0054] The nano-selenium reduced by LYB-S appears as a uniform red color, as indicated by transmission scanning electron microscopy. Figure 1The nano-selenium particles are spherical with a diameter of approximately 100–200 nm. Nano-selenium particles with a diameter of 100–500 nm exhibit biological activity, which is beneficial for the absorption of selenium by plants, animals, or humans. Therefore, this synthesized nano-selenium possesses corresponding biological activity.

[0055] Example 2:

[0056] A compound microbial agent for selenium enrichment of rice, the preparation steps of which are as follows:

[0057] YD medium: sucrose 10 g / L, K2HPO4 2 g / L, (NH4)2SO4 1 g / L, MgSO4·7H2O 0.5 g / L, yeast extract 0.5 g / L, NaCl 0.1 g / L.

[0058] LB medium: tryptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L.

[0059] The preparation of selenium-reduced LYB-S bacterial suspension includes the following steps: LYB-S bacteria are transferred to YD medium (LYB-S-specific medium) for activation, and then cultured to the logarithmic growth phase. Next, the bacteria cultured to the logarithmic growth phase are inoculated at a 2% inoculation rate into YD medium with a sodium selenite concentration of 5 mmol / L, and cultured with shaking at 30 ℃ and 180 r / min for 48 h. During the culture, LYB-S bacteria reduce sodium selenite to prepare nano-selenium. The mixed culture medium solution of reduced nano-selenium and bacteria is centrifuged at 8000 rpm for 15 min, and the precipitate is collected. 150 mL of the culture (the material before centrifugation) is resuspended in 2 mL of sterile water to obtain the selenium-reduced LYB-S bacterial suspension, i.e., the LYB-S bacterial suspension. The viable LYB-S count in this suspension is 10-1. 9 CFU / g, nano selenium concentration is 15g / L.

[0060] The preparation of Bacillus subtilis bacterial suspension includes the following steps: Bacillus subtilis is inoculated into LB medium for activation, and then the activated strain is cultured to the logarithmic growth phase. The cultured strain in the logarithmic growth phase is then inoculated into LB liquid medium at a 2% inoculum size and cultured with shaking at 30 ℃ and 180 r / min for 36 h. The culture is then centrifuged at 8000 r / min for 15 min, and the precipitate is collected. The collected precipitate is resuspended in 2 mL of sterile water to obtain the Bacillus subtilis bacterial suspension; the viable count of Bacillus subtilis in the suspension is 10-1. 9 CFU / g.

[0061] Compound preparation: Xanthan gum was dissolved in sterile water to prepare a colloidal solution. The selenium-reducing LYB-S bacterial solution, Bacillus subtilis bacterial solution, and xanthan gum colloidal solution were stirred at 500 r / min for 15 min to ensure homogeneity, resulting in a mixed solution. Finally, nano-zinc oxide (30-50 nm particle size) and nano-iron oxide (30-50 nm particle size) were added sequentially to the mixed solution, stirred again until homogeneous, and the pH was adjusted to 7.2-7.5 to obtain the compound microbial agent for selenium enrichment of rice.

[0062] Compound ratio (by mass percentage): LYB-S bacterial solution 50% + Bacillus subtilis bacterial solution 25% + Xanthan gum 0.3% + Nano zinc oxide 2% + Nano iron oxide 1% + Sterile water 21.7%.

[0063] Example 3:

[0064] Field trials

[0065] The experiment was conducted in a paddy field in Shuangliu District, Chengdu City, Sichuan Province in 2025. A randomized block design was used, with three replicates for each treatment. Dikes were built between fields and mulched to prevent water and fertilizer cross-contamination. Sowing was in May, and transplanting in June, with a plant spacing of 12 cm × 30 cm. The compound microbial agent prepared in Example 2 was applied during the tillering and booting stages. Pure water spraying served as a control group, with three replicates for each treatment. Treatment groups were sprayed with the compound microbial agent (i.e., the compound microbial agent for selenium enrichment of rice obtained in Example 2), with three replicates. The compound microbial agent was mixed with water at a mass ratio of 1:9 and applied to the water layer, avoiding contamination of leaves and stems. A total of 10 L of the compound microbial agent-water mixture was applied per acre. At maturity, soil and grain samples were collected from each treatment field, with three replicates for each treatment, and relevant indicators were measured.

[0066] (1) Soil nutrient determination

[0067] (1.1) Determination of available phosphorus content in soil (NY / T 1121.7-2014):

[0068] Accurately weigh 2.50 g of air-dried soil sample that has passed through a 1 mm sieve, place it in a 150 mL Erlenmeyer flask, and add 50 mL of NaHCO3 extraction reagent; extract at 25 ℃ with shaking at 180 r / min for 30 min, then centrifuge at 4000 r / min for 10 min, and collect the supernatant for later use; take 5 mL of the supernatant into a 50 mL test tube, add 5 mL of molybdenum antimony anti-color developing agent, shake well, and let stand at 25 ℃ in the dark for 30 min; measure the absorbance at 700 nm wavelength using a 1 cm cuvette, and simultaneously prepare a blank control; respectively pipette 0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of 5 μg / mL phosphorus standard solution into 50 mL volumetric flasks, develop the color according to the above steps, measure the absorbance, and plot a standard curve.

[0069] Soil available phosphorus content (mg / kg) =

[0070] In the formula:

[0071] ρ = Phosphorus concentration (μg / mL) obtained from the standard curve;

[0072] V = Volume of colorimetric solution (mL);

[0073] D = Dispersion ratio, the ratio of the total volume of the extract to the volume of the extract;

[0074] m = sample mass (g)

[0075] The results are as follows Figure 2 As shown, Figure 2 A graph showing the available phosphorus content in paddy soil, from... Figure 2 It can be seen that the soil treated with the compound microbial agent of this invention had a readily available phosphorus content of 19.52 mg / kg, which was significantly higher than the blank control group of 10.53 mg / kg. Therefore, it can be concluded that the compound microbial agent can effectively dissolve fixed phosphorus in the soil, significantly improve the availability of phosphorus in the soil, and provide a better growing environment for rice.

[0076] (1.2) Determination of soil organic matter content (NY / T 1121.6-2006):

[0077] Weigh 0.3 g of air-dried soil sample through a 0.25 mm sieve into a 50 mL test tube; accurately add 10.0 mL of 0.8000 mol / L K2Cr2O7 standard solution, gently shake well, then add 10 mL of concentrated H2SO4 along the tube wall and shake quickly; place the test tube in an oil bath at 170-180℃, start timing when the temperature rises back to 170℃, maintain boiling for 5 min, remove and cool; transfer to a 250 mL Erlenmeyer flask, rinse the test tube wall with about 100 mL of distilled water, and add 3 drops of o-phenanthroline indicator;

[0078] Titrate with 0.2 mol / L FeSO4 standard solution until the solution changes from orange-yellow to blue-green and then to brick-red, and record the volume consumed. For the blank test, take an equal volume of distilled water instead of soil sample, follow the above steps, and record the volume of FeSO4 consumed.

[0079] Soil organic matter content (g / kg) =

[0080] In the formula:

[0081] c = concentration of FeSO4 standard solution (mol / L);

[0082] V0 = Volume of FeSO4 consumed in the blank test (mL);

[0083] V = Volume of FeSO4 consumed by the sample (mL);

[0084] 0.003 = one-quarter of the millimolecular mass of a carbon atom (g);

[0085] 1.724 = the coefficient for converting organic carbon to organic matter;

[0086] 1.1 = Oxidation correction factor

[0087] m = sample mass (g);

[0088] The results are as follows Figure 3 As shown, Figure 3 This is a graph showing the organic matter content of rice paddy soil. Figure 3 As shown, the soil treated with the compound microbial agent of this invention had an organic matter content of 18.71 g / kg, significantly higher than the 15.02 g / kg in the blank control group. Combined with the previous data on increased available phosphorus, it can be seen that the compound microbial agent of this invention not only activates nutrients such as selenium and phosphorus, but also increases soil organic matter, comprehensively improving soil fertility and ecological function, and promoting crop growth.

[0089] (2) Agronomic traits of the plant

[0090] To investigate the effects of compound microbial inoculants on the agronomic traits of mature rice, plant height and panicle height were measured with a measuring tape after the rice plants matured, and the number of panicles and grains per mature rice seedling was counted. The mature grains were then removed, air-dried naturally, and stored at room temperature for later use.

[0091] To evaluate the effects of the compound microbial inoculant of this invention on rice growth and yield, relevant agronomic traits of rice were measured at maturity. The results are as follows: Figure 4 As shown, Figure 4 This is a graph showing the effects of compound microbial inoculants on rice growth and yield. Figure 4It can be seen that, compared with the blank control, rice treated with the compound microbial agent of this invention showed increased plant height, panicle length, number of grains per panicle, and panicle weight, with panicle length increasing by 8.88% and panicle weight increasing significantly by 22.64%. Compared with no agent, the compound microbial agent of this invention can significantly improve rice growth indicators such as panicle length, and increase the number of grains per panicle and panicle weight, thereby achieving yield improvement.

[0092] (3) Selenium content in rice

[0093] The selenium content of rice from different treatments was determined using GB 5009.93-2017 "National Food Safety Standard - Determination of Selenium in Food" (Method III). Specific results are shown in [link to results]. Figure 5 , Figure 5 This is a graph showing the relationship between the selenium content in rice treated with compound microbial agents and rice in the control group.

[0094] Depend on Figure 5 It can be seen that the total selenium content of rice treated with this compound microbial agent reached 155.3 μg / kg, which is significantly higher than the blank control of 33.98 μg / kg, and meets the national standard for selenium-enriched rice (150 μg / kg).

[0095] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

[0096] This background section is provided to generally present the context of the invention. The work of the currently named inventors, the work to the extent described in this background section, and aspects described in this section that did not constitute prior art at the time of application are neither expressly nor impliedly acknowledged as prior art to the invention.

Claims

1. A compound microbial inoculant for selenium enrichment in rice, characterized in that: The product comprises selenium-reduced LYB-S bacterial solution, Bacillus subtilis bacterial solution, xanthan gum, nano zinc oxide, nano iron oxide, and water; wherein, by mass percentage, the addition amount of selenium-reduced LYB-S bacterial solution is 40%-60%, the addition amount of Bacillus subtilis bacterial solution is 20%-30%, the addition amount of xanthan gum is 0.1%-0.5%, the addition amount of nano zinc oxide is 1%-3%, the addition amount of nano iron oxide is 0.5%-2%, and the balance is water, with a total mass percentage of 100%; the selenium-reduced LYB-S bacterial solution is Bacillus belye (… Bacillus velezensis LYB-S bacterial solution, Bacillus belyss ( Bacillus velezensis The preservation number of LYB-S is CGMCC NO.34429; the preparation method of the LYB-S bacterial culture includes the following steps: The LYB-S strain was transferred to YD medium for activation, and then cultured to the logarithmic growth phase. The cultured strain was then inoculated into YD medium containing sodium selenite and cultured. The LYB-S strain reduced sodium selenite to obtain nano-selenium. The cultured LYB-S strain and the nano-selenium produced were centrifuged, and the precipitate was collected. The precipitate was resuspended in sterile water to obtain the LYB-S bacterial culture.

2. The compound microbial agent for selenium enrichment of rice according to claim 1, characterized in that: The particle size of the nano zinc oxide and nano iron oxide is 30-50 nm.

3. The compound microbial agent for selenium enrichment in rice according to claim 1, characterized in that, The preparation method of Bacillus subtilis bacterial culture includes the following steps: Bacillus subtilis was activated by inoculating it into LB medium. The activated strain was then cultured to the logarithmic growth phase, followed by inoculation into LB liquid medium with shaking culture. The precipitate was collected by centrifugation. Finally, the precipitate was resuspended in sterile water to obtain the Bacillus subtilis bacterial suspension. The viable count of Bacillus subtilis in the suspension was 10-1. 8 -10 10 CFU / g.

4. The compound microbial agent for selenium enrichment of rice according to claim 1, characterized in that: The YD medium is a dedicated LYB-S medium; the formula of the LYB-S medium is: sucrose 10 g / L, K2HPO4 2 g / L, (NH4)2SO4 1 g / L, MgSO4·7H2O 0.5 g / L, yeast extract 0.5 g / L, NaCl 0.1 g / L; the inoculation ratio of the strain cultured to the logarithmic phase is 2%; the concentration of sodium selenite in the YD medium containing sodium selenite is 5 mmol / L; the centrifugation conditions are 8000 r / min, 15 min.

5. The compound microbial agent for selenium enrichment of rice according to claim 3, characterized in that: The inoculation ratio of the strain cultured to the logarithmic growth phase was 2%; the shaking culture conditions were 30 ℃, 180 r / min, and 36 h; the centrifugation conditions were 8000 r / min and 15 min; the viable count in the Bacillus subtilis culture was 10-1. 8 -10 10 CFU / g.

6. A method for preparing a compound microbial agent for selenium enrichment of rice according to any one of claims 1-5, characterized in that... Includes the following steps: First, xanthan gum is dissolved in sterile water to prepare a colloidal solution. Then, the obtained colloidal solution, selenium-reduced LYB-S bacterial solution, and Bacillus subtilis bacterial solution are stirred and mixed evenly in proportion to obtain a mixed solution. Finally, nano zinc oxide and nano iron oxide are added to the mixed solution in sequence, stirred and mixed evenly again, and the pH is adjusted.

7. The method for preparing a compound microbial agent for selenium enrichment in rice according to claim 6, characterized in that: All stirring conditions were 500 r / min for 15 min; pH was adjusted to 7.2-7.

5.

8. The application of the compound microbial agent for selenium enrichment of rice according to any one of claims 1-5 in improving the selenium content of rice.

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