Bacterial fertilizer for improving disease resistance of perennial rice as well as preparation method and application of bacterial fertilizer

Abstract

The invention discloses a bacterial fertilizer for improving the disease resistance of perennial rice as well as a preparation method and application of the bacterial fertilizer, and belongs to the technical field of compound microbial fertilizers. The bacterial fertilizer disclosed by the invention is prepared from 200 parts of a compound microbial agent, 50 to 60 parts of humic acid, 50 to 60 parts of urea, 70 to 80 parts of ammonium polyphosphate, 70 to 80 parts of mineral source potassium fulvate, 40 to 50 parts of potassium silicate, 40 to 50 parts of potassium magnesium sulfate, 300 to 350 parts of decomposed straw carbon, 1.0 to 1.5 parts of L-cysteine, according to the bacterial fertilizer, base fertilizer is deeply ploughed in the first season, and the fertilizing amount is 30-40 kg / mu; before overwintering, topdressing is broadcast, and the topdressing amount is 10-20 kg / mu; topdressing is conducted during tillering in the regeneration season, and the topdressing amount is 8-10 kg / mu; according to the method, the interspecific competition problem is reduced, the spore germination rate is increased, the prevention and control period is prolonged, the sheath blight resistance, rice blast resistance and bacterial blight resistance of perennial rice are improved, and the effects of increasing the yield and increasing the effective tiller number can be achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of composite microbial fertilizer, in particular to a kind of fungus fertilizer for improving the disease resistance of perennial rice and its preparation method and application. BACKGROUND

[0002] As an innovative rice planting mode, perennial rice significantly reduces labor, seed and tillage costs through one-time planting and multi-year harvesting, and has important economic and ecological values. However, its multi-year continuous growth characteristics also bring some application problems. For example, pathogenic bacteria and pest eggs and larvae in the soil are easy to accumulate year by year, which can lead to a significantly higher incidence of soil-borne diseases than annual rice. Root aging, nutrient absorption efficiency decline and soil micro-ecological imbalance further weaken the plant's own disease resistance, seriously threatening the sustainable high yield and stable yield of perennial rice.

[0003] Fungus fertilizer has the advantages of improving soil, promoting plant growth, improving nutrient utilization efficiency and enhancing plant systemic disease resistance. Among them, growth-promoting and disease-preventing fungus fertilizer is a key branch of microbial fertilizer. Using beneficial microorganisms to improve the disease resistance of perennial rice has been widely studied. For example, Bacillus subtilis is widely used in the production of fungus fertilizer for various crops due to its strong stress resistance and broad-spectrum antibacterial effect. However, single fungus fertilizer often has problems such as strain specificity and insufficient adaptability, single strain effect limitation and unstable disease prevention effect. Therefore, it is necessary to develop a new type of composite fungus fertilizer that is efficient, stable, specific, can comprehensively cope with the main disease pressure of perennial rice and adapt to its multi-year growth characteristics. SUMMARY

[0004] The purpose of the present application is to provide a fungus fertilizer for improving the disease resistance of perennial rice and its preparation method and application to solve the problems existing in the prior art. In order to improve the stability and disease resistance of fungus fertilizer, the present application provides a composite fungus fertilizer of Bacillus subtilis and Trichoderma harzianum.

[0005] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0006] The present application provides a fungus fertilizer for improving the disease resistance of perennial rice. The key is that the fungus fertilizer includes 200 parts of composite microbial agent, 50-60 parts of humic acid, 50-60 parts of urea, 70-80 parts of ammonium polyphosphate, 70-80 parts of potassium yellow humate, 40-50 parts of potassium silicate, 40-50 parts of potassium magnesium sulfate, 300-350 parts of composted straw charcoal and 1.0-1.5 parts of L-cysteine, by weight.

[0007] Specifically, the composite microbial agent consists of Bacillus subtilis agent and Trichoderma harzianum agent in a mass ratio of 1:1.1-1.3; and the ammonium polyphosphate is water-soluble ammonium polyphosphate.

[0008] Preferably, the above-mentioned Bacillus subtilis inoculant is a Bacillus subtilis dry powder inoculant embedded in aburro polysaccharide-sodium alginate hydrogel; and the above-mentioned Trichoderma harzianum inoculant is a Trichoderma harzianum dry powder inoculant embedded in sodium alginate hydrogel.

[0009] Further, the preparation process of the above-mentioned Bacillus subtilis dry powder inoculant is as follows:

[0010] S11, fermentation of Bacillus subtilis:

[0011] Commercially available Bacillus subtilis powder is activated to obtain a Bacillus subtilis seed liquid; then the Bacillus subtilis seed liquid is inoculated into a Bacillus subtilis fermentation medium at a volume ratio of 10% to 12%, and the pH value is 6.8 to 7.2, and the fermentation is carried out at 32°C to 37°C; after the Bacillus subtilis is fermented to the late logarithmic phase, the bacterial cells are collected and resuspended in physiological saline, and the bacterial cell concentration is adjusted to 10 9 ~10 10 CFU / mL to obtain a Bacillus subtilis bacterial suspension;

[0012] S12, embedding of Bacillus subtilis:

[0013] Aburro polysaccharide and chloroacetic acid are mixed in isopropyl alcohol at a mass ratio of 1:1.1 to 1.3, and reacted at 55°C to 65°C for 4 to 6 hours; after neutralization, dialysis purification is performed to obtain carboxymethylated aburro polysaccharide;

[0014] The above-mentioned carboxymethylated aburro polysaccharide and sodium alginate are mixed at a mass ratio of 1:10 to 15, and dissolved in water to obtain an aburro polysaccharide-sodium alginate mixed solution;

[0015] The Bacillus subtilis bacterial suspension is added to the aburro polysaccharide-sodium alginate mixed solution at a volume ratio of 2% to 3%, and after stirring and mixing, a calcium chloride solution is added dropwise; after the dropwise addition is completed, the mixture is allowed to stand for 20 to 30 minutes to obtain a mixed solution containing Bacillus subtilis microspheres;

[0016] S13, freeze-drying of the Bacillus subtilis dry powder inoculant:

[0017] Trehalose, glycerol and nano-silicon oxide are added to the mixed solution containing Bacillus subtilis microspheres, pre-cooled at 4°C to 6°C for 12 to 14 hours, then rapidly frozen at -45°C to -40°C for 1.5 to 2.0 hours, and finally vacuum freeze-dried at -55°C to -50°C and 0.1 mbar to 0.2 mbar for 20 to 24 hours to obtain the above-mentioned Bacillus subtilis dry powder inoculant.

[0018] Preferably, the amount of isopropanol used is 10 to 12 times the volume of okra polysaccharide; the amount of water used is 30 to 40 times the volume of carboxymethylated okra polysaccharide; the mass concentration of the calcium chloride solution is 2% to 4%, and the amount of calcium chloride solution used is 1 to 2 times the volume of the okra polysaccharide-sodium alginate mixed solution; the mass ratio of the mixed solution containing Bacillus subtilis microspheres, trehalose, glycerol, and nano-silicon oxide is 100:8 to 12:1.5 to 2.0:0.1 to 0.2.

[0019] Furthermore, the preparation process of the Trichoderma harzianum dry powder fungicide is as follows:

[0020] S21, fermentation of Trichoderma harzianum:

[0021] Commercial Trichoderma harzianum powder is activated to obtain Trichoderma harzianum seed liquid; the Trichoderma harzianum seed liquid is inoculated into Trichoderma harzianum fermentation medium at an inoculation amount of 5% to 8% by volume, the pH value is 6.8 to 7.2, and fermentation is carried out at 32°C to 37°C; after the Trichoderma harzianum is fermented to the late logarithmic phase, the bacterial cells are collected and resuspended in physiological saline to adjust the bacterial cell concentration to 10 9 ~ 10 10 CFU / mL to obtain a Trichoderma harzianum bacterial suspension;

[0022] S22, embedding of Trichoderma harzianum:

[0023] The Trichoderma harzianum bacterial suspension is added to a sodium alginate solution at a volume ratio of 2% to 3%, rice husk ash is added, and after stirring and mixing, a calcium chloride solution is added dropwise; after the dropwise addition is completed, it is left to stand for 1 to 2 hours to obtain a mixed solution containing Trichoderma harzianum microspheres;

[0024] S23, freeze-drying of the Trichoderma harzianum dry powder fungicide:

[0025] Non-ionic surfactant, trehalose, glycerol, and nano-silicon oxide are added to the mixed solution containing Trichoderma harzianum microspheres, pre-cooled at 4°C to 6°C for 12 to 14 hours, then rapidly frozen at -45°C to -40°C for 1.5 to 2.0 hours, and finally vacuum freeze-dried at -55°C to -50°C and 0.1 mbar to 0.2 mbar for 20 to 24 hours to obtain the Trichoderma harzianum dry powder fungicide.

[0026] Preferably, the mass concentration of the calcium chloride solution is 2% to 4%, the amount of calcium chloride solution used is 1 to 2 times the volume of the sodium alginate solution, and the mass concentration of the sodium alginate solution is 2.5% to 3.5%; the mass ratio of the mixture containing Trichoderma harzianum microspheres, nonionic surfactant, trehalose, glycerol, and nano-silica is 100:3 to 8:8 to 12:1.5 to 2.0:0.1 to 0.2, and the nonionic surfactant includes any one of Tween 80, Tween 60, or soybean lecithin; the amount of rice husk ash used is 5% to 10% of the mass of the Trichoderma harzianum suspension.

[0027] A method for preparing a microbial fertilizer to improve the disease resistance of perennial rice involves, by weight, mixing and packaging 200 parts of compound microbial agent, 50-60 parts of humic acid, 50-60 parts of urea, 70-80 parts of ammonium polyphosphate, 70-80 parts of mineral-derived potassium fulvate, 40-50 parts of potassium silicate, 40-50 parts of potassium magnesium sulfate and 300-350 parts of decomposed straw charcoal to obtain component A, and separately packaging 1.0-1.5 parts of L-cysteine ​​to obtain component B.

[0028] The key to the application of a microbial fertilizer that enhances the disease resistance of perennial rice lies in the fact that the aforementioned microbial fertilizer is used to improve the resistance of perennial rice to sheath blight, rice blast, and bacterial blight.

[0029] Specifically, before fertilization, components A and B are mixed. For the first season's base fertilizer, the soil is deeply tilled to a depth of 8-10 cm, and the amount of the above-mentioned microbial fertilizer applied as base fertilizer is 30-40 kg / mu. Before winter, topdressing is applied by broadcasting, and the amount of the above-mentioned microbial fertilizer applied before winter is 10-20 kg / mu. During the regeneration season, topdressing is applied by broadcasting during tillering, and the amount of the above-mentioned microbial fertilizer applied during the regeneration season is 8-10 kg / mu.

[0030] The present invention discloses the following technical effects:

[0031] The microbial fertilizer of this invention uses a compound microbial agent of Bacillus subtilis and Trichoderma harzianum, and single-species encapsulation is carried out according to the different characteristics of different microbial species. Specifically, Bacillus subtilis is encapsulated in okra polysaccharide-sodium alginate hydrogel, and Trichoderma harzianum loaded with rice husk ash is encapsulated in sodium alginate. This not only forms physical isolation, but also forms a spatiotemporal synergistic effect between the two. That is, Bacillus subtilis plays a leading role in prevention and control, while Trichoderma harzianum plays a continuous role, covering the threat of diseases throughout the entire growth period.

[0032] The microbial fertilizer of this invention not only has a scientifically formulated composition of basic elements such as N, P, K, and C, but its components also have certain synergistic effects. For example, L-cysteine ​​can enhance the colonization ability of bacterial communities. Adding it before fertilization can activate dormant cells and increase spore germination rate. In addition, the combination of L-cysteine ​​and potassium silicate can also improve the lodging resistance of rice. Okra polysaccharide encapsulated with Bacillus subtilis cross-links with sodium alginate to form a pH-responsive gel, which slowly releases live bacteria in the acidic environment of the roots, prolonging the action period. At the same time, okra polysaccharide can also act as a plant immune elicitor, activate the rice PR protein pathway, and enhance systemic resistance. Trichoderma harzianum loaded with rice husk ash not only increases the oxygen flux of the encapsulated Trichoderma harzianum microspheres, but the porous structure of rice husk ash can also efficiently adsorb hyphae such as those of sheath blight, providing "targets" for Trichoderma harzianum, increasing its chitinase secretion, accelerating the decomposition of parasitic pathogen cell walls, and the active silicon dissolved from rice husk ash enhances the silicification of rice cell walls, forming a physical barrier, which, together with the parasitic effect of Trichoderma harzianum, forms a "physical + biological" dual defense.

[0033] In summary, this invention significantly reduces interspecific competition between Bacillus subtilis and Trichoderma harzianum, increases the spore germination rate of Trichoderma harzianum, enhances the synergistic effect between the two, and extends the control cycle. It demonstrates that it can improve the resistance of perennial rice to sheath blight, rice blast, and bacterial blight in both the first and second cropping seasons, and can also increase yield and the number of effective tillers, ultimately achieving the green planting goal of "fewer diseases and higher yield". Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Example 1

[0040] In this embodiment, the preparation of Bacillus subtilis dry powder inoculum is carried out. The specific process is as follows:

[0041] S11, Fermentation by Bacillus subtilis:

[0042] Commercially available Bacillus subtilis powder was activated to obtain Bacillus subtilis seed culture. The Bacillus subtilis seed culture was then inoculated into Bacillus subtilis fermentation medium at a volume ratio of 11%, with a pH of 7.0. Fermentation was carried out at 35°C. Once the Bacillus subtilis reached the late logarithmic fermentation stage, the cells were collected, resuspended in physiological saline, and the cell concentration was adjusted to 3.6 × 10⁻⁶. 9 CFU / mL was used to obtain a Bacillus subtilis suspension.

[0043] S12, Encapsulation of Bacillus subtilis:

[0044] Okra polysaccharide and chloroacetic acid were mixed in isopropanol at a mass ratio of 1:1.2, wherein the amount of isopropanol used was 11 times the volume of okra polysaccharide; the mixture was reacted at 60°C for 5 h, and after neutralization, it was purified by dialysis to obtain carboxymethylated okra polysaccharide.

[0045] Carboxymethylated okra polysaccharide and sodium alginate were mixed at a mass ratio of 1:12, dissolved in water, and the amount of water used was 35 times the volume of carboxymethylated okra polysaccharide. The mixture was stirred and mixed evenly to obtain an okra polysaccharide-sodium alginate mixture.

[0046] Bacillus subtilis suspension was added to okra polysaccharide-sodium alginate mixture at a volume ratio of 2.5%. After stirring and mixing, calcium chloride solution with a mass concentration of 3% was added dropwise. The amount of calcium chloride solution used was 1.5 times the volume of okra polysaccharide-sodium alginate mixture. After the addition was completed, the mixture was allowed to stand for 25 minutes to obtain a mixture containing Bacillus subtilis microspheres.

[0047] S13, Freeze-drying of Bacillus subtilis dry powder inoculum:

[0048] Trehalose, glycerol, and nano-silica were added to a mixture containing Bacillus subtilis microspheres. The mass ratio of the mixture containing Bacillus subtilis microspheres, trehalose, glycerol, and nano-silica was 100:10:1.8:0.15.

[0049] Pre-cooled at 5℃ for 13h, then quick-frozen at -42℃ for 1h45min, and finally freeze-dried under vacuum at -52℃ and 0.15mbar for 22h to obtain Bacillus subtilis dry powder inoculant, which is recorded as Bacillus subtilis dry powder inoculant sample 1.

[0050] Example 2

[0051] In this embodiment, the preparation of Bacillus subtilis dry powder inoculum is carried out. The specific process is as follows:

[0052] S11, Fermentation by Bacillus subtilis:

[0053] Commercially available Bacillus subtilis powder was activated to obtain Bacillus subtilis seed culture. The Bacillus subtilis seed culture was then inoculated into Bacillus subtilis fermentation medium at a volume ratio of 10%, with a pH of 7.2. Fermentation was carried out at 32℃. After the Bacillus subtilis reached the late logarithmic fermentation stage, the cells were collected, resuspended in physiological saline, and the cell concentration was adjusted to 9.8 × 10⁻⁶. 9 CFU / mL was used to obtain a Bacillus subtilis suspension.

[0054] S12, Encapsulation of Bacillus subtilis:

[0055] Okra polysaccharide and chloroacetic acid were mixed in isopropanol at a mass ratio of 1:1.3, wherein the amount of isopropanol used was 10 times the volume of okra polysaccharide; the mixture was reacted at 55°C for 6 hours, and after neutralization, it was purified by dialysis to obtain carboxymethylated okra polysaccharide.

[0056] Carboxymethylated okra polysaccharide and sodium alginate were mixed at a mass ratio of 1:10, dissolved in water, and the amount of water used was 40 times the volume of carboxymethylated okra polysaccharide. The mixture was stirred and mixed evenly to obtain an okra polysaccharide-sodium alginate mixture.

[0057] Add 3% by volume of Bacillus subtilis suspension to okra polysaccharide-sodium alginate mixture, stir and mix, then add 2% calcium chloride solution dropwise. The amount of calcium chloride solution used is twice the volume of okra polysaccharide-sodium alginate mixture. After the addition is complete, let stand for 30 minutes to obtain a mixture containing Bacillus subtilis microspheres.

[0058] S13, Freeze-drying of Bacillus subtilis dry powder inoculum:

[0059] Trehalose, glycerol, and nano-silica were added to a mixture containing Bacillus subtilis microspheres. The mass ratio of the mixture containing Bacillus subtilis microspheres, trehalose, glycerol, and nano-silica was 100:8:2.0:0.1.

[0060] Pre-cooled at 4℃ for 12h, then quick-frozen at -40℃ for 2.0h, and finally freeze-dried under vacuum at -55℃ and 0.1mbar for 20h to obtain Bacillus subtilis dry powder inoculant, which is recorded as Bacillus subtilis dry powder inoculant sample 2.

[0061] Example 3

[0062] In this embodiment, the preparation of Bacillus subtilis dry powder inoculum is carried out. The specific process is as follows:

[0063] S11, Fermentation by Bacillus subtilis:

[0064] Commercially available Bacillus subtilis powder was activated to obtain Bacillus subtilis seed culture. The Bacillus subtilis seed culture was then inoculated into Bacillus subtilis fermentation medium at a volume ratio of 12%, with a pH of 6.8, and fermented at 37°C. After the Bacillus subtilis reached the late logarithmic fermentation stage, the cells were collected, resuspended in physiological saline, and the cell concentration was adjusted to 1.2 × 10⁻⁶. 9 CFU / mL was used to obtain a Bacillus subtilis suspension.

[0065] S12, Encapsulation of Bacillus subtilis:

[0066] Okra polysaccharide and chloroacetic acid were mixed in isopropanol at a mass ratio of 1:1.1, wherein the amount of isopropanol used was 12 times the volume of okra polysaccharide; the mixture was reacted at 65°C for 4 hours, and after neutralization, it was purified by dialysis to obtain carboxymethylated okra polysaccharide.

[0067] Carboxymethylated okra polysaccharide and sodium alginate were mixed at a mass ratio of 1:15, dissolved in water, and the amount of water used was 30 times the volume of carboxymethylated okra polysaccharide. The mixture was stirred and mixed evenly to obtain an okra polysaccharide-sodium alginate mixture.

[0068] Bacillus subtilis suspension was added to okra polysaccharide-sodium alginate mixture at a volume ratio of 2%. After stirring and mixing, calcium chloride solution with a mass concentration of 4% was added dropwise. The amount of calcium chloride solution used was 1 times the volume of okra polysaccharide-sodium alginate mixture. After the addition was completed, the mixture was allowed to stand for 20 minutes to obtain a mixture containing Bacillus subtilis microspheres.

[0069] S13, Freeze-drying of Bacillus subtilis dry powder inoculum:

[0070] Trehalose, glycerol, and nano-silica were added to a mixture containing Bacillus subtilis microspheres. The mass ratio of the mixture containing Bacillus subtilis microspheres, trehalose, glycerol, and nano-silica was 100:12:1.5:0.2.

[0071] Pre-cooled at 6℃ for 14h, then quick-frozen at -45℃ for 1.5h, and finally freeze-dried under vacuum at -50℃ and 0.2mbar for 24h to obtain Bacillus subtilis dry powder inoculant, which is recorded as Bacillus subtilis dry powder inoculant sample 3.

[0072] Example 4

[0073] In this embodiment, the preparation of Trichoderma harzianum dry powder inoculant is carried out, and the specific process is as follows:

[0074] S21, Fermentation by Trichoderma harzianum:

[0075] Commercially available *Trichoderma harzianum* powder was activated to obtain *Trichoderma harzianum* seed culture. This seed culture was then inoculated into *Trichoderma harzianum* fermentation medium at a volume ratio of 6.5%, with a pH of 7.0. Fermentation was carried out at 35°C. Once the *Trichoderma harzianum* reached the late logarithmic fermentation stage, the cells were collected, resuspended in physiological saline, and the cell concentration was adjusted to 2.6 × 10⁻⁶. 9 CFU / mL was used to obtain a Trichoderma harzianum suspension.

[0076] S22, Embedding of Trichoderma harzianum:

[0077] The Trichoderma harzianum suspension was added to a 3.0% sodium alginate solution at a volume ratio of 2.5%. Rice husk ash (8% of the mass of the Trichoderma harzianum suspension) was added, and the mixture was stirred. Then, a 2.8% calcium chloride solution was added dropwise. The volume of calcium chloride solution used was 1.5 times that of the sodium alginate solution. After the addition was completed, the mixture was allowed to stand for 1.5 hours to obtain a mixture containing Trichoderma harzianum microspheres.

[0078] S23, Freeze-drying of Trichoderma harzianum dry powder inoculum:

[0079] Nonionic surfactants soybean lecithin, trehalose, glycerol, and nano-silica were added to a mixture containing Trichoderma harzianum microspheres. The mass ratio of the mixture containing Trichoderma harzianum microspheres, nonionic surfactants, trehalose, glycerol, and nano-silica was 100:5:10:1.8:0.15.

[0080] Pre-cooled at 5℃ for 13h, then quick-frozen at -42℃ for 1h40min, and finally freeze-dried under vacuum at -52℃ and 0.15mbar for 22h to obtain Trichoderma harzianum dry powder inoculant, which is designated as Trichoderma harzianum dry powder inoculant sample 1.

[0081] Example 5

[0082] In this embodiment, the preparation of Trichoderma harzianum dry powder inoculant is carried out, and the specific process is as follows:

[0083] S21, Fermentation by Trichoderma harzianum:

[0084] Commercially available *Trichoderma harzianum* powder was activated to obtain *Trichoderma harzianum* seed culture. The seed culture was then inoculated into *Trichoderma harzianum* fermentation medium at a volume ratio of 8%, with a pH of 6.8, and fermented at 37°C. After fermentation reached the late logarithmic phase, the cells were collected, resuspended in physiological saline, and the cell concentration was adjusted to 9.6 × 10⁻⁶. 9 CFU / mL was used to obtain a Trichoderma harzianum suspension.

[0085] S22, Embedding of Trichoderma harzianum:

[0086] Add 2% by volume of Trichoderma harzianum suspension to a 3.5% sodium alginate solution, add 5% by mass of rice husk ash of Trichoderma harzianum suspension, stir and mix, then add dropwise a 2% calcium chloride solution. The amount of calcium chloride solution used is 1 times the volume of sodium alginate solution. After the addition is complete, let stand for 2 hours to obtain a mixture containing Trichoderma harzianum microspheres.

[0087] S23, Freeze-drying of Trichoderma harzianum dry powder inoculum:

[0088] Nonionic surfactant Tween 80, trehalose, glycerol, and nano-silica were added to a mixture containing Trichoderma harzianum microspheres. The mass ratio of the mixture containing Trichoderma harzianum microspheres, nonionic surfactant, trehalose, glycerol, and nano-silica was 100:3:12:2.0:0.1.

[0089] Pre-cooled at 4℃ for 14h, then quick-frozen at -40℃ for 2.0h, and finally freeze-dried under vacuum at -50℃ and 0.2mbar for 20h to obtain Trichoderma harzianum dry powder inoculant, which is designated as Trichoderma harzianum dry powder inoculant sample 2.

[0090] Example 6

[0091] In this embodiment, the preparation of Trichoderma harzianum dry powder inoculant is carried out, and the specific process is as follows:

[0092] S21, Fermentation by Trichoderma harzianum:

[0093] Commercially available *Trichoderma harzianum* powder was activated to obtain *Trichoderma harzianum* seed culture. This seed culture was then inoculated into *Trichoderma harzianum* fermentation medium at a volume ratio of 5%, with a pH of 7.2. Fermentation was carried out at 32°C. Once the fermentation reached the late logarithmic phase, the cells were collected, resuspended in physiological saline, and the cell concentration was adjusted to 1.4 × 10⁻⁶. 9 CFU / mL was used to obtain a Trichoderma harzianum suspension.

[0094] S22, Embedding of Trichoderma harzianum:

[0095] Add 3% by volume of Trichoderma harzianum suspension to a 2.5% sodium alginate solution, add 10% by mass of rice husk ash of Trichoderma harzianum suspension, stir and mix, then add dropwise a 4% calcium chloride solution. The amount of calcium chloride solution used is twice the volume of the sodium alginate solution. After the addition is complete, let stand for 1 hour to obtain a mixture containing Trichoderma harzianum microspheres.

[0096] S23, Freeze-drying of Trichoderma harzianum dry powder inoculum:

[0097] Nonionic surfactant Tween 60, trehalose, glycerol, and nano-silica were added to a mixture containing Trichoderma harzianum microspheres. The mass ratio of the mixture containing Trichoderma harzianum microspheres, nonionic surfactant, trehalose, glycerol, and nano-silica was 100:8:8:1.5:0.2.

[0098] Pre-cooled at 6℃ for 12h, then quick-frozen at -45℃ for 1.5h, and finally freeze-dried under vacuum at -50℃ and 0.2mbar for 24h to obtain Trichoderma harzianum dry powder inoculant, which is designated as Trichoderma harzianum dry powder inoculant sample 3.

[0099] Example 7

[0100] This embodiment describes the preparation of a microbial fertilizer to enhance the disease resistance of perennial rice. The specific process is as follows:

[0101] 200 parts of compound microbial inoculant, 55 parts of humic acid, 55 parts of urea, 75 parts of water-soluble ammonium polyphosphate, 75 parts of mineral-derived potassium fulvate, 45 parts of potassium silicate, 45 parts of potassium magnesium sulfate and 325 parts of decomposed straw charcoal were weighed and placed in a mixing tank. The stirring speed was controlled at 120 r / min and the stirring time was 15 min. After mixing, the mixture was packaged to obtain component A of the microbial fertilizer.

[0102] In this embodiment, the 200-part compound microbial agent consists of 90 parts of Bacillus subtilis dry powder agent sample 1 and 110 parts of Trichoderma harzianum dry powder agent sample 1.

[0103] 1.2 portions of L-cysteine ​​are packaged separately as component B of the microbial fertilizer;

[0104] Components A and B together form the prepared microbial fertilizer, denoted as microbial fertilizer sample 1.

[0105] Example 8

[0106] This embodiment describes the preparation of a microbial fertilizer to enhance the disease resistance of perennial rice. The specific process is as follows:

[0107] 200 parts of compound microbial inoculant, 60 parts of humic acid, 50 parts of urea, 80 parts of water-soluble ammonium polyphosphate, 70 parts of mineral-derived potassium fulvate, 40 parts of potassium silicate, 50 parts of potassium magnesium sulfate and 350 parts of decomposed straw charcoal were weighed and placed in a mixing tank. The stirring speed was controlled at 100 r / min and the stirring time was 20 min. After mixing, the mixture was packaged to obtain component A of the microbial fertilizer.

[0108] In this embodiment, the 200 samples of compound microbial inoculant consist of 87 samples of Bacillus subtilis dry powder inoculant sample 2 and 113 samples of Trichoderma harzianum dry powder inoculant sample 3.

[0109] 1.5 portions of L-cysteine ​​are packaged separately as component B of the microbial fertilizer;

[0110] Components A and B together form the prepared microbial fertilizer, which is designated as microbial fertilizer sample 2.

[0111] Example 9

[0112] This embodiment describes the preparation of a microbial fertilizer to enhance the disease resistance of perennial rice. The specific process is as follows:

[0113] Weigh out 200 parts of compound microbial inoculant, 50 parts of humic acid, 60 parts of urea, 70 parts of water-soluble ammonium polyphosphate, 80 parts of mineral-derived potassium fulvate, 50 parts of potassium silicate, 40 parts of potassium magnesium sulfate and 300 parts of decomposed straw charcoal, place them in a mixing tank, control the stirring speed at 150 r / min and the stirring time at 8 min, mix well and then package to obtain component A of the microbial fertilizer;

[0114] In this embodiment, the 200-part compound microbial agent consists of 95 parts of Bacillus subtilis dry powder agent sample 3 and 105 parts of Trichoderma harzianum dry powder agent sample 2.

[0115] 1.0 part of L-cysteine ​​is packaged separately as component B of the microbial fertilizer;

[0116] Components A and B together form the prepared microbial fertilizer, which is designated as microbial fertilizer sample 3.

[0117] Comparative Example 1

[0118] The preparation process of the microbial fertilizer to improve the disease resistance of perennial rice in this comparative example is as follows:

[0119] 200 parts of the comparative compound microbial agent 1, 55 parts of humic acid, 55 parts of urea, 75 parts of water-soluble ammonium polyphosphate, 75 parts of mineral-derived potassium fulvate, 45 parts of potassium silicate, 45 parts of potassium magnesium sulfate and 325 parts of decomposed straw charcoal were weighed and placed in a mixing tank. The stirring speed was controlled at 120 r / min and the stirring time was 15 min. After mixing, the mixture was packaged to obtain component A of the microbial fertilizer.

[0120] Among them, the 200 comparative compound microbial agent 1 consisted of 90 parts of ordinary commercially available Bacillus subtilis powder and 110 parts of ordinary commercially available Trichoderma harzianum powder, and the effective substance mass of the commercially available Bacillus subtilis powder and Trichoderma harzianum powder used was >99%.

[0121] 1.2 portions of L-cysteine ​​are packaged separately as component B of the microbial fertilizer;

[0122] Components A and B together form the prepared microbial fertilizer, denoted as microbial fertilizer control standard 1.

[0123] Comparative Example 2

[0124] The preparation process of the microbial fertilizer to improve the disease resistance of perennial rice in this comparative example is as follows:

[0125] 200 parts of the comparative compound microbial agent 2, 55 parts of humic acid, 55 parts of urea, 75 parts of water-soluble ammonium polyphosphate, 75 parts of mineral-derived potassium fulvate, 45 parts of potassium silicate, 45 parts of potassium magnesium sulfate and 325 parts of decomposed straw charcoal were weighed and placed in a mixing tank. The stirring speed was controlled at 120 r / min and the stirring time was 15 min. After mixing, the mixture was packaged to obtain component A of the microbial fertilizer.

[0126] Among them, the 200 comparative compound microbial agents 2 consisted of 90 Bacillus subtilis dry powder agent samples 1 and 110 Trichoderma harzianum dry powder agent control samples 2.

[0127] 1.2 portions of L-cysteine ​​are packaged separately as component B of the microbial fertilizer;

[0128] Components A and B together form the prepared microbial fertilizer, which is designated as microbial fertilizer control standard 2.

[0129] The preparation method of the *Trichoderma harzianum* dry powder inoculum reference standard in this comparative example is as follows:

[0130] S11, Fermentation by Trichoderma harzianum:

[0131] Commercially available *Trichoderma harzianum* powder was activated to obtain *Trichoderma harzianum* seed culture. This seed culture was then inoculated into *Trichoderma harzianum* fermentation medium at a volume ratio of 6.5%, with a pH of 7.0. Fermentation was carried out at 35°C. Once the *Trichoderma harzianum* reached the late logarithmic fermentation stage, the cells were collected, resuspended in physiological saline, and the cell concentration was adjusted to 2.8 × 10⁻⁶. 9 CFU / mL was used to obtain a Trichoderma harzianum suspension.

[0132] S12, Embedding of Trichoderma harzianum:

[0133] Okra polysaccharide and chloroacetic acid were mixed in isopropanol at a mass ratio of 1:1.2, wherein the amount of isopropanol used was 11 times the volume of okra polysaccharide; the mixture was reacted at 60°C for 5 h, and after neutralization, it was purified by dialysis to obtain carboxymethylated okra polysaccharide.

[0134] Carboxymethylated okra polysaccharide and sodium alginate were mixed at a mass ratio of 1:12, dissolved in water, and the amount of water used was 35 times the volume of carboxymethylated okra polysaccharide. The mixture was stirred and mixed evenly to obtain an okra polysaccharide-sodium alginate mixture.

[0135] The Trichoderma harzianum suspension was added to the okra polysaccharide-sodium alginate mixture at a volume ratio of 2.5%. After stirring and mixing, a 3% calcium chloride solution was added dropwise. The amount of calcium chloride solution used was 1.5 times the volume of the okra polysaccharide-sodium alginate mixture. After the addition was completed, the mixture was allowed to stand for 25 minutes to obtain a mixture containing Trichoderma harzianum microspheres.

[0136] S13, Freeze-drying of Trichoderma harzianum dry powder inoculum:

[0137] Trehalose, glycerol, and nano-silica were added to a mixture containing Trichoderma harzianum microspheres. The mass ratio of the mixture containing Trichoderma harzianum microspheres, trehalose, glycerol, and nano-silica was 100:10:1.8:0.15.

[0138] Pre-cooled at 5℃ for 13h, then quick-frozen at -42℃ for 1h45min, and finally freeze-dried under vacuum at -52℃ and 0.15mbar for 22h to obtain Trichoderma harzianum dry powder inoculant, which is recorded as Trichoderma harzianum dry powder inoculant control.

[0139] Comparative Example 3

[0140] The preparation process of the microbial fertilizer to improve the disease resistance of perennial rice in this comparative example is as follows:

[0141] 200 parts of the comparative compound microbial agent 3, 55 parts of humic acid, 55 parts of urea, 75 parts of water-soluble ammonium polyphosphate, 75 parts of mineral-derived potassium fulvate, 45 parts of potassium silicate, 45 parts of potassium magnesium sulfate and 325 parts of decomposed straw charcoal were weighed and placed in a mixing tank. The stirring speed was controlled at 120 r / min and the stirring time was 15 min. After mixing, the mixture was packaged to obtain component A of the microbial fertilizer.

[0142] Among them, the 200 comparative compound microbial inoculants 3 consisted of 90 Bacillus subtilis dry powder inoculant control and 110 Trichoderma harzianum dry powder inoculant sample 1.

[0143] 1.2 portions of L-cysteine ​​are packaged separately as component B of the microbial fertilizer;

[0144] Components A and B together form the prepared microbial fertilizer, which is designated as microbial fertilizer control standard 3.

[0145] The preparation method of the Bacillus subtilis dry powder inoculum reference standard in this comparative example is as follows:

[0146] S21, Fermentation by Bacillus subtilis:

[0147] Commercially available Bacillus subtilis powder was activated to obtain Bacillus subtilis seed culture. The Bacillus subtilis seed culture was then inoculated into Bacillus subtilis fermentation medium at a volume ratio of 11%, with a pH of 7.0. Fermentation was carried out at 35°C. Once the Bacillus subtilis reached the late logarithmic fermentation stage, the cells were collected, resuspended in physiological saline, and the cell concentration was adjusted to 3.9 × 10⁻⁶. 9 CFU / mL was used to obtain a Bacillus subtilis suspension.

[0148] S22, Encapsulation of Bacillus subtilis:

[0149] Add 4% by volume of Bacillus subtilis suspension to a 5.0% sodium alginate solution, stir and mix, then add dropwise a 3% calcium chloride solution. The amount of calcium chloride solution used is 1.5 times the volume of the sodium alginate solution. After the addition is complete, let stand for 25 minutes to obtain a mixture containing Bacillus subtilis microspheres.

[0150] S23, Freeze-drying of Bacillus subtilis dry powder inoculum:

[0151] Trehalose, glycerol, and nano-silica were added to a mixture containing Bacillus subtilis microspheres. The mass ratio of the mixture containing Bacillus subtilis microspheres, trehalose, glycerol, and nano-silica was 100:10:1.8:0.15.

[0152] Pre-cooled at 5℃ for 13 hours, then quick-frozen at -42℃ for 1 hour and 40 minutes, and finally freeze-dried under vacuum at -52℃ and 0.15 mbar for 22 hours. The dried Bacillus subtilis powder was sprayed onto the powder and recorded as a control.

[0153] Implementation effect

[0154] Perennial paddy fields were selected as experimental areas, with each experimental group or control group having an area greater than 1 mu (approximately 0.16 acres). Protective rows were set up between different experimental groups. Before applying the microbial fertilizer sample or microbial fertilizer control, components A and B needed to be mixed. For the crop blank control group applying ordinary ternary compound fertilizer without microbial agents, fertilization was carried out according to the records in Table 1.

[0155] Table 1: Fertilization status of each experimental or control group

[0156]

[0157]

[0158] The experiment lasted for 3 years. The average yield, effective tiller number, disease incidence and disease type of each experimental group and control group were calculated at each harvest. The results are shown in Tables 2 and 3.

[0159] Table 2: Average yield and number of effective tillers in each experimental or control group

[0160]

[0161] Table 3: Incidence rate and types of disease in each experimental or control group

[0162]

[0163]

[0164] As shown in Tables 2 and 3, after three consecutive years and six seasons of field trials, the experimental group applying the microbial fertilizer of this invention showed a significant increase in yield. Compared with the blank control group, the yield in the first season increased by 10.3%–12.0%, and the yield in the second season increased by more than 20%, indicating that the microbial fertilizer of this invention is more conducive to the sustained release of ratoon dominance. In addition, the number of effective tillers in the first and second seasons also increased from 5.0–6.5 in the blank control group to 9–10, a relative increase of 25%–30%. This indicates that the microbial fertilizer provides a foundation for increased yield by promoting tiller formation. More importantly, the incidence of rice sheath blight, rice blast, and bacterial leaf blight was significantly reduced in the experimental group applying this invention, indicating that this invention can improve the disease resistance of perennial rice.

[0165] While the experimental results of controls 1-3 were significantly better than the blank control group, they were still inferior to the experimental group. For example, control group 1, which used a mixture of commercially available Bacillus subtilis powder and Trichoderma harzianum powder as the inoculant, showed the worst effect, indicating that although the two inoculants could have a synergistic effect, the result was not ideal. This may be because there is interspecific competition between Bacillus subtilis and Trichoderma harzianum, inhibiting the spore germination rate of Trichoderma harzianum and reducing their synergistic effect. Control groups 2 and 3 used different methods to encapsulate the inoculants, and while the results were improved compared to the unencapsulated group, they were still not as good as the experimental group. This shows that the experimental group, by encapsulating the inoculants according to the different characteristics of Bacillus subtilis and Trichoderma harzianum, was more conducive to spore germination and improved the utilization efficiency of the inoculant.

[0166] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microbial fertilizer for improving the disease resistance of perennial rice, characterized in that, By weight, the microbial fertilizer comprises 200 parts of compound microbial agent, 50-60 parts of humic acid, 50-60 parts of urea, 70-80 parts of ammonium polyphosphate, 70-80 parts of mineral-derived potassium fulvate, 40-50 parts of potassium silicate, 40-50 parts of potassium magnesium sulfate, 300-350 parts of decomposed straw charcoal, 1.0-1.5 parts of L-cysteine.

2. The microbial fertilizer for improving the disease resistance of perennial rice according to claim 1, characterized in that, The compound microbial agent is composed of Bacillus subtilis agent and Trichoderma harzianum agent in a mass ratio of 1:1.1 to 1.3; the ammonium polyphosphate is water-soluble ammonium polyphosphate.

3. The microbial fertilizer for improving the disease resistance of perennial rice according to claim 2, characterized in that, The Bacillus subtilis inoculant is a Bacillus subtilis dry powder inoculant embedded in okra polysaccharide-sodium alginate hydrogel; the Trichoderma harzianum inoculant is a Trichoderma harzianum dry powder inoculant embedded in sodium alginate hydrogel.

4. The microbial fertilizer for improving the disease resistance of perennial rice according to claim 3, characterized in that, The preparation process of the Bacillus subtilis dry powder inoculant is as follows: S11, Fermentation by Bacillus subtilis: Commercially available Bacillus subtilis powder was activated to obtain Bacillus subtilis seed culture. The Bacillus subtilis seed culture was then inoculated into Bacillus subtilis fermentation medium at a volume ratio of 10%–12%, with a pH of 6.8–7.

2. Fermentation was carried out at 32℃–37℃. Once the Bacillus subtilis reached the late logarithmic fermentation stage, the cells were collected, resuspended in physiological saline, and the cell concentration was adjusted to 10⁻⁶. 9 ~10 10 CFU / mL was used to obtain a Bacillus subtilis suspension. S12, Encapsulation of Bacillus subtilis: Okra polysaccharide in a mass ratio of 1:1.1 to 1.3 was mixed with chloroacetic acid in isopropanol and reacted at 55℃ to 65℃ for 4 to 6 hours. After neutralization, the mixture was purified by dialysis to obtain carboxymethylated okra polysaccharide. The carboxymethylated okra polysaccharide and sodium alginate were mixed at a mass ratio of 1:10-15, dissolved in water, and stirred to obtain an okra polysaccharide-sodium alginate mixture. Add 2%–3% by volume of Bacillus subtilis suspension to okra polysaccharide-sodium alginate mixture, stir and mix, then add calcium chloride solution dropwise. After the addition is complete, let stand for 20–30 minutes to obtain a mixture containing Bacillus subtilis microspheres. S13, Freeze-drying of Bacillus subtilis dry powder inoculum: Trehalose, glycerol, and nano-silica were added to a mixture containing Bacillus subtilis microspheres. The mixture was pre-cooled at 4℃~6℃ for 12h~14h, then rapidly frozen at -45℃~-40℃ for 1.5h~2.0h, and finally freeze-dried under vacuum at -55℃~-50℃ and 0.1mbar~0.2mbar for 20h~24h to obtain the Bacillus subtilis dry powder inoculant.

5. The microbial fertilizer for improving the disease resistance of perennial rice according to claim 4, characterized in that, The amount of isopropanol used is 10 to 12 times the volume of okra polysaccharide; the amount of water used is 30 to 40 times the volume of carboxymethylated okra polysaccharide; the mass concentration of the calcium chloride solution is 2% to 4%, and the amount of calcium chloride solution used is 1 to 2 times the volume of the okra polysaccharide-sodium alginate mixture; the mass ratio of the mixture containing Bacillus subtilis microspheres, trehalose, glycerol, and nano-silica is 100:8 to 12:1.5 to 2.0:0.1 to 0.

2.

6. The microbial fertilizer for improving the disease resistance of perennial rice according to claim 3, characterized in that, The preparation process of the aforementioned Trichoderma harzianum dry powder inoculant is as follows: S21, Fermentation by Trichoderma harzianum: Commercially available *Trichoderma harzianum* powder was activated to obtain *Trichoderma harzianum* seed culture. The seed culture was then inoculated into *Trichoderma harzianum* fermentation medium at a volume ratio of 5%–8%, with a pH of 6.8–7.

2. Fermentation was carried out at 32℃–37℃. Once the fermentation reached the late logarithmic phase, the cells were collected, resuspended in physiological saline, and the cell concentration was adjusted to 10⁻⁶. 9 ~10 10 CFU / mL was used to obtain a Trichoderma harzianum suspension. S22, Embedding of Trichoderma harzianum: Add 2%–3% by volume of Trichoderma harzianum suspension to sodium alginate solution, add rice husk ash, stir and mix, then add calcium chloride solution dropwise. After the addition is complete, let stand for 1–2 hours to obtain a mixture containing Trichoderma harzianum microspheres. S23, Freeze-drying of Trichoderma harzianum dry powder inoculum: A nonionic surfactant, trehalose, glycerol, and nano-silica were added to a mixture containing Trichoderma harzianum microspheres. The mixture was pre-cooled at 4℃~6℃ for 12h~14h, then rapidly frozen at -45℃~-40℃ for 1.5h~2.0h, and finally freeze-dried under vacuum at -55℃~-50℃ and 0.1mbar~0.2mbar for 20h~24h to obtain the Trichoderma harzianum dry powder inoculant.

7. The microbial fertilizer for improving the disease resistance of perennial rice according to claim 6, characterized in that, The calcium chloride solution has a mass concentration of 2% to 4%, and the amount of calcium chloride solution used is 1 to 2 times the volume of the sodium alginate solution. The mass concentration of the sodium alginate solution is 2.5% to 3.5%. The mass ratio of the mixture containing Trichoderma harzianum microspheres, nonionic surfactant, trehalose, glycerol, and nano-silica is 100:3 to 8:8 to 12:1.5 to 2.0:0.1 to 0.

2. The nonionic surfactant includes any one of Tween 80, Tween 60, or soybean lecithin. The amount of rice husk ash used is 5% to 10% of the mass of the Trichoderma harzianum suspension.

8. The method for preparing a microbial fertilizer to improve the disease resistance of perennial rice according to claim 1, characterized in that, By weight, 200 parts of compound microbial agent, 50-60 parts of humic acid, 50-60 parts of urea, 70-80 parts of ammonium polyphosphate, 70-80 parts of mineral-derived potassium fulvate, 40-50 parts of potassium silicate, 40-50 parts of potassium magnesium sulfate, and 300-350 parts of decomposed straw charcoal are mixed and packaged to obtain component A, and 1.0-1.5 parts of L-cysteine ​​are packaged separately to obtain component B.

9. The application of the microbial fertilizer for improving the disease resistance of perennial rice according to claim 1, characterized in that, The microbial fertilizer is used to improve the resistance of perennial rice to sheath blight, rice blast, and bacterial blight.

10. The application according to claim 8, characterized in that, Before fertilization, components A and B are mixed. For the first season, the base fertilizer is applied by deep plowing to a depth of 8-10 cm, and the amount of the microbial fertilizer applied as base fertilizer is 30-40 kg / mu. Before winter, the topdressing is applied by broadcasting, and the amount of the microbial fertilizer applied before winter is 10-20 kg / mu. During the regeneration season, the topdressing is applied by broadcasting, and the amount of the microbial fertilizer applied during the regeneration season is 8-10 kg / mu.

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