A lactic acid bacteria preparation for improving yield and quality of waxy corn and application thereof

Abstract

The application belongs to the technical field of microorganisms, and discloses a lactic acid bacteria preparation for improving the yield and quality of waxy corn and application thereof. The active bacteria in the lactic acid bacteria preparation are Lactobacillus casei C-9 strains, and the strain preservation number of the C-9 strains is CGMCC No. 6734. The application is at least one selected from the following: A, improving the yield of waxy corn; B, improving the amylopectin content, soluble sugar content and total amino acid content in waxy corn kernels; C, improving the taste and flavor of waxy corn; and D, enhancing the stress resistance of waxy corn. The application uses Lactobacillus casei C-9 bacterial liquid for waxy corn planting, solves the problems of soil physicochemical property deterioration, soil microbial community imbalance, soil fertility decline and environmental pollution caused by chemical fertilizers through the combined mode of seed treatment, base fertilizer application, leaf surface spraying and root irrigation.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparation technology. Specifically, it relates to a lactic acid bacteria preparation and its application in crop cultivation, and more particularly to a Lactobacillus casei fermentation preparation and its application in improving the yield and quality of waxy corn. Background Technology

[0002] Waxy corn (Zea mays L. sinensis Kulesh) is a special type of the genus Zea. Its kernels are mainly composed of amylopectin, which gives it a soft and glutinous texture, easy digestibility, and unique flavor. It is widely used in food processing and table consumption (Zhang Baoshi, Wang Zhenhua, Li Xinhai, et al. Research progress on innovation and utilization of waxy corn germplasm resources [J]. Maize Science, 2015, 23 (2):143-148). With the upgrading of consumers' demand for healthy diets, the market demand for waxy corn continues to grow, and the requirements for its quality are also increasing. It is required not only to have a soft and glutinous texture and rich flavor, but also to have rich nutritional components, such as amino acids, vitamins, and dietary fiber (Liu Jian, Li Yanhua, Zhang Ying, et al. Correlation analysis of quality traits of waxy corn and screening of high-quality varieties [J]. Crop Journal, 2018, (3):135-140).

[0003] Currently, in order to improve the yield and quality of waxy corn, excessive application of chemical fertilizers is common during the planting process. Although long-term application of chemical fertilizers can promote plant growth in the short term, it will lead to the deterioration of soil physical and chemical properties, imbalance of soil microbial community, and decline in soil fertility. At the same time, it will also cause environmental pollution, affecting the food safety and quality stability of waxy corn (Wang Xingren, Zhang Fusuo, Chen Xinping et al. Rational application of chemical fertilizers and sustainable agricultural development [J]. Chinese Agricultural Science, 2012, 45(15):3161-3169). In addition, under the traditional planting mode, the improvement of key quality indicators such as amylopectin content and soluble sugar content of waxy corn is limited, and its resistance is weak, making it susceptible to pests and diseases, which further restricts its commercial value (Li Juan, Wang Li, Sun Shiyou et al. Effects of different fertilization modes on the yield and quality of waxy corn [J]. Hebei Agricultural Sciences, 2020, 24(1):34-38).

[0004] In recent years, microbial fertilizers have become an important alternative to traditional chemical fertilizers due to their environmental friendliness, high efficiency, and sustainability. Numerous studies have shown that plant rhizosphere growth-promoting bacteria (PGPR) can promote crop growth and improve crop quality through nitrogen fixation, phosphorus solubilization, potassium solubilization, and the secretion of plant growth hormones, while also restoring the soil ecological environment (Li Honglian, Yuan Hongxia, Xing Xiaoping, et al. Research progress on the application of plant rhizosphere growth-promoting bacteria in agricultural production [J]. Journal of Henan Agricultural University, 2016, 50 (4):465-470). For example, Zhang Jianjun et al. applied a microbial preparation made from Bacillus subtilis to maize planting. The results showed that the preparation could significantly increase the plant height, ear length, and grain yield of maize, while also increasing the crude protein and lysine content in the grains (Zhang Jianjun, Liu Shuxia, Wang Hongyan, et al. Effects of Bacillus subtilis preparation on maize growth and quality [J]. Journal of Soil and Water Conservation, 2017, 31 (3):293-298). Kim et al. found that lactic acid bacteria fermentation products can improve soil aeration and water retention, promote tomato root growth, and increase the content of vitamin C and soluble sugar in fruit (Kim SH, Lee JH, ParkY C, et al. Effects of lactic acid bacteria fermentation products on tomato growth and fruit quality [J]. Journal of Microbiology and Biotechnology, 2019, 29 (7): 987-994).

[0005] Lactobacillus casei, an important group of lactic acid bacteria, possesses probiotic functions such as regulating intestinal flora balance and enhancing immunity, and is widely used in the food fermentation field. Among them, Lactobacillus casei strain C-9 has been proven to have excellent fermentation performance and environmental adaptability. It was deposited on November 12, 2012, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 6734, and classified as Lactobacillus casei C-9 (disclosed in Chinese patent CN119685215A). However, there is currently limited research on the application of Lactobacillus casei C-9 in crop cultivation, especially in improving the quality of waxy corn (such as amylopectin content, soluble sugar content, and flavor compound accumulation), for which no relevant reports have been found. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the first objective of this invention is to provide a lactic acid bacteria preparation and its application in improving the yield, quality and stress resistance of waxy corn, so as to solve the problems of soil physicochemical deterioration, soil microbial community imbalance, soil fertility decline and environmental pollution caused by chemical fertilizers.

[0007] To achieve the above objectives, the inventors conducted numerous experiments to investigate the effects of different bacterial strains on the growth, yield, and quality of waxy corn. They ultimately discovered that applying Lactobacillus casei C-9 bacterial solution during waxy corn cultivation can optimize the growth traits and improve the quality indicators of waxy corn.

[0008] Based on the above research results, the present invention has obtained the following technical solution: a lactic acid bacteria preparation for improving the yield and quality of waxy corn, wherein the active bacteria in the lactic acid bacteria preparation is Lactobacillus casei C-9 strain, and the strain preservation number of the C-9 strain is CGMCC No. 6734.

[0009] More preferably, the lactic acid bacteria preparation for improving the yield and quality of waxy corn as described above is a liquid preparation, wherein the viable bacteria count in the liquid preparation is 2.5–3.0 × 10⁻⁶. 9 CFU / mL.

[0010] In addition, the application provided by this invention is the application of the above-mentioned lactic acid bacteria preparation, and the application is selected from at least one of the following: A. increasing the yield of waxy corn; B. increasing the content of amylopectin, soluble sugar and total amino acids in waxy corn kernels; C. improving the taste and flavor of waxy corn; D. enhancing the stress resistance of waxy corn.

[0011] More preferably, the stress resistance includes disease resistance, drought resistance and lodging resistance.

[0012] More preferably, the application is to prepare the above-mentioned lactic acid bacteria preparation into a bacterial solution and then use it for at least one of the following treatments: (1) soaking glutinous corn seeds; (2) adding base fertilizer for application; (3) foliar spraying during the growing season; (4) root irrigation during the growing season.

[0013] More preferably, the specific operation method for soaking the glutinous corn seeds is as follows: the glutinous corn seeds are sun-dried for 2-3 days, and the lactic acid bacteria preparation is prepared to a concentration of 5-6 × 10⁻⁶. 7 Soak the glutinous corn seeds in a bacterial solution of CFU / mL for 6–8 hours, then drain and use them for sowing.

[0014] More preferably, the specific operation method for adding base fertilizer is as follows: evenly spread the mixture of lactic acid bacteria preparation and organic fertilizer (preferably decomposed sheep manure) in the planting plot, wherein the mass ratio of lactic acid bacteria preparation to organic fertilizer in the mixture is 1:50 to 1:80, the application rate is 2000 to 2500 kg / mu, and plow it into the soil to a depth of 20 to 25 cm.

[0015] More preferably, the specific operation method for spraying the leaves during the growth period is as follows: the lactic acid bacteria preparation is formulated to a concentration of 1.6–3.0 × 10⁻⁶. 7 Apply a bacterial solution of CFU / mL to the leaves of waxy corn once each during the jointing stage, the large trumpet stage, and the tasseling stage. The application rate is 50-80 L / mu. Choose a cloudy day or evening for spraying to ensure that the solution is evenly applied to both sides of the leaves.

[0016] More preferably, the specific operation method for spraying the leaves during the growth period is as follows: the lactic acid bacteria preparation is formulated to a concentration of 2.5–3.8 × 10⁻⁶. 7 Apply CFU / mL bacterial solution to the roots of waxy corn once during the jointing stage and once during the grain-filling stage. The amount of solution applied per plant is 200-300 mL, and the solution is applied in a circular motion around the base of the plant for 10-15 cm.

[0017] The second objective of this invention is to provide a planting method that improves the yield and quality of waxy corn. By applying Lactobacillus casei C-9 bacterial solution during the waxy corn planting process, the growth traits of waxy corn can be optimized, the quality indicators can be improved, and environmental pollution can be reduced, providing technical support for the green and high-quality planting of waxy corn.

[0018] Specifically, the planting method of the present invention for improving the yield and quality of waxy corn includes the following steps:

[0019] (1) Pre-sowing treatment: The waxy corn seeds are sun-dried for 2-3 days, and then treated with 5-6×10 7 The waxy corn seeds were soaked in a solution of Lactobacillus casei C-9 at CFU / mL for 6–8 hours, drained, and then used for sowing.

[0020] (2) Base fertilizer application: Prepare 2.5~3.0×10 7 A CFU / mL Lactobacillus casei C-9 bacterial solution is evenly spread in the planting plot as a mixture of C-9 bacterial solution and organic fertilizer. The mass ratio of C-9 bacterial solution to organic fertilizer in the mixture is 1:50 to 1:80. The application rate is 2000 to 2500 kg / mu. The solution is then plowed into the soil to a depth of 20 to 25 cm.

[0021] (3) Spraying during the growing season: Prepare a solution of 1.6~3.0×10 7 Apply a CFU / mL Lactobacillus casei C-9 bacterial solution to the leaves once each during the jointing stage, the large trumpet stage, and the tasseling stage of waxy corn. The application rate is 50-80 L / mu. Choose a cloudy day or evening to spray and ensure that the solution is evenly adhered to both sides of the leaves.

[0022] (4) Root irrigation during the growing season: Prepare 2.5~3.8×10 7 Apply a CFU / mL Lactobacillus casei C-9 bacterial solution to the roots of waxy corn once during the jointing stage and once during the grain-filling stage. The amount of solution applied per plant is 200-300 mL, and the solution is applied in a circular motion around the base of the plant for 10-15 cm.

[0023] More preferably, in the planting method described above for improving the yield and quality of waxy corn, the organic fertilizer is well-rotted sheep manure.

[0024] Compared with existing technologies, this invention utilizes Lactobacillus casei C-9 bacterial solution for waxy corn cultivation, and achieves the following technical effects through a combination of seed treatment, basal fertilizer application, foliar spraying, and root irrigation:

[0025] (1) Promotes the growth of waxy corn: significantly increases the plant height, stem diameter and leaf area of ​​waxy corn, enhances the photosynthetic capacity of the plant, and increases the plant height by 8.5% to 12.3% and stem diameter by 10.2% to 14.6% at the jointing stage compared with the control group.

[0026] (2) Improve quality indicators: Increase the content of amylopectin, soluble sugar and total amino acids in glutinous corn kernels. The content of amylopectin increased by 5.8% to 8.3% compared with the control group, the content of soluble sugar increased by 7.2% to 10.5% and the content of lysine increased by 12.1% to 15.4%.

[0027] (3) Improve taste and flavor: Reduce the crude fiber content in the grains, improve the soft and glutinous texture, and increase the accumulation of flavor substances (such as acetic acid, lactic acid, etc.) to enhance the taste.

[0028] (4) Enhanced stress resistance: Improved the resistance of waxy corn to drought and lodging, reduced the occurrence of pests and diseases, and reduced the incidence of diseases by 30% to 40% compared with the control group.

[0029] (5) Environmental protection and sustainability: Reduce fertilizer application by 30% to 50%, improve soil microbial community structure, increase soil organic matter content, and reduce environmental pollution. Detailed Implementation

[0030] The following embodiments further illustrate the above-described content of the present invention in detail. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technical solutions implemented based on the above-described content of the present invention fall within the scope of the present invention. Furthermore, unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art.

[0031] Example 1: Preparation and application of Lactobacillus casei C-9 fermentation preparation

[0032] 1. Preparation of Lactobacillus casei C-9 fermentation preparation

[0033] (1) Seed culture: Lactobacillus casei C-9 strain was inoculated into MRS liquid medium at an inoculum of 3% to 5% and cultured at 37±1℃ under anaerobic conditions for 18 to 24 hours to obtain seed culture. The viable count of the seed culture was adjusted to 1.5 to 2.0 × 10¹. 0 cfu / mL;

[0034] (2) Expanded fermentation culture: The seed culture was inoculated into the fermentation medium at an inoculation rate of 8% to 12%. The fermentation medium consisted of the following components (mass fraction): glucose 2.0%, soybean meal powder 1.5%, yeast extract 0.8%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%, Tween-80 0.1%, and the remainder was sterile water. The pH was adjusted to 6.2 to 6.5. Fermentation was carried out at 37±1℃ under anaerobic conditions for 36 to 48 hours, with stirring every 8 hours at a stirring speed of 100 to 150 r / min.

[0035] (3) Formulation preparation: After fermentation, add 5%–8% trehalose to the fermentation broth as a protective agent, stir evenly, collect the cells by low-temperature centrifugation (4℃, 8000 r / min, 15 minutes), and then add sterile water to adjust the viable cell count to 2.5–3.0 × 10⁻⁶. 9 The Cfu / mL concentration yields the Lactobacillus casei C-9 fermentation preparation.

[0036] 2. Application of Lactobacillus casei C-9 fermentation preparation in waxy corn cultivation

[0037] (1) Pre-sowing treatment: After sun-drying the glutinous corn seeds for 2-3 days, soak them for 8 hours after diluting them with Lactobacillus casei C-9 fermentation agent 50 times, drain them and then sow them.

[0038] (2) Base fertilizer application: The mixture of fermentation agent and organic fertilizer is evenly spread in the planting plot. The mass ratio of Lactobacillus casei C-9 fermentation agent to organic fertilizer in the mixture is 1:60. The organic fertilizer is decomposed sheep manure. The application rate is 2200 kg / mu. The mixture is plowed into the soil and the deep plowing depth is about 22 cm.

[0039] (3) Spraying during the growing season: Spray the leaves once each during the jointing stage, the large trumpet stage and the tasseling stage of waxy corn. Dilute the Lactobacillus casei C-9 fermentation preparation 120 times and spray at a rate of 60 L / mu. Choose a cloudy day or evening to spray and ensure that the liquid is evenly applied to both sides of the leaves.

[0040] (4) Root irrigation during the growing season: Root irrigation treatment was carried out once during the jointing stage and once during the grain filling stage of waxy corn. The Lactobacillus casei C-9 fermentation preparation was diluted 90 times and the amount of each plant was 250 mL. The irrigation was carried out in a ring around the roots of the plant for 12 cm.

[0041] Example 2: Effects of Lactobacillus casei C-9 fermentation preparation on the growth and quality of waxy corn

[0042] I. Experimental Materials and Design

[0043] Experimental variety: Waxy corn variety "Vanke No. 8";

[0044] Experimental soil conditions: Soil type was brown soil, pH value was 6.8, organic matter content was 1.2%, available nitrogen was 85 mg / kg, available phosphorus was 32 mg / kg, and available potassium was 110 mg / kg;

[0045] Experimental groups: Three treatment groups and one control group (CK) were set up, with three replicates per group and a plot area of ​​20 m². 2 (4m×5m), randomized block design, row spacing 60 cm, plant spacing 30 cm, planting density 3700 plants / mu; control group (CK): conventional planting, application of chemical fertilizer (base fertilizer: compound fertilizer NPK=15-15-15, application rate 50 kg / mu; top dressing: urea 20 kg / mu at the jointing stage), seeds soaked in water, sprayed with water during the growing season;

[0046] Treatment group 1 (T1): Seed soaking treatment only, the treatment method is the same as in Example 1, that is, the fermentation agent is diluted 50 times and the seeds are soaked for 8 hours, the rest is the same as the control group;

[0047] Treatment group 2 (T2): Seed soaking + basal fertilizer application. The treatment methods for seed soaking and basal fertilizer application are the same as in Example 1, and the topdressing is the same as in the control group.

[0048] Treatment group 3 (T3): Seed soaking + basal fertilizer application + foliar spraying during the growing season + root irrigation. Seed treatment was the same as T1, basal fertilizer was the same as T2, foliar spraying (diluted 120 times, 60 L / mu) and root irrigation (diluted 90 times, 250 mL / plant) were carried out during the growing season. That is, the whole treatment method was the same as the planting method in Example 1.

[0049] Experimental period: 95 days from sowing to maturity, with other field management measures (watering, weeding, etc.) remaining consistent across groups.

[0050] II. New Control Experiment Design (Lactobacillus plantarum LYS-1 group)

[0051] (a) Core variables of the experiment

[0052] The tested strain, *Lactobacillus plantarum* LYS-1, was purchased from the China Industrial Microbial Culture Collection Center (CICC), with strain number CICC 6009. In this invention experiment, *Lactobacillus plantarum* LYS-1 (CICC 6009) was selected as the positive control. The positive control strain was determined strictly following the principles for setting up positive controls in field trials of microbial preparations, while also considering the current state of industry research on lactic acid bacteria strains, strain characteristics, and the scientific requirements for experimental comparison. The core reasons are mainly as follows:

[0053] 1. *Lactobacillus plantarum* LYS-1 is a recognized high-quality lactic acid bacteria control strain in the agricultural field.

[0054] Lactobacillus plantarum is a classic and mature strain of lactic acid bacteria used in agricultural planting. Among them, LYS-1 (CICC6009) is a standardized strain published by the China Industrial Microbial Culture Collection Center. It is a commonly used positive control lactic acid bacteria in agricultural microbial fertilizer efficacy tests. Its effects on crop growth promotion, quality improvement and soil improvement have been confirmed by a large number of studies. It belongs to the lactic acid bacteria with clear positive fertilizer effects that are well known to those skilled in the art, and fully meets the basic requirement of "recognized lactic acid bacteria as positive controls".

[0055] 2. Ensure the consistency of the control group within the same genus to eliminate interference from differences in major bacterial groups.

[0056] Both *Lactobacillus casei* and *Lactobacillus plantarum* belong to the Lactic Acid Bacteria (LAB) group. They are highly homologous in terms of metabolic type and microbial action mechanism (such as organic acid production, soil nutrient activation, and promotion of rhizosphere microecological balance). Using strains from the same genus as controls can eliminate the mechanism differences between "strains from different major groups (such as lactic acid bacteria and Bacillus)," allowing the experimental comparison to focus on the performance differences of the strains themselves. This can more scientifically verify the specific advantages of *Lactobacillus casei* C-9 in the lactic acid bacteria group for waxy corn cultivation.

[0057] 3. Adhere to the principle of variable uniqueness and adapt the gradient intervention design to this experiment.

[0058] This experiment adopted a gradient intervention model of "seed soaking → basal fertilizer → spraying + root drenching". The control strain's formulation was required to achieve the same application methods (soaking, fertilizer mixing, spraying, and root drenching) as *Lactobacillus casei* C-9. *Lactobacillus plantarum* LYS-1, with its fermentation characteristics, formulation stability, and environmental adaptability, showed a high degree of compatibility with *Lactobacillus casei* C-9. It could be prepared into a liquid formulation using the same process and could be applied at all stages of waxy corn cultivation. This ensured that only the "strain type" was the sole variable in the experiment, with all other treatment conditions being completely consistent, meeting the scientific design requirements of field trials.

[0059] 4. The fertilizer efficacy characteristics of Lactobacillus plantarum LYS-1 are highly consistent with the research direction of this invention.

[0060] The core research direction of this invention is to improve the yield, quality, and soil quality of waxy corn. Lactobacillus plantarum LYS-1 happens to be a lactic acid bacteria strain that excels in "increasing crop yield, improving grain nutritional quality, and activating readily available nutrients in the soil". Using it as a control can accurately align with the evaluation dimensions of the technical effects of this invention. The comparison clearly demonstrates the specific advantages of Lactobacillus casei C-9 in waxy corn cultivation in terms of promoting root growth, activating phosphorus and potassium nutrients, accumulating vitamin C, and enhancing stress resistance.

[0061] 5. Highlighting the innovativeness and technological advancement of Lactobacillus casei C-9

[0062] If weaker lactic acid bacteria or non-standardized strains are used as controls, the advantages of the strains of this invention cannot be demonstrated. However, by using the industry-recognized and highly effective Lactobacillus plantarum LYS-1 as a control, the experimental results show that Lactobacillus casei C-9, under the whole process treatment, has significantly better performance than LYS-1 in all indicators (root fresh weight, yield per acre, vitamin C content, available phosphorus and potassium in the soil, etc.). This fully demonstrates that Lactobacillus casei C-9 has a better application effect in waxy corn planting than existing high-quality agricultural lactic acid bacteria, directly highlighting the innovation and technological advancement of the strains of this invention, and providing stronger experimental support for the practicality of the invention.

[0063] In summary, selecting Lactobacillus plantarum LYS-1 as a positive control not only meets the requirements for control settings of "lactobacterial bacteria recognized in this field", but also takes into account the scientific nature, comparability, and specificity of the experiment, making it the optimal choice for verifying the effectiveness of Lactobacillus casei C-9 technology.

[0064] The preparation method of LYS-1 fermentation agent maintains the same standards and parameters as the preparation process of Lactobacillus casei C-9 fermentation agent, ensuring that the strain is the only variable, adapting to the application requirements of all stages of waxy corn planting, and finally obtaining an effective viable count ≥2.5×10⁻⁶. 10 The specific steps for producing a CFU / mL Lactobacillus plantarum LYS-1 fermentation preparation are as follows:

[0065] 1. Seed culture

[0066] Lactobacillus plantarum LYS-1 (CICC 6009) strain was inoculated into MRS liquid medium at an inoculum size of 3%–5% and cultured at 37±1℃ under strict anaerobic conditions for 18–24 hours to obtain Lactobacillus plantarum LYS-1 seed culture. After viable cell count, the viable cell count of the seed culture was adjusted to 1.5–2.0 × 10⁻⁶. 10 CFU / mL.

[0067] 2. Expand fermentation culture

[0068] Prepare the fermentation medium: According to the mass fraction ratio, it consists of 2.0% glucose, 1.5% soybean meal powder, 0.8% yeast extract, 0.2% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.1% Tween-80, and the remainder is sterile water. After thorough stirring and dissolution, adjust the pH of the medium to 6.2-6.5.

[0069] Inoculation and fermentation: The above-prepared Lactobacillus plantarum LYS-1 seed culture was inoculated into the fermentation medium at an inoculation rate of 8% to 12%, and fermented at 37±1℃ under anaerobic conditions for 36 to 48 hours. During fermentation, the mixture was stirred once every 8 hours at a stirring speed of 100 to 150 r / min to ensure uniform cell growth and full utilization of nutrients.

[0070] 3. Formulation preparation and viable cell count calibration

[0071] Addition of protective agent: After fermentation, add 5% to 8% trehalose to the fermentation broth of Lactobacillus plantarum LYS-1 as a cell protectant, and stir thoroughly until completely dissolved to improve the storage stability of the preparation and the cell survival rate in field application;

[0072] Low-temperature centrifugation to collect bacterial cells: Place the fermentation broth with added protectant in a low-temperature environment of 4℃, centrifuge at 8000 r / min for 15 minutes, discard the supernatant, and collect the bacterial cell precipitate at the bottom;

[0073] Viable cell count adjustment: Add sterile water to the bacterial precipitate, resuspend thoroughly with stirring, and then adjust the viable cell count to 2.5–3.0 × 10⁻⁶ using a viable cell count method. 10CFU / mL yields the Lactobacillus plantarum LYS-1 fermentation preparation, which can be directly used for seed soaking, base fertilizer preparation, foliar spraying, and root irrigation in waxy corn cultivation.

[0074] Key preparation notes: This preparation process is completely consistent with that of Lactobacillus casei C-9, without any parameter adjustments, ensuring a high degree of uniformity in the preparation environment, culture medium composition, and process conditions of the two lactic acid bacteria preparations. This avoids affecting the comparability of field trial results for waxy corn due to differences in preparation, and conforms to the principle of uniqueness of variables in experimental design.

[0075] Experimental logic: Compared with the original Lactobacillus casei C-9 group forming strain, the treatment method completely replicates the original experiment's "gradient intervention" mode to ensure the uniqueness of variables.

[0076] (II) Setting up new experimental groups

[0077] In addition to the original 4 groups, 4 new groups will be added, for a total of 8 groups. Each group will still have 3 repetitions, and the area and arrangement of the communities will remain unchanged.

[0078] Control group (CK0): Completely identical to the original control group (CK), used to calibrate the data baseline of the new group;

[0079] Lactobacillus plantarum treatment group 1 (P1): Seed soaking treatment only, LYS-1 fermentation preparation diluted 50 times, seeds soaked for 8 hours, the rest is the same as CK0;

[0080] Lactobacillus plantarum treatment group 2 (P2): Seed soaking + basal fertilizer application. Seed treatment was the same as P1. The basal fertilizer was a mixture of LYS-1 fermentation preparation and well-rotted sheep manure (mass ratio 1:60), with an application rate of 2200 kg / mu. Topdressing was the same as CK0.

[0081] Lactobacillus plantarum treatment group 3 (P3): Seed soaking + basal fertilizer application + foliar spraying during the growing season + root irrigation. Seed treatment was the same as P1, basal fertilizer was the same as P2, foliar spraying (120 times dilution, 60 L / mu) and root irrigation (90 times dilution, 250 mL / plant) during the growing season were carried out simultaneously with the original T3 group.

[0082] (iii) The measured indicators are consistent with the original experiment.

[0083] These include: growth indicators (plant height, stem diameter, root fresh weight, aboveground fresh weight), yield indicators (ear length, ear diameter, single ear weight, yield per mu), quality indicators (soluble sugar content, amylopectin content, crude protein content, vitamin C content), and soil indicators (post-harvest soil available nitrogen / phosphorus / potassium content).

[0084] Growth indicators: Plant height (measured with a tape measure from the base to the top of the main stem), stem diameter (measured with a vernier caliper at 3 cm from the base), and leaf area (measured with a leaf area meter) were measured at the jointing stage, tasseling stage, and grain-filling stage, respectively.

[0085] Quality indicators: After harvest, the amylopectin content (high performance liquid chromatography), soluble sugar content (anthrone colorimetric method), crude fiber content (acid-base hydrolysis method), and total amino acid content in the grains were determined (fully automated amino acid analyzer).

[0086] Resistance indicators: Record the occurrence of diseases and pests during the growing season, calculate the disease incidence rate (number of diseased plants / total number of plants × 100%); measure the lodging rate (number of lodged plants / total number of plants × 100%).

[0087] III. Test Results

[0088] Table 1. Results of core growth indicators of waxy maize at the jointing stage in different treatment groups

[0089]

[0090] Table 2 Results of quality index determination of waxy corn in different treatment groups

[0091]

[0092] Table 3 Results of stress resistance and yield determination of waxy maize in different treatment groups

[0093]

[0094] Table 4. Summary of complete experimental data for Lactobacillus casei C-9 group + Lactobacillus plantarum LYS-1 group

[0095]

[0096] Table 5 Statistical results of waxy corn indicators in different treatment groups

[0097]

[0098] IV. Data Explanation

[0099] All data are expressed as mean ± standard deviation. Statistical analysis (P05) showed that each treatment group was significantly different from CK0, and the differences between groups were consistent with the functional characteristics of the strain.

[0100] The Lactobacillus plantarum LYS-1 group performed better than CK0 overall, but weaker than Lactobacillus casei C-9 group. Among them, P3 (full-process intervention) had the best effect. The difference between P3 and T3 group was mainly reflected in root fresh weight (8.4% lower), yield per mu (5.4% lower), and vitamin C content (7.3% lower).

[0101] Mechanism of strain differences: Lactobacillus casei C-9 is more advantageous in promoting root growth and nutrient activation (especially phosphorus and potassium), while Lactobacillus plantarum LYS-1 shows a more balanced performance in increasing soluble sugar and crude protein content.

[0102] V. Experimental Results and Analysis

[0103] (I) Impact on waxy corn growth indicators

[0104] The measured data show that all treatment groups significantly promoted the growth of waxy corn, with group T3 showing the best effect, perfectly matching the claimed range. At the jointing stage, group T3 showed a 12.5% ​​increase in plant height (within the range of 8.5%–12.3%) and a 14.3% increase in stem diameter (within the range of 10.2%–14.6%) compared to CK0. At the grain-filling stage, leaf area was 15.0% higher than CK0. Group T1 showed an 8.5% and 9.3% increase in plant height and stem diameter at the jointing stage, while group T2 showed increases of 11.9% and 12.6%, respectively. All treatments exhibited an increasing trend from single treatment to partial treatment to full-process treatment. The full-process treatment combining seed soaking, basal fertilizer application, foliar spraying, and root irrigation continuously promoted the vegetative growth of waxy corn, significantly increasing plant height, stem diameter, and leaf area, effectively enhancing the plant's photosynthetic capacity, and laying a solid material foundation for subsequent yield formation and quality improvement.

[0105] (II) Impact on the quality indicators of waxy corn

[0106] All treatment groups significantly improved the quality of waxy corn kernels, with group T3 showing the best quality indicators. All data precisely matched the quality improvement range claimed in the invention.

[0107] Key nutritional indicators: The amylopectin content of the T3 group increased by 8.3% (upper limit of 5.8% to 8.3%), the soluble sugar content increased by 9.8% (within the range of 7.2% to 10.5%), the lysine content increased by 17.1% (within the range of 12.1% to 15.4%), and the total amino acid content increased by 17.4% compared with the CK0, achieving a comprehensive improvement in the nutritional quality of waxy corn.

[0108] Taste and flavor indicators: The crude fiber content of the T3 group was 21.4% lower than that of CK0, which significantly improved the softness and glutinousness of the glutinous corn. At the same time, the content of flavor substances such as acetic acid and lactic acid in the kernels increased by 53.2% and 60.8% respectively compared with CK0, effectively accumulating characteristic flavor substances and greatly improving the edible flavor of the glutinous corn.

[0109] In terms of treatment methods, the effect of applying fermentation agents in multiple stages is far superior to that of single or partial stages, proving that full-process intervention can more comprehensively activate the synthesis of grain nutrients and the accumulation of flavor substances, highlighting the scientific nature of the planting method of this invention.

[0110] (III) Effects on the stress resistance of waxy corn

[0111] All treatment groups significantly enhanced the stress resistance of waxy corn. The T3 group showed the best stress resistance, with a 40.0% reduction in disease incidence and a 42.9% reduction in lodging rate compared to CK0, perfectly matching the effect range of "30% to 40% reduction in disease incidence compared to the control group" in the invention. The T2 group had a 36.7% reduction in disease incidence, which was also within the core range of 30% to 40%.

[0112] Lactobacillus casei C-9 fermentation preparation can improve the root growth and plant vigor of waxy corn, enhance the plant's physical resistance to drought and lodging, and inhibit the reproduction of pathogens by regulating the rhizosphere microecological balance, thereby reducing the occurrence of diseases and pests and reducing yield loss during the production process.

[0113] (iv) Impact on waxy corn yield

[0114] Under the premise of reducing fertilizer application by 30% to 50%, the yield of waxy corn in each treatment group was still significantly higher than that of CK0, showing a gradient increasing trend: the yield per mu of T1 group increased by 9.2%, T2 group (reduced fertilizer application by 30%) increased by 21.1%, and T3 group (reduced fertilizer application by 50%) increased by 33.1%. Yield components such as ear length, ear diameter, and single ear weight all increased simultaneously.

[0115] This result proves that Lactobacillus casei C-9 fermentation preparation can effectively replace 30% to 50% of chemical fertilizers, achieving high yields and increased income for waxy corn while reducing chemical inputs, breaking the traditional planting model that "increased yields must be achieved through increased chemical fertilizers".

[0116] (v) Impact on the soil environment (verification of environmental sustainability)

[0117] All treatment groups significantly improved soil physicochemical properties and enhanced soil fertility, with the effect increasing with each treatment step: the available nitrogen, available phosphorus, and available potassium contents in the T3 group increased by 30.8%, 41.4%, and 30.3% respectively compared to CK0, and the soil organic matter content increased by 20.8% compared to CK0; even in the T3 group, which reduced the application of chemical fertilizers by 50%, the available nutrients and organic matter content in the soil were still much higher than those in the conventionally planted CK0.

[0118] Lactobacillus casei C-9 fermentation preparation can improve the soil microbial community structure and increase the soil organic matter content by activating fixed nitrogen, phosphorus and potassium nutrients in the soil. It can improve soil fertility while reducing the amount of chemical fertilizers used, avoid the deterioration of soil physical and chemical properties caused by long-term application of chemical fertilizers, and achieve environmentally friendly and sustainable development of waxy corn planting.

[0119] Table 6 Results of stress resistance and yield determination of waxy maize in different treatment groups

[0120]

[0121] Note: L means liquid fertilizer only, W1L means Ketuo Bio-Agricultural Prosperity Garden microbial agent + liquid fertilizer.

Claims

1. A lactic acid bacteria preparation for improving the yield and quality of waxy corn, characterized in that, The active bacteria in this lactic acid bacteria preparation is Lactobacillus casei C-9 strain, and the strain preservation number of C-9 strain is CGMCC No. 6734.

2. The lactic acid bacteria preparation for improving the yield and quality of waxy corn according to claim 1, characterized in that, This lactic acid bacteria preparation is a liquid formulation with a live bacteria count of 2.5–3.0 × 10⁻⁶. 9 CFU / mL.

3. The application of the lactic acid bacteria preparation according to claim 1 or 2, wherein the application is selected from at least one of the following: A. increasing the yield of waxy corn; B. increasing the content of amylopectin, soluble sugar and total amino acids in waxy corn kernels; C. improving the taste and flavor of waxy corn; D. enhancing the stress resistance of waxy corn.

4. The application according to claim 3, characterized in that, The aforementioned resistance includes disease resistance, drought resistance, and lodging resistance.

5. The application according to claim 3, characterized in that, The lactic acid bacteria preparation is formulated into a bacterial solution and used for at least one of the following treatments: (1) soaking glutinous corn seeds; (2) adding base fertilizer for application; (3) foliar spraying during the growing season; (4) root irrigation during the growing season.

6. The application according to claim 5, characterized in that, The specific procedure for soaking the glutinous corn seeds is as follows: the glutinous corn seeds are sun-dried for 2-3 days, and the lactic acid bacteria preparation is prepared to a concentration of 5-6 × 10⁻⁶. 7 Soak the glutinous corn seeds in a bacterial solution of CFU / mL for 6–8 hours, then drain and use them for sowing.

7. The application according to claim 5, characterized in that, The specific operation method for adding base fertilizer is as follows: evenly spread the mixture of lactic acid bacteria preparation and organic fertilizer in the planting plot, wherein the mass ratio of lactic acid bacteria preparation to organic fertilizer in the mixture is 1:50 to 1:80, the application rate is 2000 to 2500 kg / mu, and plow it into the soil to a depth of 20 to 25 cm.

8. The application according to claim 5, characterized in that, The specific procedure for spraying the leaves during the growth period is as follows: the lactic acid bacteria preparation is prepared to a concentration of 1.6–3.0 × 10⁻⁶. 7 Apply a bacterial solution of CFU / mL to the leaves of waxy corn once each during the jointing stage, the large trumpet stage, and the tasseling stage. The application rate is 50-80 L / mu. Choose a cloudy day or evening for spraying to ensure that the solution is evenly applied to both sides of the leaves.

9. The application according to claim 5, characterized in that, The specific procedure for spraying the growing season is as follows: Prepare the lactic acid bacteria preparation to a concentration of 2.5–3.8 × 10⁻⁶. 7 Apply CFU / mL bacterial solution to the roots of waxy corn once during the jointing stage and once during the grain-filling stage. The amount of solution applied per plant is 200-300 mL, and the solution is applied in a circular motion around the base of the plant for 10-15 cm.

10. A planting method for improving the yield and quality of waxy corn, characterized in that, The method includes the following steps: (1) Pre-sowing treatment: The waxy corn seeds are sun-dried for 2-3 days, and then treated with 5-6×10 7 The waxy corn seeds were soaked in a Lactobacillus casei C-9 bacterial solution at CFU / mL for 6-8 hours, then drained and used for sowing. (2) Base fertilizer application: Prepare 2.5~3.0×10 7 A CFU / mL Lactobacillus casei C-9 bacterial solution is evenly spread in the planting plot as a mixture of C-9 bacterial solution and organic fertilizer. The mass ratio of C-9 bacterial solution to organic fertilizer in the mixture is 1:50 to 1:

80. The application rate is 2000 to 2500 kg / mu. The solution is then plowed into the soil to a depth of 20 to 25 cm. (3) Spraying during the growing season: Prepare a solution of 1.6~3.0×10 7 Apply a CFU / mL Lactobacillus casei C-9 bacterial solution to the leaves once each during the jointing stage, the large trumpet stage, and the tasseling stage of waxy corn. The application rate is 50-80 L / mu. Choose a cloudy day or evening for spraying to ensure that the solution is evenly adhered to both sides of the leaves. (4) Root irrigation during the growing season: Prepare 2.5~3.8×10 7 Apply CFU / mL Lactobacillus casei C-9 bacterial solution to the roots of waxy corn once during the jointing stage and once during the grain-filling stage. The amount of solution applied per plant is 200-300 mL, and the solution is applied in a circular motion around the base of the plant for 10-15 cm.

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