Microbial coating agent for relieving salt stress of alfalfa, and preparation method and application thereof

Abstract

The present application belongs to the field of grass planting, and particularly relates to a microbial coating agent for relieving salt and alkali stress of alfalfa, a preparation method and application thereof. The microbial coating agent comprises endomycorrhizal fungus powder, Trichoderma harzianum powder, talcum powder, diatomite and carboxymethyl cellulose sodium aqueous solution. The endomycorrhizal fungus and Trichoderma harzianum are combined to coat alfalfa seeds, with Trichoderma harzianum as the main component and endomycorrhizal fungus as the auxiliary component. A small amount of endomycorrhizal fungus and Trichoderma harzianum are used to coat alfalfa seeds, which can relieve salt and alkali stress, significantly improve the emergence rate, promote the increase of alfalfa plant height, root length and biomass, promote the growth of alfalfa, and improve the stress resistance. The microbial coating agent has few components, low fungus content and low cost, and is suitable for large-scale popularization and use.

Description

Technical Field

[0001] This invention belongs to the field of forage cultivation and provides a green and efficient technical path for the alfalfa industry in saline-alkali areas. Specifically, it relates to a microbial coating agent for alleviating alfalfa saline-alkali stress, its preparation method, and its application. Background Technology

[0002] Seed coating is a high-tech method developed in the mid-1980s to promote increased agricultural production and harvests. It involves using adhesives or film-forming agents to coat the seed surface with non-seed materials such as fungicides, insecticides, micronutrients, and plant growth regulators, forming a protective film. Seeds coated with this agent absorb water and swell rapidly after sowing. As the embryo develops and the seedling grows, the active ingredients are slowly released and absorbed, effectively preventing seedling diseases and pests and promoting crop growth. There are many existing studies on coating agents suitable for alfalfa seeds in saline-alkali land. For example, Chinese patent CN201711455606.4 discloses a method for increasing the yield of alfalfa intercropping in saline-alkali land, including the following steps: A. Land preparation; B. Seed treatment: first, soak alfalfa seeds in ABT rooting powder solution for 2.5 hours; then coat the soaked alfalfa seeds with a fungicide; then coat the alfalfa seeds coated with the fungicide with a coating agent, the weight percentage of which is: urea 18-23%, monoammonium phosphate 16-21%, polyaspartic acid 22-27%, vinegar residue 8-12%, water-retaining agent 3-8%, desalination agent 5-9%, boric acid 2-5%, water 3-6%, talc powder 2-5%, and clay powder 2-5%; C. Sowing; D. Harvesting; thereby improving the salt and alkali resistance of the alfalfa seeds and increasing the yield of intercropped alfalfa. Chinese patent CN201711456302.X discloses a seed coating agent for alfalfa in saline-alkali soil and its preparation method. The weight percentages of the components in the coating agent are as follows: urea 15-19%, monoammonium phosphate 13-17%, polyaspartic acid 9-12%, alfalfa rhizobium 6-10%, ABT rooting powder 9-12%, vinegar residue 6-9%, desalination agent 5-9%, water-retaining agent 4-8%, boric acid 4-7%, water 4-7%, clay powder 2-4%, and talc powder 2-4%. This agent can improve the germination rate and yield of alfalfa seeds in saline-alkali soil.

[0003] However, there are many different types of alfalfa seed coating agents available at present. It is necessary to find a simple coating agent to coat alfalfa seeds, so as to alleviate the damage of salt and alkali stress to alfalfa and promote alfalfa growth. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a microbial coating agent for alleviating salt and alkali stress in alfalfa, as well as its preparation method and application.

[0005] To achieve the above objectives, this invention provides a microbial coating agent for alleviating salt-alkali stress in alfalfa, comprising endomycorrhizal fungal powder, Trichoderma harzianum powder, talc, diatomaceous earth, and an aqueous solution of sodium carboxymethyl cellulose. The ratio of the amounts of Trichoderma harzianum powder, endomycorrhizal fungal powder, diatomaceous earth, talc, and sodium carboxymethyl cellulose aqueous solution is 0.13g : 0.02g : 0.6g : 0.3g : 1-3mL.

[0006] The concentration of the sodium carboxymethyl cellulose aqueous solution is 0.01 g / mL, which is obtained by mixing and dissolving 1 g of sodium carboxymethyl cellulose with purified water and then making up to 100 mL.

[0007] The present invention also provides a method for preparing the microbial coating agent for alleviating salt and alkali stress in alfalfa. The specific steps are as follows: endomycorrhizal fungal powder, Trichoderma harzianum powder, talc powder and diatomaceous earth are mixed in a mass ratio. After the mixture is homogeneous, 1-3 mL of sodium carboxymethyl cellulose aqueous solution with a mass-volume ratio concentration of 0.01 g / mL is added to obtain the microbial coating agent.

[0008] The microbial coating agent of this invention contains 0.49-0.97% endomycorrhizal fungal powder, 3.2-6.3% Trichoderma harzianum powder, 14.7-29.1% diatomaceous earth, and 7.4-14.6% talc powder; the remainder is an aqueous solution of sodium carboxymethyl cellulose; it can alleviate 150 mmol / L salt and alkali stress.

[0009] The present invention also provides the application of the microbial coating agent, which can effectively alleviate 150 mmol / L salt and alkali stress, improve germination rate, promote alfalfa growth, and enhance stress resistance when alfalfa seeds are coated with the microbial coating agent.

[0010] Compared with existing technologies, this invention is the first to combine endomycorrhizal fungi and Trichoderma harzianum for seed coating of alfalfa. Using Trichoderma harzianum as the main component and endomycorrhizal fungi as a supplement, even a very small amount of both can effectively alleviate salt-alkali stress, significantly improve germination rate, promote alfalfa plant height, root length, and biomass, and enhance alfalfa growth and stress resistance. The microbial coating agent of this invention has few components, low bacterial content, and low cost, making it suitable for large-scale application. Attached Figure Description

[0011] Figure 1 The present invention relates to alfalfa seeds coated with different inoculant ratios, wherein A is treatment 1; B is treatment 2; C is treatment 3; D is treatment 4; E is treatment 5; F is treatment 6; G is treatment 7; H is treatment 8; I is treatment 9; and J is CK.

[0012] Figure 2 This invention relates to the germination of alfalfa seeds under 25℃ cultivation conditions.

[0013] Figure 3 This invention relates to the germination of alfalfa seeds under 25℃ growing conditions, where the red circle indicates the death of alfalfa seedlings.

[0014] Figure 4 The effect of the microbial coating agent involved in this invention on the growth of alfalfa seedlings is shown in the red circle, where the red circle indicates the branching of alfalfa seedlings. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0016] Example 1:

[0017] This embodiment relates to a microbial coating agent for alleviating salt and alkali stress in alfalfa, its preparation method, and its application. The microbial coating agent comprises a fungal agent, a powder, and a binder. The fungal agent is endomycorrhizal fungi and Trichoderma harzianum powder. The powder is talc powder and diatomaceous earth. The binder is an aqueous solution of sodium carboxymethyl cellulose. The endomycorrhizal fungi are Rhizocystis fungi strains purchased from Nanjing Cuijingyuan Biotechnology Co., Ltd.; Trichoderma harzianum is Trichoderma harzianum MCBC3 strain, a preserved strain with accession number CGMCC No. 41745, deposited at the China General Microbiological Culture Collection Center; the talc powder is pharmaceutical grade 325 mesh, and the diatomaceous earth is 200 mesh.

[0018] The preparation method of the microbial coating agent described in this embodiment is as follows: endomycorrhizal fungal powder, Trichoderma harzianum powder, talc powder, and diatomaceous earth are mixed in a mass ratio. After the mixture is homogeneous, 1-3 mL of sodium carboxymethyl cellulose aqueous solution with a concentration of 0.01 g / mL is added and mixed evenly to obtain the composite microbial coating agent.

[0019] The method for coating seeds with the microbial coating agent is as follows: place the seeds in a 20 ml beaker, add the inoculum (endomycorrhizal fungus powder and Trichoderma harzianum powder) and powder (talc powder and diatomaceous earth), place the beaker on a shaker, add sodium carboxymethyl cellulose aqueous solution during the shaking process, and stir with a glass rod until the inoculum and powder coat the seeds.

[0020] Example 2:

[0021] To verify the effect of the microbial coating agent described in Example 1 on alfalfa seeds, this example sets up 9 experimental groups and one blank control (CK) to conduct an experiment on the effect of the microbial coating agent on alfalfa seeds. The test seeds were alfalfa (Zhongmu 10 alfalfa), and the specific formulation of the coating agent is shown in Table 1. The coated seeds and the control group seeds are shown in the table below. Figure 1 As shown.

[0022] Table 1. Raw material ratio for alfalfa seed coating agent

[0023] (1) Methods for determining thousand-grain weight and disintegration rate

[0024] 1000-seed weight: Randomly select 500 seeds from each treatment group and accurately weigh them in grams, accurate to 0.01 g. Record the number of seeds (N) in each sample. Each sample should be measured in triplicate, and the average of the three measurements is taken as the final 1000-seed weight. 1000-seed weight (mH): Where m0 represents the weight of the sample weighed. The results are shown in Table 2.

[0025] Disintegration rate: 30 coated seeds were randomly selected and neatly placed on filter paper in a 9 cm diameter petri dish, separated from each other. 5 mL of purified water was slowly added along the wall of the dish. The disintegration of the coated seeds was observed within 1 minute; the shedding of the seed coat was considered disintegration. This was repeated 3 times. Disintegration rate (%) = (number of seed coat particles cracking within 1 minute / total number of seeds tested) × 100. The results are shown in Table 2.

[0026] Table 2 Effects of coating on single seed rate and disintegration rate of alfalfa seeds

[0027] Note: Different lowercase letters in the same column of the table indicate significant differences (P < 0.05).

[0028] Table 2 shows that there were significant differences in thousand-seed weight and disintegration rate among different treatments (P < 0.05). The thousand-seed weight of alfalfa seeds coated with different coating agents varied, with treatments 2 and 4 showing the highest thousand-seed weight (2.67 g), an increase of 20.81% compared to the control (CK). Regarding the disintegration rate of coated alfalfa seeds, treatment 4 had the highest disintegration rate at 91%, while treatment 3 had the lowest at only 54%. Treatments 7, 8, and 9 showed the highest disintegration rate. Figure 1 It can be seen that the ratio was unreasonable and the alfalfa seeds were not evenly coated, so no disintegration rate data was obtained.

[0029] (3) Germination test

[0030] Seeds of similar size were selected from alfalfa coated seeds treated with different methods and evenly placed in germination dishes lined with two layers of moistened filter paper. Each treatment had three replicates, with 30 seeds per replicate. Germination conditions were 16 h light / 25℃, 8 h darkness / 15℃, light intensity of 6000 lx, and humidity of 60%. Germination dishes were placed in a light incubator for the germination experiment. Water was added daily at fixed times to maintain consistent treatment concentrations. Germination was defined as the seed radicle penetrating more than 1 mm from the seed coat. The number of germinated seeds was observed and recorded daily for 9 consecutive days. Germination potential was calculated on day 5, and germination rate, germination index, and vigor index were calculated on day 9. Germination rate (%) = (number of germinated seeds on 9 days / number of tested seeds) × 100; Germination potential (%) = (number of germinated seeds on 5 days / number of tested seeds) × 100; Germination index (GI) = Σ(G t / t); Vitality Index (VI) = GI × Root Bud Length of Seedlings on Day 9. Where, G t Let be the number of germinated seeds on day t. The results are shown in Table 3 and... Figure 2 As shown.

[0031] Table 3 Germination potential, germination rate, germination index, and vigor index of coated alfalfa seeds

[0032] Note: Different lowercase letters in the same column of the table indicate significant differences (P < 0.05).

[0033] From Table 3 and Figure 2 It can be seen that under normal temperature (25℃) cultivation conditions, alfalfa seeds treated with different coatings were affected to varying degrees compared with the control (CK). The germination rate and germination potential of coated alfalfa seeds decreased compared with the CK. Among them, treatment 4 had the highest germination rate and germination potential, at 81.11% and 77.78%, respectively. In terms of germination index and vigor index, there were no significant differences between the treatments and the CK (P<0.05), but treatment 4 had a vigor index that was 9.93% higher than the CK, indicating that the coating agent in treatment 4 had a certain promoting effect on the germination of alfalfa seeds.

[0034] (4) Pot experiment

[0035] Alfalfa seeds with similar coated particle sizes under different treatments were selected and sown in pots. The soil used in the experiment was strongly alkaline soil, taken from the 0-20cm topsoil layer of Mengcao Seed Industry Center, Xincheng District, Hohhot (40.904273°N, 111.778852°E). The soil composition was pH 8.41, organic matter 9.3863 g / kg, total phosphorus 1.282 g / kg, total potassium 14.4298 g / kg, total nitrogen 0.38 g / kg, available nitrogen 77.35 mg / kg, available phosphorus 7.076 mg / kg, and available potassium 144.7 mg / kg.

[0036] Seed germination conditions were as follows: 60% humidity in a light-incubator, 16 h light at 25℃, 8 h darkness at 15℃, and a light intensity of 6000 lx. Ten treatment groups were set up, with each treatment replicated three times, and nine seeds sown per replicate. For each treatment, day 0 was the day of sowing. On day 10, each treatment was subjected to 100 mL of a 150 mmol / L (NaCl:NaHCO3 = 1:1) saline-alkali solution. Rehydration was performed three days after the saline-alkali stress period, with 100 mL of purified water added every two days for a total of 63 days. Germination rates were statistically analyzed and measured, and the results are shown in Table 4. Figure 3 As shown.

[0037] Table 4. Effects of seed coating on alfalfa seed emergence

[0038] Note: Different lowercase letters in the same column of the table indicate significant differences (P < 0.05).

[0039] From Table 4 and Figure 3 It was found that alfalfa emergence was significantly inhibited under salt-alkali stress, and seedling death occurred. Different coating agents effectively alleviated the impact of salt-alkali stress on alfalfa emergence. On day 7, seeds treated with different coating agents all emerged normally, with treatment 4 showing the highest emergence rate at 74.08%, which was not significantly different from the control (P < 0.05). On day 20, the emergence rate of treatment 4 after salt-alkali stress was significantly different from that of the control (P < 0.05), increasing by 1.29 times. On day 60, the emergence rate of treatment 4 was 59.26%, increasing by 1.67 times compared to the control, reaching a significant level (P < 0.05). This indicates that the coating agent in treatment 4 significantly enhanced the alfalfa's resistance to stress.

[0040] To evaluate the effects of different coating agent ratios on alfalfa seedling growth under salt-alkali stress, plant height, root length, and biomass (aboveground + belowground) of alfalfa seedlings were measured at day 60. The results are shown in Table 5. Figure 4 As shown.

[0041] Table 5 Effects of seed coating on alfalfa seedling growth

[0042] Note: Different lowercase letters in the same column of the table indicate significant differences (P < 0.05).

[0043] Table 5 shows that, compared with the control (CK), treatments 1-7 and treatment 9 promoted plant growth to varying degrees. Regarding plant height and root length, the differences between the different coating treatments and the CK were not statistically significant (P < 0.05). Measurements showed that the plant height and root length of alfalfa treated with treatments 1-7 and treatment 9 were increased. Specifically, compared with the CK, treatment 1 increased plant height and root length by 18.6% and 42.92%, respectively; treatment 4 increased plant height and root length by 9.5% and 33.3%, respectively, compared with the CK.

[0044] Regarding biomass, Table 5 shows that treatments 2 and 4, affected by the coating agent, had significantly higher average aboveground and belowground biomass than the control (CK) (P < 0.05). The average aboveground biomass increased by 1.84 times and 1.95 times compared to the CK, respectively, with treatment 4 showing the most significant difference. Figure 4 Alfalfa plants already showed branching; among them, treatment 4 showed a 2.29-fold increase in average underground biomass compared to the control (CK), and a 2.02-fold increase in the average total aboveground and underground biomass. These results indicate that the coating agent in treatment 4 significantly promoted plant height, root length, and biomass (aboveground + belowground) of alfalfa under salt-alkali stress, enhancing the plant's resistance to adverse conditions.

[0045] In this invention, when the mass-to-volume ratio of Trichoderma harzianum powder, endomycorrhizal fungus powder, diatomaceous earth, talc powder, and sodium carboxymethyl cellulose aqueous solution in the coating agent is 0.13g : 0.02g : 0.6g : 0.3g : 1-3ml, alfalfa seeds are coated with the following formula: thousand-seed weight, disintegration rate, germination index, and growth index are optimal. This formula effectively alleviates the damage caused by salt-alkali stress to alfalfa, promotes increased plant height, root length, and biomass, and enhances alfalfa growth and stress resistance. It is a coating formula worthy of widespread application.

Claims

1. A microbial coating agent for alleviating salt-alkali stress in alfalfa, characterized in that, It is composed of endomycorrhizal fungus powder, Trichoderma harzianum powder, talc powder, diatomaceous earth, and sodium carboxymethyl cellulose aqueous solution; the ratio of Trichoderma harzianum powder, endomycorrhizal fungus powder, diatomaceous earth, talc powder, and sodium carboxymethyl cellulose aqueous solution is 0.13g : 0.02g : 0.6g : 0.3g : 1-3mL.

2. The microbial coating agent for alleviating alfalfa salinity stress according to claim 1, characterized in that, The concentration of the sodium carboxymethyl cellulose aqueous solution is 0.01 g / mL.

3. The microbial coating agent for alleviating alfalfa salinity stress according to claim 1, characterized in that, The microbial coating agent contains 0.49-0.97% endomycorrhizal fungal powder, 3.2-6.3% Trichoderma harzianum powder, 14.7-29.1% diatomaceous earth, and 7.4-14.6% talc; the remainder is an aqueous solution of sodium carboxymethyl cellulose; it can alleviate 150 mmol / L salt and alkali stress.

4. The method for preparing the microbial coating agent for alleviating alfalfa salinity stress as described in claim 1, characterized in that, The specific steps are as follows: Mix endomycorrhizal fungus powder, Trichoderma harzianum powder, talc powder, and diatomaceous earth according to the mass ratio. After the mixture is homogeneous, add 1-3 mL of sodium carboxymethyl cellulose aqueous solution with a mass-volume ratio concentration of 0.01 g / mL to obtain the microbial coating agent.

5. The application of the microbial coating agent according to claim 1, characterized in that, Coating alfalfa seeds with the aforementioned microbial coating agent can effectively alleviate 150 mmol / L salt-alkali stress, increase germination rate, promote alfalfa growth, and enhance stress resistance.

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