A biological seed coating agent to enhance seed drought resistance, its preparation method and application

Abstract

This invention belongs to the field of seed treatment technology and discloses a biological seed coating agent for improving seed drought resistance, its preparation method, and its application. The biological seed coating agent is composed of drought-resistant bacterial strains, sodium alginate, and carboxymethyl chitosan, wherein the drought-resistant bacterial strain is... Acinetobacter The strain, sp., is numbered CICC 24296. When the mass ratio of sodium alginate to carboxymethyl chitosan solution in the biological seed coating agent was 6:1, the biocompatibility with the strain was good, and the effective viable count of microorganisms in the biological seed coating agent reached 2.30 × 10⁻⁶. 8 Compared to other treatment groups, the effective viable bacteria count increased by 36.09–265.08%. When this biological seed coating agent was used for seed coating, the seed water retention capacity, rehydration capacity after dehydration, seed vigor, and seedling drought resistance were optimal when the seed weight gain rate was 40–60%. This weight gain rate index can effectively reflect the stability of the coating and the physiological performance of the seeds, and has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of seed treatment technology, and relates to a biological seed coating agent for improving seed drought resistance, its preparation method and application. Background Technology

[0002] Seed coating technology is a technique that uses mechanical or manual methods to uniformly coat the seed surface with a mixture of active ingredients such as pesticides, fungicides, plant growth regulators, fertilizers, and biological agents, along with inactive ingredients such as film-forming agents and dispersants, forming a protective film. This technology can simultaneously exert multiple effects, including seedling pest and disease control, growth regulation, and stress resistance. It has significant application value in improving seed vigor, ensuring uniform emergence, reducing pesticide use in the field, and achieving cost reduction and yield increase, and has become a widely used technique in crop cultivation.

[0003] Existing seed coating agents mainly fall into two categories: chemical seed coating agents and biological seed coating agents. Chemical seed coating agents, with chemical pesticides as their core active ingredient, offer advantages such as rapid onset of action, stable efficacy, and low cost. However, they suffer from drawbacks including soil residue, non-target biotoxicity, and the potential to induce pesticide resistance in pathogens. Furthermore, chemical fungicides and insecticides exhibit inherent antagonistic effects when directly combined with beneficial microorganisms, making it difficult to achieve a synergistic effect of rapid chemical protection and long-lasting biological growth promotion through conventional mixing. Biological seed coating agents offer advantages such as environmental friendliness, no residue, and crop safety, but they generally suffer from low viable bacterial counts, poor application stability, and short-lasting effects. Therefore, developing a biological seed coating agent containing microorganisms, with a substrate exhibiting good biocompatibility and a high initial viable bacterial count, is of great significance.

[0004] Meanwhile, quality control after seed coating is also a crucial factor limiting the effectiveness of seed coating agents. Existing evaluation methods for seed coating quality mainly include the following indicators: physical indicators, such as coating uniformity, coating coverage, coating peeling rate, and film formation time; chemical indicators, such as pH value, suspension rate, active ingredient content, and low-temperature stability of the seed coating agent; and physiological and field indicators, such as seed germination rate, seedling emergence rate, and field pest and disease control effectiveness. However, these quality control methods are generally cumbersome and time-consuming, making them difficult to meet the online quality control needs of seed processing lines. Therefore, providing a simple, convenient, and efficient method for evaluating seed coating quality is of great significance for ensuring the commercial quality of coated seeds and improving production efficiency. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a biological seed coating agent for improving seed drought resistance, its preparation method, and its application. This biological seed coating agent is composed of drought-resistant bacterial strains, sodium alginate, and carboxymethyl chitosan, wherein the drought-resistant bacterial strain is... AcinetobacterThe strain, sp., is numbered CICC 24296. In this invention, the bio-coating agent containing sodium alginate and carboxymethyl chitosan solution at a mass ratio of 6:1 exhibits good biocompatibility with drought-resistant strains, and the effective viable count of microorganisms in the bio-coating agent reaches 2.30 × 10⁻⁶. 8 Compared to other quality groups, the effective viable bacteria count increased by 36.09–265.08%. When the above-mentioned biological seed coating agent was used for seed coating, the seed weight gain rate was 40–60%, resulting in optimal water retention capacity, rehydration capacity after dehydration, seed vigor, and drought resistance of seedlings developed from these seeds. This weight gain rate is directly related to the amount of seed coating agent loaded on the seed surface, effectively reflecting the stability of the coating process. It can also serve as a convenient evaluation indicator for predicting seed germination water regulation capacity and seedling establishment quality, showing promising application prospects in the quality control of biological seed coating agents.

[0006] On the one hand, the present invention provides a biological seed coating agent to improve the drought resistance of seeds, wherein the effective components of the biological seed coating agent are composed of drought-resistant strains, sodium alginate and carboxymethyl chitosan;

[0007] The drought-resistant strain is Acinetobacter sp., strain number CICC 24296; The mass ratio of sodium alginate to carboxymethyl chitosan in the biological seed coating agent is 6:1 to 20:27. The effective viable count in the system after the addition of the drought-resistant strain was 6.3 × 10⁻⁶. 7 ~2.3×10 8 CFU / g biological seed coating agent.

[0008] Furthermore, the preparation of the drought-resistant strain includes: adding 5 mL of OD... 600 A value of 0.8 Acinetobacter The drought-resistant strain was obtained by centrifuging the bacterial culture at 6000 rpm for 5 min.

[0009] Furthermore, the mass ratio of sodium alginate to carboxymethyl chitosan in the biological seed coating agent is 6:1.

[0010] On the other hand, the present invention seeks protection for a coating method of the above-mentioned biological seed coating agent, comprising: Sodium alginate solution and carboxymethyl chitosan solution were prepared, mixed, and then drought-resistant bacterial strains were added to obtain a coating solution; Add the seeds to the coating solution, soak and stir for 2-3 minutes to obtain a coating mixture; The coating mixture is added to a calcium chloride solution for cross-linking and curing for 4-5 minutes to obtain coated seeds.

[0011] Furthermore, the concentration of the sodium alginate solution is 3-5 wt%; The concentration of the carboxymethyl chitosan solution is 0.5~0.7 wt%; The concentration of the calcium chloride solution is 0.1~0.2 mol / L.

[0012] Furthermore, the mass ratio of sodium alginate solution to carboxymethyl chitosan solution in the mixture is 9:1 to 1:9.

[0013] Furthermore, the mass ratio of sodium alginate solution to carboxymethyl chitosan solution in the mixture is 9:1.

[0014] On the other hand, the present invention seeks protection for the application of the above-mentioned biological seed coating agent in seed coating, wherein the weight gain rate after seed coating is 40-60%.

[0015] Furthermore, the biological seed coating agent improves the water retention capacity and rehydration capacity of coated seeds after dehydration.

[0016] Furthermore, the biological seed coating agent enhances seed vigor.

[0017] Furthermore, the seedlings developed from the seeds treated with the biological seed coating agent exhibit improved drought resistance.

[0018] Specifically, this invention, through experiments, found that the coated seeds in the 50% weight gain treatment group (S50) exhibited excellent combined water retention and rehydration performance. During the hygroscopic phase, the water absorption after 48 hours was 188.9% of that of uncoated seeds, and the coating layer did not hinder water absorption. After 48 hours of dehydration, the S50 treatment group retained the highest standardized moisture content, at 0.072 g water / g dry weight. During the rehydration phase, the S50 treatment group also exhibited the highest standardized moisture content, at 0.358 g water / g dry weight, significantly higher than other treatment groups. The S50 treatment group also had a higher viable bacterial load, at 8.8 × 10⁻⁶. 8 CFU / g. The vigor index of the S50 treatment group was 11.0–12.1% higher than that of uncoated seeds, 4.6–10.3% higher than that of S25, and 26.6–32.1% higher than that of S10, showing the best seed vigor performance. After index normalization, S50 was found to be the best-performing seed coating. These results indicate that weight gain rate can effectively characterize the coating effect of the coating agent on seeds. When the weight gain rate is 40–60%, the seed's water retention capacity, rehydration capacity after dehydration, seed vigor, and drought resistance of seedlings developed from these seeds are optimal.

[0019] Furthermore, this invention seeks protection for a method for evaluating the coating quality of the aforementioned biological seed coating agent, comprising: Weigh the seeds, weigh the seeds after coating, and calculate the weight gain rate after coating. When the weight gain rate is within the range of 40% to 60%, the coating quality is qualified. The seeds include corn seeds.

[0020] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) This invention provides a biological seed coating agent to improve the drought resistance of seeds, which is composed of drought-resistant strains, sodium alginate and carboxymethyl chitosan, wherein the drought-resistant strains are Acinetobacter sp., drought-resistant strains in the biological seed coating agent of this invention Acinetobacter *Sp.* exhibits superior drought resistance, nitrogen fixation, phosphorus solubilization, and growth-promoting properties. In this invention, when the mass ratio of sodium alginate solution to carboxymethyl chitosan solution in the biological seed coating agent is 9:1, the biocompatibility of the drought-resistant strain is good, and the effective viable count of microorganisms in the biological seed coating agent reaches 2.30 × 10⁻⁶. 8 Compared with other quality groups, the number of effective viable bacteria increased by 36.09~265.08%.

[0021] (2) Based on this biological seed coating agent, this invention also provides a quality evaluation index for the seed coating effect, and the coating effect is optimal for seeds with a seed weight gain rate of 40-60%. Through experiments, this invention found that the coated seeds in the 50% weight gain rate (S50) treatment group have good combined water retention and rehydration performance. During the hygroscopic stage, the water absorption rate after 48 hours was 188.9% of that of uncoated seeds, and the coating layer did not hinder water absorption. The standardized moisture content retained after 48 hours of dehydration was the highest, at 0.072 g water / g dry weight. During the rehydration stage, the S50 treatment group had the highest standardized moisture content, at 0.358 g water / g dry weight, significantly higher than other treatment groups. The viable bacterial load of the S50 treatment group was relatively high, at 8.8 × 10⁻⁶. 8 CFU / g. The vigor index of the S50 treatment group was 11.0–12.1% higher than that of uncoated seeds, 4.6–10.3% higher than that of S25, and 26.6–32.1% higher than that of S10, showing the best seed vigor performance. After index normalization, S50 was found to be the best-performing seed coating. These results indicate that weight gain rate can effectively characterize the coating effect of the coating agent on seeds. When the weight gain rate is 40–60%, the seed's water retention capacity, rehydration capacity after dehydration, seed vigor, and drought resistance of seedlings developed from these seeds are optimal. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 for Acinetobacter The growth-promoting properties of sp. Figure 1In the diagram, A represents the culture of the strain on nitrogen-fixing solid medium; Figure 1 B in the diagram represents the culture of the strain on organic phosphorus solid medium; Figure 1 C in the figure represents the culture of the strain on inorganic phosphorus solid medium.

[0024] Figure 2 The moisture dynamics curves of coated seeds in different load groups during the hygroscopic-dehydration-rehydration stages are shown.

[0025] Figure 3 The results of the vigor index I for coated seeds under different load groups are shown in the figure.

[0026] Figure 4 The results of the Vigor Index II for coated seeds under different load groups are shown in the figure.

[0027] Figure 5 The results of chlorophyll content in maize plants under different treatment groups are shown in the figure. Detailed Implementation

[0028] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially. Unless otherwise specified, the percentages in the following embodiments refer to mass percentages.

[0029] Beef extract peptone medium (LB): tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0030] Inorganic phosphorus culture medium: glucose 10 g / L, ammonium sulfate 0.5 g / L, yeast extract 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, tricalcium phosphate 5 g / L, pH 7.0~7.5, 25℃.

[0031] Organophosphorus culture medium: glucose 10 g / L, ammonium sulfate 0.5 g / L, yeast extract 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, lecithin 0.2 g / L, calcium carbonate 1 g / L, pH 7.0~7.5, 25℃.

[0032] Nitrogen-fixing medium: potassium dihydrogen phosphate 0.2 g / L, magnesium sulfate 0.2 g / L, sodium chloride 0.2 g / L, calcium carbonate 5.0 g / L, mannitol 10.0 g / L, calcium sulfate 0.1 g / L, pH 7.0 ± 0.1, 25℃.

[0033] Example 1 This embodiment provides the selection and preparation of microbial strains for biological seed coating agents.

[0034] 1. Selection of microbial strains Two existing microorganisms in the laboratory that may possess drought-resistant and growth-promoting properties were used as experimental materials, namely... Acinetobacter sp. (CICC 24296) and Pseudomonas resinovorans (ATCC 14235). 20% PEG-6000 was selected as the drought treatment, with no PEG-6000 added as the control. Each treatment was repeated in triplicate. Candidate strains were picked and added to 20 mL of LB liquid medium and cultured on a shaker at 28°C and 180 rpm for 3 days. OD was then adjusted. 600 The bacterial suspension was inoculated at a rate of 1% (v / v) into 20 mL of LB liquid medium in both the drought treatment group and the control group, and cultured on a shaker for 3 days under the same conditions. OD was then measured. 600 The absorbance values ​​were used to determine the extent of the decrease in microbial cell count, and the results are shown in Table 1.

[0035] OD 600 Bacterial suspensions with a concentration of approximately 0.6 μL were added dropwise to nitrogen-fixing, organic phosphorus, and inorganic phosphorus LB solid media, respectively. Each plate was added three times, 2.5 μL each time. After the bacterial suspensions were completely dry, they were incubated upside down in a 28°C incubator for 3 days. The presence or absence of a clear zone was observed. Acinetobacter The culture results of sp. are as follows Figure 1 As shown. Strains with a clear zone are considered growth-promoting strains; the larger the clear zone, the stronger the growth-promoting properties of the strain. Culture revealed... Pseudomonas resinovorans A small clear zone was observed only on nitrogen-fixing medium; no clear zone was observed on either organic or inorganic phosphorus solid media. Acinetobacter sp. showed a clear transparent zone in nitrogen-fixing, organic phosphorus, and inorganic phosphorus solid media.

[0036] Table 1 Growth characteristics of candidate strains under drought conditions

[0037] In conclusion, choose Acinetobacter sp. to conduct subsequent seed coating experiments.

[0038] Example 2 This embodiment provides a biological seed coating agent for... Acinetobacter The effect of sp. effective viable bacteria count.

[0039] 1. Preparation of biological seed coating agents Microbial cell preparation: OD 600The bacterial suspension with an OD value of approximately 0.6 was inoculated into a new 20 mL LB liquid medium at an inoculation rate of 1% (v / v). The medium was cultured for 3 days under constant temperature shaking conditions at 28℃ and 180 rpm. After the OD value at a wavelength of 600 nm was measured to be approximately 0.8, 5 mL of the bacterial suspension was taken and centrifuged at 6000 rpm for 5 min. The supernatant was discarded, and the precipitate was used to obtain 1 part of microbial cells (0.3 g).

[0040] 12g of sodium alginate (SA) powder was dissolved in 300mL of water to achieve a concentration of 4% (wt / vol) and sterilized at 121℃ under high temperature and autoclave for 15min to obtain a sodium alginate solution. 0.6g of carboxymethyl chitosan (CMCS) powder was dissolved in 100mL of aqueous solution to achieve a concentration of 0.6% (wt / vol) and sterilized at 121℃ under high temperature and autoclave for 15min to obtain a carboxymethyl chitosan solution. The sodium alginate solution and carboxymethyl chitosan solution were mixed at mass ratios of 1:9, 3:7, 5:5, 7:3, and 9:1, respectively, and then one part of microbial cells was added to each to obtain a coating solution. The coating solution was injected into a 0.15mol / L CaCl2 solution using a syringe to obtain a biological seed coating agent.

[0041] 2. Detection of the number of viable bacteria in microspheres Accurately weigh 1.0 g of microspheres into a centrifuge tube, add 0.2 mol / L sterile sodium citrate solution (sterilized at 121℃ for 30 min) to a final volume of 10 mL. This is a 10:10 dilution. -1 Gradient mixing, inverting and shaking for 5-10 minutes to fully dissolve the embedded particles. Take 10 μL of each sample. -3 10 -4 10 -5 10 -6 10 μL of each of the four dilution gradients was added dropwise to LB solid medium and spread evenly with a sterile spreader until the surface moisture disappeared. The medium was incubated at 32°C for 24–48 h. Plate counts were performed on medium with colony counts of 30–300. Each treatment was replicated in three places. The treatment group with the highest average viable count was used as the target amount. The effective viable count was calculated as follows. The statistical results of the effective viable counts of different treatment groups are shown in Table 2.

[0042]

[0043] Table 2. Effective viable counts of microorganisms in biological seed coating agents with different mass ratios of carboxymethyl chitosan solution and sodium alginate solution.

[0044] Note: Taking SA:CMCS=9:1 as an example, 230 single colonies represent the microorganisms contained in 10 μL of bacterial culture, diluted 10... -4Next, 1 mL should have been taken to check the colony count, but only 10 μL was actually taken, a reduction of 100 times. Therefore, the number of viable bacteria per gram of microspheres was 230 × 100 × 10⁻⁶. 4 =2.3×10 8 .

[0045] Table 2 shows that the highest number of viable microorganisms was achieved when the mass ratio of sodium alginate solution to carboxymethyl chitosan solution was 9:1. Therefore, this mass ratio was used for subsequent experiments. Beyond the 1:9 to 9:1 ratio range, the system approached a single polysaccharide system, and the synergistic effect of the two polysaccharides completely disappeared, exhibiting only the effect of a single material.

[0046] Example 3 This embodiment provides a method for quality control of biological seed coating agents.

[0047] 1. Seed coating treatment Take a batch of uniformly sized corn seeds (approximately 0.35 g per seed), weigh 10 g of seeds for each treatment, and perform three biological replicates per group. Pre-treat the corn seeds with 75% ethanol for 45 s, rinse three times with sterile water, then soak in a NaClO solution containing 4% available chlorine for 2 min, and rinse thoroughly with sterile water. Mix the sodium alginate solution and carboxymethyl chitosan solution from Example 2 at a mass ratio of 9:1, add one part of microbial cells, and shake to mix thoroughly to obtain the coating solution. Add the sterilized seeds to the coating solution, soak and stir for 2 min, then transfer to a 0.15 mol / L sterile calcium chloride solution, cross-link and solidify for 5 min, and weigh to calculate the weight gain rate. By adjusting the coating process parameters (the leaching time before transferring to the 0.15 mol / L sterile calcium chloride solution after stirring; shorter leaching time results in a higher coating weight gain rate), three coated seeds with different weight gain rate gradients were prepared, with three replicates for each treatment. The weight gain groups were: low-load group S10 (total weight before coating: 9.5~10.5g, total weight after coating: 10.45~11.5g, weight gain rate: approximately 10%), medium-load group S25 (total weight before coating: 9.5~10.5g, total weight after coating: 11.9~12.9g, weight gain rate: approximately 25%), and high-load group S50 (total weight before coating: 9.5~10.5g, total weight after coating: 14.3~15.8g, weight gain rate: approximately 50%).

[0048] Weight gain rate = (Total weight after coating - Total weight before coating) / Total weight before coating × 100% Observation of coated seeds in different load groups revealed that the coating layer in the low load group (weight gain rate of about 10%) was extremely thin and almost invisible to the naked eye; the coating layer in the medium load group (weight gain rate of about 25%) was visible, and the seed surface had a noticeable luster; the coating layer in the high load group (weight gain rate of about 50%) was the thickest, and the seed surface was evenly and completely covered.

[0049] 2. Moisture dynamics determination of coated seeds under different load groups Moisture absorption stage: Ten coated seeds of different load groups were placed in petri dishes containing filter paper pre-soaked in 10 mL of distilled water. The petri dishes were placed in a constant temperature incubator at 25℃. Fresh weight was measured at 6 h, 12 h, 24 h, 36 h and 48 h after moisture absorption. The weight of each seed at each time point was recorded. Each treatment was repeated three times.

[0050] Dehydration stage: After absorbing moisture for 48 hours, the seeds were removed from the petri dish, placed in an open petri dish, and put into a 35℃ constant temperature incubator. The seed weight was recorded at 6h, 12h, 24h, 36h, and 48h after dehydration, and the moisture loss rate was calculated.

[0051] Rehydration stage: After dehydration for 48 hours, the seeds were placed again in a petri dish containing 10 mL of distilled water pre-soaked filter paper and incubated at a constant temperature of 25°C. Fresh weight was measured at the same time intervals as in the hygroscopic stage (6 h, 12 h, 24 h, 36 h, 48 h).

[0052] Final dry weight determination: After rehydration for 48 hours, the seeds were dried at 130℃ for 1 hour, and the final dry weight was determined.

[0053] The moisture dynamics curves of the three stages of hygroscopic absorption, dehydration, and rehydration of coated seeds under different load groups are shown below. Figure 2 As shown.

[0054] Depend on Figure 2 The results showed that after 48 hours of moisture absorption, the standardized moisture content (SMC) of the uncoated control group (CK) seeds was 0.323 g water / g dry weight, the S10 (10% weight gain rate) coated seeds had a SMC of 0.364 g water / g dry weight (112.7% of CK), the S25 (25% weight gain rate) coated seeds had a SMC of 0.455 g water / g dry weight (140.9% of CK), and the S50 (50% weight gain rate) coated seeds had a SMC of 0.610 g water / g dry weight (188.9% of CK). These results indicate that the SMC of coated seeds significantly increased after 48 hours of moisture absorption with increasing weight gain rate, with the 50% weight gain rate coated seeds exhibiting the highest moisture content, providing sufficient water reserves for seed germination.

[0055] Depend on Figure 2It can be seen that after dehydration at 35℃ for 48 hours, the standardized moisture content of the uncoated control group seeds (CK) decreased from 0.323 g water / g dry weight after moisture absorption to 0.062 g water / g dry weight, with a moisture loss rate of 80.8%. The standardized moisture content of S10 (10% weight gain) coated seeds after 48 hours of dehydration was 0.052 g water / g dry weight, with a moisture loss rate of 85.7%. The standardized moisture content of S25 (25% weight gain) coated seeds after 48 hours of dehydration was 0.071 g water / g dry weight, with a moisture loss rate of 84.4%. The standardized moisture content of S50 (50% weight gain) coated seeds after 48 hours of dehydration was the highest among all groups, at 0.072 g water / g dry weight, 16.1% higher than CK, with a moisture loss rate of 88.2%. The results showed that the coating layer with a 50% weight gain rate could retain the most moisture (by unit dry weight) after 48 hours of dehydration, and had the best water retention effect.

[0056] Depend on Figure 2 It can be seen that after one wet-dry cycle, the standardized moisture content of the uncoated control group seeds (CK) recovered from 0.062 g water / g dry weight after dehydration to 0.328 g water / g dry weight, with a moisture recovery rate of 101.1%, almost completely recovered. The standardized moisture content of the S10 (10% weight gain) coated seeds after 48 hours of rehydration was 0.318 g water / g dry weight, with a moisture recovery rate of 83.3%. The standardized moisture content of the S25 (25% weight gain) coated seeds after 48 hours of rehydration was 0.344 g water / g dry weight, with a moisture recovery rate of 72.1%. The standardized moisture content of the S50 (50% weight gain) coated seeds after 48 hours of rehydration was 0.358 g water / g dry weight, the highest among all groups, 9.1% higher than the CK, with a moisture recovery rate of 54.6%. Although the moisture recovery rate of S50 (54.6%) was lower than that of other groups, it had the highest standardized moisture content (0.072 g water / g dry weight) after 48 h of dehydration and the highest standardized moisture content (0.358 g water / g dry weight) after 48 h of rehydration. This indicates that the coated seeds with a 50% weight gain rate have good overall water retention and rehydration performance after undergoing a wet-dry cycle.

[0057] 3. Determination of seed vigor index of coated seeds in different load groups Twenty seeds were collected from each of the S10, S25, and S50 coated seeds and uncoated seeds (CK), with three replicates for a total of 60 seeds. Before the experiment, each seed was individually weighed and its initial mass recorded. Each seed was also numbered to track its germination progress. Subsequent analysis will use an analysis of covariance with initial seed weight as a covariate to eliminate the influence of seed size on growth and detect true differences between treatments. Coated and uncoated seeds were placed in sterile petri dishes, with sterile filter paper thoroughly moistened with sterile water placed on top as a germination bed. The seeds were cultured at a constant temperature of 25°C with a 12-hour photoperiod. To ensure the filter paper remained moist, 10 mL of sterile water was added to each petri dish daily. Germination was defined as a radicle length greater than 1 mm. Germination status of each seed was observed and recorded daily, including the germination date and seed number. The cumulative germination rate for each treatment group was also calculated. Germination experiments continued until no new seeds germinated for three consecutive days. The effect of seed coating on breaking dormancy and initiating germination was assessed by calculating the final germination rate. Seed coating weight gain exceeding 70% and excessively thick coating significantly reduced seed germination rate.

[0058] After seed germination was confirmed, the germination time was recorded. Samples were taken from each seed 48 hours after germination to standardize the physiological age of each seedling. Seedlings were carefully removed, avoiding damage to the root system. The shoot length (from seed attachment point to seedling tip) and root length (from seed attachment point to root tip) of each seedling were measured, and the total seedling length was calculated. The seedlings were then carefully separated and placed in an oven to dry at 70℃ for 12 hours. The dry weight of each seedling was then recorded using an analytical balance. The root system is a key organ for early nutrient absorption in crops, and dry matter accumulation directly reflects the photosynthetic production capacity and establishment quality of seedlings. Seedling length and dry weight together determine the robustness of the seedlings. Two vigor index formulas (Vigor Index I and Vigor Index II) were used. These indices combine the germination rate / ratio with the seedling growth potential and are reliable indicators for evaluating seed emergence and establishment potential under field conditions. They can more comprehensively reflect the overall effect of coating treatment and comprehensively evaluate seed vigor from different perspectives. The results are as follows: Figure 3 , Figure 4 As shown.

[0059] Vitality Index I = Germination Rate % × Seedling Length (cm) Vitality Index II = Germination Rate % × Seedling Dry Weight (mg) Depend on Figure 3The vigor index I showed certain differences among the treatment groups. The S50 treatment had the highest vigor index I at 502.6, significantly higher than the other treatment groups. The S25 treatment was second highest, with a vigor index I of 480.4, slightly lower than S50 but still at a high level. The uncoated control group (CK) had a vigor index I of 448.5, showing moderate performance. The S10 treatment had the lowest vigor index I at only 380.4, lower than the other groups. This result indicates that the S50 and S25 coating treatments have a positive effect on promoting seed germination and seedling root growth, while the S10 treatment showed a certain inhibitory effect. Overall, the vigor index I is quite sensitive to coating treatment and can effectively distinguish the combined effects of different treatments on seed germination and root formation.

[0060] Depend on Figure 4 It can be seen that the S50 treatment had the highest vitality index II, reaching 1505.1, which was about 11.0% higher than the control group. The S25 treatment had a vitality index II of 1364.8, which was basically the same as the control group, only slightly higher by 0.6%. The S10 treatment had the lowest vitality index II, at 1188.9, which was about 12.3% lower than the control group.

[0061] In summary, the seed coating weight gain rate is a good indicator of the seed coating effect, with the S50 treatment showing the best coating effect. The S50 treatment significantly promoted seedling dry matter accumulation, demonstrating the best coating effect; the S25 treatment showed no significant difference from the control group, failing to exhibit a clear promoting or inhibiting effect; while the S10 treatment showed an inhibitory effect, resulting in seedling dry matter accumulation lower than the control level. Overall, S50 is the optimal concentration of this coating agent under the experimental conditions, effectively improving the overall vigor of maize seeds.

[0062] 4. Determination of the number of viable bacteria embedded in seeds coated with different load groups Add 0.2 mol / L sterile sodium citrate solution (sterilized at 121℃ for 30 min) to the coated seeds of S50, S25, and S10, bringing the volume to 10 mL. This is a 10:10 dilution. -1 Gradient, inverted shaking for 50-60 minutes to fully dissolve the embedded particles. Add 10 -5 10 -6 10 -7 10 μL of each of the three dilution gradients was added dropwise to LB solid medium, allowed to stand for 30 seconds until the surface moisture disappeared, and incubated at 32°C for 24–48 h. Plate counts were performed on medium with colony counts of 20–300. Each treatment was replicated three times. The viable count was calculated as follows: viable count (CFU) = number of colonies in medium × dilution factor. The results are shown in Table 3.

[0063] Table 3. Number of viable bacteria embedded in seeds coated with different loading groups

[0064] As shown in Table 3, the effective viable cell count in treatment S25 was significantly higher than that in other treatment groups. Since the bacteria in the coated seeds were not pushed through the syringe, there was no high-pressure extrusion or rapid deformation, resulting in minimal physical impact and virtually no mechanical damage to the microorganisms. Therefore, the effective viable cell count was significantly higher than in Table 2. During syringe extrusion, the bacteria are forced through the narrow needle, generating strong fluid shear forces. This physical force directly tears or crushes the bacterial cell membrane / wall, leading to the immediate death of a large number of bacteria during preparation. Therefore, the initial viable cell count in Table 2 was lower.

[0065] 5. Indicator normalization processing The effective viable bacterial count, seed viability index, and moisture content (g water / g seed dry weight) data at three key points (hygroscopic endpoint, dehydration endpoint, and rehydration endpoint) during the hydration-dehydration-rehydration cycle of the three types of coated seeds were processed using a weighted comprehensive scoring method. First, each indicator was normalized to 0-1 to eliminate the influence of dimensions. The seed viability comprehensive score was the arithmetic mean of the two viability indices. Moisture content performance was calculated using a weighted average, with the weights allocated as follows: 0.2 for the hygroscopic endpoint, 0.4 for the dehydration endpoint, and 0.4 for the rehydration endpoint, to highlight water retention and recovery capabilities. The final comprehensive score weights were set as follows: seed viability 50%, effective viable bacterial count 30%, and moisture content performance 20%. The calculation results are shown in Table 4.

[0066] Table 4. Normalization results of each indicator

[0067] Table 4 shows that the final scores for the three treatments were: S50 0.824, S25 0.749, and S10 performed the worst in terms of overall viability, viable bacteria count, and water cycle. After normalization, all scores were 0.000. Among them, S50 performed best in both overall viability and water cycle, while S25 had the highest effective viable bacteria count and excellent overall viability. Considering all indicators, S50 is the best-performing seed coating and will be used for subsequent pot experiments.

[0068] Example 4 This embodiment provides a greenhouse pot experiment of coated seeds.

[0069] Pretreatment of potting soil: The soil material for the potted experiment was collected from Qinniu Agricultural Machinery Experimental Base in Wugong County, Xianyang City, Shaanxi Province. After the soil was sieved through a 2mm sieve, it was divided into PE flat bags, 5kg per bag, and sterilized by high-pressure steam at 121℃ for 1 hour.

[0070] Experimental groups: Two factors were set up: moisture and coating. The moisture factor included drought treatment and watering treatment. The coating factor was divided into uncoated (CK), mycelium root irrigation treatment (BS), S50 coating treatment (SMC, mycelium embedded in the coating agent), and S50 coating treatment (SSC, inactivated mycelium embedded in the coating agent). Each treatment was set up with three replicates, for a total of 24 pots of corn. The mycelium was applied around the seeds when the seeds were planted in the pots.

[0071] Corn Planting: Fill each flowerpot (19cm in diameter; 20cm in height) with soil from a separate bag. Plant 5 corn seeds with initial roots of about 2cm in length in each flowerpot and water with 300mL. Cover the flowerpot with a flat PE bag to prevent bacterial contamination. After two true leaves have emerged, remove the weaker seedlings, leaving 3 healthy and uniform seedlings in each pot, and add 50mL of sterile water. Water management begins one week after planting, with watering treatment (soil relative moisture content 70%~80%) and drought treatment (soil relative moisture content 40%~50%). Measure the soil relative moisture with a soil moisture meter every evening and replenish the moisture as needed to maintain the soil relative moisture. Continue water management for 30 days, then harvest the plant samples.

[0072] Measurement of maize growth indicators: Chlorophyll content was measured using a chlorophyll meter ( Figure 5 ).

[0073] Depend on Figure 5 It was found that, compared with the uncoated control group, the SMC treatment increased the SPAD value of maize leaves. Under drought conditions, the SMC treatment had the most significant effect on increasing plant height, reaching 29.42%, while the BS treatment increased it by 18.85% and the SSC treatment by 4.33%. Under normal irrigation conditions, the SMC treatment increased it by 23.32%, the BS treatment by 8.10%, and the SSC treatment by 1.92%, with the SMC treatment showing the strongest promoting effect.

[0074] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A biological seed coating agent for improving seed drought resistance, characterized in that, The active ingredients of the biological seed coating agent consist of drought-resistant bacterial strains, sodium alginate, and carboxymethyl chitosan. The drought-resistant strain is Acinetobacter sp., strain number CICC 24296; The mass ratio of sodium alginate to carboxymethyl chitosan in the biological seed coating agent is 6:1 to 20:

27. The effective viable count in the system after the addition of the drought-resistant strain was 6.3 × 10⁻⁶. 8 ~2.3×10 8 CFU / g biological seed coating agent.

2. The biological seed coating agent according to claim 1, characterized in that, The mass ratio of sodium alginate to carboxymethyl chitosan in the biological seed coating agent is 6:

1.

3. The coating method of the biological seed coating agent according to any one of claims 1 to 2, characterized in that, include: Sodium alginate solution and carboxymethyl chitosan solution were prepared, mixed, and then drought-resistant bacterial strains were added to obtain a coating solution; Add the seeds to the coating solution, soak and stir for 2-3 minutes to obtain a coating mixture; The coating mixture is added to a calcium chloride solution for cross-linking and curing for 4-5 minutes to obtain coated seeds.

4. The coating method for the biological seed coating agent according to claim 3, characterized in that, The concentration of the sodium alginate solution is 3-5 wt%; The concentration of the carboxymethyl chitosan solution is 0.5~0.7 wt%; The concentration of the calcium chloride solution is 0.1~0.2 mol / L.

5. The coating method for the biological seed coating agent according to claim 3, characterized in that, The mass ratio of sodium alginate solution to carboxymethyl chitosan solution in the mixture is 9:1 to 1:

9.

6. The application of the biological seed coating agent according to any one of claims 1 to 2 in seed coating, characterized in that, The weight gain rate after seed coating is 40-60%.

7. The application according to claim 6, characterized in that, The biological seed coating agent improves the water retention capacity of coated seeds and their rehydration capacity after dehydration.

8. The application according to claim 6, characterized in that, The biological seed coating agent enhances seed vigor.

9. The application according to claim 6, characterized in that, The biological seed coating agent enhances the drought resistance of seedlings during seed development.

10. The method for evaluating the coating quality of the biological seed coating agent according to any one of claims 1 to 2, characterized in that, include: Weigh the seeds, weigh the seeds after coating, and calculate the weight gain rate after coating. When the weight gain rate is within the range of 40% to 60%, the coating quality is qualified. The seeds include corn seeds.

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