A microbial compound inoculant and its application in blueberry growth

Abstract

This invention discloses a microbial compound inoculant and its application in blueberry growth, belonging to the field of microbial inoculant technology. The compound inoculant is composed of *Bacillus cereus*, *Pseudomonas koreensis*, and *Rhodococcus qingshengii* in a viable count-to-mass ratio of 1:1:1. The three strains exhibit significant synergistic effects, not only improving growth indicators such as stem length, root length, fresh weight, and dry weight of blueberries, but also increasing the content of soluble sugars, soluble proteins, and proline in blueberry leaves. The microbial compound inoculant constructed in this invention is adapted to the acidic growth environment of blueberries, is environmentally friendly, and low in cost, solving the problems of fertilizer dependence, slow growth, and lack of specialized inoculants in blueberry cultivation, and providing technical support for large-scale artificial cultivation of blueberries.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, specifically relating to a microbial compound inoculant and its application in blueberry growth. Background Technology

[0002] In fertilizer reduction, increasing the application of organic fertilizer and microbial fertilizer are the main methods. However, while increasing the application of organic fertilizer has many advantages, it also leads to a significant increase in production costs. Microbial inoculants, due to their green and environmentally friendly properties, ability to improve the soil microenvironment, and ability to enhance plant stress resistance, are gradually becoming an important research direction for replacing traditional chemical agents.

[0003] Blueberry, a plant belonging to the genus Vaccinium in the family Ericaceae, typically lacks root hairs in its root system, resulting in slow plant growth. Wild blueberries often grow in acidic environments due to soil exudates, a soil characteristic that significantly impacts the application of conventional microbial agents, limiting their effectiveness. Consequently, there has long been a lack of dedicated microbial agents for blueberry production. Therefore, developing microbial agents to promote blueberry growth and alleviate the problem of slow growth despite widespread cultivation is of paramount importance.

[0004] Existing technologies mostly focus on organic fertilizers or traditional single microbial agents. There is an urgent need to develop new specialized microbial compound agents for blueberry production to achieve more efficient growth promotion effects and thus overcome current technological obstacles. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a compound microbial agent that can promote the growth of blueberries, so as to solve the problems of chemical fertilizer pollution, lack of special microbial agents, slow growth and low yield and quality in blueberry planting. At the same time, it provides its preparation method and application, providing technical support for the large-scale planting of blueberries.

[0006] This invention is achieved through the following technical solution:

[0007] A microbial compound inoculant, composed of Bacillus cereus, Pseudomonas koreensis, and Rhodococcus qingshengii.

[0008] Furthermore, the compound bacterial agent is composed of Bacillus cereus, Pseudomonas kansui, and Rhodococcus qingshengensis in a live bacteria mass ratio of 1:1:1.

[0009] The compound microbial agent is prepared according to the following steps:

[0010] (1) Liquid fermentation of a single strain: Bacillus cereus, Pseudomonas kansui and Rhodococcus qingsheng were prepared into seed liquids, which were then inoculated into fermentation medium and cultured on a shaker to obtain fermentation broth;

[0011] (2) Preparation of compound microbial agent: The fermentation broth from the fermentation culture was mixed at a ratio of viable cell count of 1:1:1 to prepare a compound microbial agent with a total effective viable cell count of 1.0 × 10⁻⁶. 8 ~3.0×10 8 CFU / mL.

[0012] The application of the above-mentioned compound microbial agent in the growth of blueberries involves inoculating the compound microbial agent into the roots of blueberries and evaluating its growth-promoting effect by detecting the growth indicators (stem length, root length, fresh weight, dry weight) and physiological and biochemical indicators (content of soluble sugar, soluble protein, proline, and malondialdehyde).

[0013] Compared with the prior art, the present invention has the following technical effects:

[0014] 1. Significant synergistic growth-promoting effect: The combination of the three strains produced a synergistic effect, which significantly increased the stem length, root length, fresh weight and dry weight of blueberry compared with the blank control group, single inoculation group and the combination of two strains, thus promoting the vigorous growth of the plants.

[0015] 2. Improve plant quality and stress resistance: It can increase the content of soluble sugar, soluble protein and proline in blueberries, reduce the content of malondialdehyde, and enhance the plant's stress resistance;

[0016] 3. Green economy and easy to scale up: It does not rely on chemical fertilizers, is environmentally friendly, the strains are easy to preserve and can be produced on a large scale by liquid fermentation, the production cost is low, and it is suitable for commercial promotion and application.

[0017] The Bacillus cereus of this invention has the accession number CCTCC AB 93238, Pseudomonas koreensis has the accession number CCTCC FB 2024177, and Rhodococcus qingshengii has the accession number CCTCC AB 207271. All of these were purchased from the China Center for Type Culture Collection. Attached Figure Description

[0018] Figure 1 The effects of single-agent and compound-agent inoculation on malondialdehyde content in blueberry leaves.

[0019] Figure 2 The effects of single-agent and compound-agent inoculation on proline content in blueberry leaves.

[0020] Figure 3The effects of inoculation with single and compound microbial agents on the soluble sugar content of blueberry leaves.

[0021] Figure 4 The effects of inoculation with single and compound microbial agents on the soluble protein content of blueberry leaves. Detailed Implementation

[0022] Unless otherwise specified, the test methods used in the following examples are conventional test methods; the materials and reagents used are commercially available unless otherwise specified.

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0024] Example 1: Preparation of Compound Microbial Agent

[0025] (1) Liquid fermentation of a single strain: Bacillus cereus (accession number CCTCC AB93238), Pseudomonas koreensis (accession number CCTCC FB 2024177), and Rhodococcus qingshengii (accession number CCTCC AB 207271) were prepared as seed cultures, inoculated into fermentation medium, and cultured with shaking at 37°C and 180 rpm to obtain fermentation broth.

[0026] (2) Preparation of compound bacterial agent: The above strains were inoculated into fresh TSB liquid medium for activation, and the OD of the fermentation culture was measured using a UV spectrophotometer. 600 =1, mix the fermentation broth with the fermentation culture medium in a ratio of 1:1:1 for live bacteria count to prepare a microbial compound agent.

[0027] TSB liquid culture medium formulation: tryptone: 17.0 g / L, soybean papain hydrolysate: 3.0 g / L, sodium chloride: 5.0 g / L, dipotassium hydrogen phosphate: 2.5 g / L, glucose: 2.5 g / L.

[0028] Example 2: Verification of the growth-promoting effect of compound microbial agents

[0029] The OD value of the microbial agent prepared in Example 1 was measured using a UV spectrophotometer. 600 =1, dilute the bacterial count to 1×10⁻⁶ 8CFU / mL. Apply 2 mL of the bacterial agent to the roots of blueberry seedlings, once. Verify the growth-promoting effect of the compound bacterial agent application on blueberry seedlings 30 days later. To compare the growth-promoting effect of the compound bacterial agent in Example 1, a treatment without any bacterial agent inoculation (blank control group) and seven treatment combinations with bacterial agents were established, as follows:

[0030] (1) Blank control group (CK);

[0031] (2) Single inoculation with SH-192 (Bacillus cereus, code SH-192);

[0032] (3) Single inoculation with BL-340 (Korean Pseudomonas aeruginosa, code BL-340).

[0033] (4) Single inoculation of T16-7 group (Rhodococcus qingsheng, code T16-7).

[0034] (5) Combined inoculation with SH-192 (Bacillus cereus) + BL-340 (Pseudomonas aeruginosa);

[0035] (6) Combined inoculation with SH-192 (Bacillus cereus) + T16-7 (Rhodococcus qingsheng) group;

[0036] (7) Group inoculated with BL-340 (Pseudomonas aeruginosa) + T16-7 (Rhodococcus kyngsei);

[0037] (8) The group was inoculated with a combination of SH-192 (Bacillus cereus) + BL-340 (Pseudomonas kansui) + T16-7 (Rhodococcus qingsheng).

[0038] Experimental testing:

[0039] After 30 days of culturing blueberry, three intact plants were randomly selected from each group to measure morphological indicators such as plant height and root length. After washing with deionized water, the aboveground parts and roots of the plants were separated for analysis of physiological and biochemical indicators.

[0040] (a) Measurement of plant growth indicators

[0041] Gently pull up the blueberry plant, trying not to break the roots, wash away any remaining soil from the roots, and dry them. Use calipers to measure the plant's height and root length. Use an analytical balance to accurately weigh the fresh weight of the whole plant. Place the plant in an 80°C oven to dry until the weight no longer changes, and then measure its dry weight.

[0042] The effects of 30 days of inoculation with the compound microbial agent on the root and stem length of blueberry (Table 1) and the effects of 30 days of inoculation with the compound microbial agent on the growth indicators of blueberry (Table 2) show that the stem length (64.73±2.44 mm), root length (22.47±4.79 mm), fresh root weight (19.40±0.66 mg), fresh stem weight (19.80±5.33 mg), fresh leaf weight (64.05±4.45 mg), and dry weight of each part of the blueberry treated with the compound microbial agent of the three strains of this invention were significantly higher than those of the blank control group and other single-inoculation and compound-inoculation groups, indicating that the synergistic effect of the three strains can effectively promote the growth of blueberry.

[0043] (II) Effects of inoculation with compound microbial agents on plant physiological indicators

[0044] (1) Determination of malondialdehyde content

[0045] Mix 500 μL of enzyme solution with 1 mL of malondialdehyde reaction solution in a test tube, react thoroughly in a boiling water bath, centrifuge at 4000 rpm for 10 min, and measure the absorbance of the reaction solution at 600 nm, 532 nm and 450 nm. Each sample is measured three times.

[0046] The effect of compound microbial inoculation for 30 days on malondialdehyde content in blueberry leaves is as follows: Figure 1 As shown, the malondialdehyde (MDA) content significantly increased under the treatment with the three strains of the compound inoculant, indicating a significant acceleration in the metabolic rate of blueberry and an increase in membrane lipid metabolism activity. However, MDA, as an intermediate product of normal membrane lipid metabolism, accumulates slightly with increasing metabolic rate, and this abnormal increase is not due to membrane damage. The increase in MDA content suggests that the plant activates its stress resistance mechanism through "moderate adjustment of membrane lipid metabolism."

[0047] (2) Determination of proline content

[0048] Weigh 0.5 g of leaf material from different culture conditions, cut it into small pieces and put it into test tubes. Add 5 mL of 3% sulfosalicylic acid to each tube, seal the tubes, boil in a water bath for 10 min, and let them cool naturally to room temperature. Transfer them to 50 mL centrifuge tubes and centrifuge at 4℃ and 4000 r / min for 10 min. The supernatant is the proline extract.

[0049] Pipette 2 mL of the extract into a glass test tube, add 2 mL of glacial acetic acid and 3 mL of 2.5% acidic ninhydrin colorimetric solution, and react in a boiling water bath for 30 min. The solution turns red.

[0050] Cool to room temperature, then add 4 mL of toluene to each test tube, shake for 30 s, let stand for a period of time, then transfer the supernatant to a new 50 mL centrifuge tube, centrifuge at 3000 r / min for 5 min, transfer the upper layer solution to a cuvette, and measure the absorbance at 520 nm wavelength using toluene as a control.

[0051] Proline is an important osmotic regulator and antioxidant in plants. The effect of compound microbial inoculation for 30 days on the proline content of blueberry leaves is as follows: Figure 2 As shown, the proline content in the leaves of blueberries significantly increased under the treatment with the compound inoculant containing the three bacterial strains. Blueberries grow in acidic soil, and their roots are susceptible to water loss due to low pH. Proline can help maintain cell turgor pressure, reduce water loss, and ensure normal root absorption by increasing intracellular solute concentration.

[0052] (3) Soluble sugar content

[0053] Weigh 0.5 g of leaf material from different culture conditions, cut it into small pieces and put it into 20 mL glass test tubes. Add 5 mL of water to the glass test tubes, stopper them and boil them in a water bath for 30 min. Let them cool naturally to room temperature, filter the solution into a 25 mL volumetric flask, and dilute to volume with distilled water. The result is the soluble sugar extract.

[0054] Pipette 0.5 mL of the extract into separate glass test tubes, add 1.5 mL of distilled water and 0.5 mL of anthrone ethyl acetate solution, then slowly add 5 mL of concentrated sulfuric acid. React in a boiling water bath for 1 min. After cooling to room temperature, measure the absorbance at 620 nm using a UV-Vis spectrophotometer.

[0055] Soluble sugars are essential for blueberry's stress resistance and energy reserves. The effect of 30 days of inoculation with a compound microbial agent on the soluble sugar content of blueberry leaves is as follows: Figure 3 As shown, its content increased significantly under the treatment of the compound inoculant of the three strains. Soluble sugars, as osmotic regulators, can increase cell osmotic pressure and enhance the tolerance of blueberries to potential stress in acidic soils; at the same time, after the strains promote the increase of photosynthetic rate, in addition to some carbohydrates being used for growth, another part is converted into soluble sugars and stored in the leaves as an emergency energy source.

[0056] (4) Soluble protein content

[0057] Weigh 0.1 g of fresh leaves, add a small amount of quartz sand and 1 mL of 0.1 mol / L PBS buffer, and grind into a homogenate. Transfer the homogenate to a 10 mL volumetric flask, rinse the mortar and pestle three times with 1 mL of PBS buffer, and transfer the rinsing solution to the volumetric flask as well, ensuring minimal damage during the transfer. Make up to 5 mL. Take 2 mL of the liquid from the volumetric flask into a centrifuge tube, centrifuge at 5000 r / min for 10 min. Take 0.1 mL of the supernatant, add 0.9 mL of distilled water and 5 mL of Coomassie Brilliant Blue G-250 solution, mix well, and let stand for 2 min for color development. Measure the OD value at a wavelength of 595 nm using a UV-Vis spectrophotometer.

[0058] Soluble protein is a core substance for the metabolic activity of blueberry. The effect of inoculation with compound microbial agents for 30 days on the soluble protein content of blueberry leaves is as follows: Figure 4 It is evident that the content of the compound inoculum of the three strains was significantly higher than that of the control group and other single-inoculation and compound-inoculation treatment groups after inoculation. The metabolic products of the strains stimulate the synthesis of enzymes related to nutrient metabolism in blueberries. These enzymes are all soluble proteins, and their increased content can further improve nutrient utilization efficiency.

[0059] In summary, the three-strain compound microbial agent of the present invention can significantly promote the growth and stress resistance of blueberries, is suitable for the acidic soil environment of blueberry planting, is green and economical and easy to apply on a large scale, and provides important theoretical basis and technical support for the sustainable planting and commercial development of blueberries.

[0060] Table 1. Effects of compound microbial inoculation on root and stem length of blueberry after 30 days.

[0061]

[0062] Table 2 Effects of compound microbial inoculation on growth indicators of blueberry 30 days after inoculation.

[0063]

Claims

1. Application of a microbial complex microbial inoculant in promoting growth of blueberries, characterized in that, The compound bacterial agent is composed of Bacillus cereus, Pseudomonas koreensis, and Rhodococcus qingshengii. The Bacillus cereus accession number is CCTCC AB 93238, the Pseudomonas koreensis accession number is CCTCC FB 2024177, and the Rhodococcus qingshengii accession number is CCTCC AB 207271.

2. Use according to claim 1, characterized in that, The compound bacterial agent is composed of Bacillus cereus, Pseudomonas kansui, and Rhodococcus qingshengensis in a live bacteria mass ratio of 1:1:

1.

3. Use according to claim 1, characterized in that, The total effective viable cell number of the three bacteria in the microbial complex microbial agent is 1.0 x 10 8 ~ 3.0 x 10 8 CFU / mL.

4. Use according to claim 1, characterized in that, 2 mL of compound bacterial agent was inoculated at the roots of blueberry seedlings.

5. The use according to claim 1, characterized in that, Thirty days after inoculation with compound microbial agent, blueberry seedlings showed increased height, root length, and fresh weight of roots, stems, and leaves.

6. Use according to claim 1, characterized in that, Thirty days after inoculation with the compound microbial agent, the content of soluble sugar, soluble protein, and proline in blueberry leaves increased.

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