Microbial complex microbial inoculant and application thereof in improving yield and quality of persimmons

Abstract

The invention discloses a microbial complex microbial inoculant and application thereof in improving the yield and quality of persimmons, the microbial complex microbial inoculant comprises lactobacillus plantarum P9 and lactobacillus casei ZHANG, and the viable bacteria value ratio of the lactobacillus plantarum P9 to the lactobacillus casei ZHANG is (1-3): 1. The compound microbial inoculant has remarkable advantages in the aspects of increasing the yield of the five-original yellow persimmons, improving the fruit quality, improving the disease resistance and promoting the soil health, and provides powerful support for sustainable development of persimmon planting in the five-original region.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a microbial composite agent and application thereof in improving the yield and quality of persimmons. Background Art

[0002] In recent years, with the rapid development of my country's agricultural economy, agricultural production has generally faced problems such as the intensification of soil-borne diseases, imbalance of soil microbial populations, decline in soil fertility, serious heavy metal pollution and pesticide residues, and low utilization rate of chemical fertilizers, which have led to a decline in the yield and quality of crops and cash crops.

[0003] Wuyuan yellow persimmons, a specialty agricultural product of Bayannur City, have a significant impact on local farmers' income and agricultural economic development due to their yield and quality. In recent years, persimmon cultivation in the Wuyuan region has been plagued by declining soil fertility, pests and diseases, and other issues. While traditional methods like spraying pesticides and applying chemical fertilizers have increased persimmon yields to a certain extent, these methods often lead to further declines in soil fertility, significant pesticide residues, and low fertilizer utilization rates. Therefore, scientific methods are urgently needed to improve yield and fruit quality. Summary of the Invention

[0004] The present invention aims to provide a microbial composite agent and its application in improving the yield and quality of persimmons. The microbial composite agent has the functions of reducing the use of chemical fertilizers, promoting crop growth, and improving crop yield and quality.

[0005] The technical solutions provided by the present invention are as follows: A first aspect of the present invention provides a microbial composite agent, comprising Lactobacillus plantarum P9 and Lactobacillus casei ZHANG, wherein the ratio of viable bacteria of Lactobacillus plantarum P9 to Lactobacillus casei ZHANG is (1-3):1.

[0006] Furthermore, the preservation number of the Lactobacillus plantarum P9 is CGMCC NO.16662; the preservation number of the Lactobacillus casei ZHANG is CGMCC NO.5469.

[0007] Furthermore, the ratio of the viable bacteria count of the Lactobacillus plantarum P9 to the Lactobacillus casei ZHANG is 2:1.

[0008] Furthermore, the bacterial concentration in the microbial composite agent is not less than 1×10 9 CFU / mL.

[0009] Furthermore, the microbial composite agent is preserved in a freeze-dried protective agent, and the components of the freeze-dried protective agent are: trehalose 12-18%, skim milk powder 2-3%, glutathione 0.5-0.8 mmol / L, nano-silicon dioxide 0.05-0.1% and phosphate.

[0010] A second aspect of the present invention provides a method for preparing the above-mentioned microbial composite agent, comprising the following steps: Purified Lactobacillus plantarum P9 and Lactobacillus casei ZHANG were inoculated onto MRS agar plates, and different colonies were formed after cultivation. Inoculating colonies of Lactobacillus plantarum P9 and Lactobacillus casei ZHANG into a first MRS liquid culture medium and culturing for 24-48 hours; Take 2-5% of the culture medium and inoculate it into the second MRS liquid medium for expansion culture. When the pH value of the culture medium is stable and the bacterial growth reaches the exponential phase, use a high-speed centrifuge at 4-8 rpm for 1-15 minutes to precipitate the bacteria. The bacterial precipitate is taken out and washed with physiological saline for multiple times. The washed Lactobacillus plantarum P9 and Lactobacillus casei ZHANG are concentrated to obtain the microbial composite agent.

[0011] Furthermore, the first MRS liquid culture medium and the second MRS liquid culture medium include the following components by mass: 1 g / L peptone, 2 g / L glucose, 2 g / L bile salt, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, and 2 g / L magnesium sulfate, the volume is adjusted to 1000 mL with distilled water, and the pH is adjusted to 6.4-6.6.

[0012] A third aspect of the present invention provides a use of the above-mentioned microbial composite agent in improving the quality and yield of Wuyuan persimmon.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The composite microbial agent provided by the present invention can be applied to the cultivation of agricultural or cash crops. It reduces fertilizer usage, promotes crop growth, and increases crop yield and quality. This composite microbial agent has demonstrated significant advantages in increasing Wuyuanhuang persimmon yield, improving fruit quality, enhancing disease resistance, and promoting soil health, providing strong support for the sustainable development of persimmon cultivation in the Wuyuan region. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the process for preparing the microbial composite agent in an embodiment of the present invention. DETAILED DESCRIPTION

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the embodiments described below are part of the embodiments of the present application, not all of the embodiments. Generally, the components of the embodiments of the present application described and shown in the drawings herein can be arranged and designed in various different configurations.

[0016] Therefore, the detailed description of the embodiments of the present application provided below in conjunction with the accompanying drawings is intended to represent only selected embodiments of the present application and is not intended to limit the scope of protection claimed in the present application. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present application without making any creative efforts shall fall within the scope of protection claimed in the present application.

[0017] Example 1 Preparation of microbial composite agent The present invention provides a microbial composite agent, comprising Lactobacillus plantarum P9 and Lactobacillus casei. The ratio of viable bacteria of Lactobacillus plantarum P9 to Lactobacillus casei is (1-3):1. Lactobacillus plantarum P9 was deposited with the General Microorganism Center of the China National Culture Collection Administration on October 31, 2018, with a deposit number of CGMCC No. 16662. Lactobacillus casei was deposited with the General Microorganism Center of the China National Culture Collection Administration on November 18, 2011, with a deposit number of CGMCC No. 5469.

[0018] This microbial composite agent can be stored in a lyoprotectant containing 12-18% (by mass) trehalose, 2-3% (by mass) skim milk powder, 0.5-0.8 mmol / L glutathione, 0.05-0.1% nano-silicon dioxide, and phosphate. While the survival rate of live bacteria in this microbial composite agent is approximately 78% in conventional lyoprotectants, the survival rate in this lyoprotectant exceeds 94%.

[0019] Figure 1 A method for preparing a microbial composite agent is shown, which is used to prepare the above-mentioned microbial composite agent, comprising the following steps: S1. Purified Lactobacillus plantarum P9 and Lactobacillus casei ZHANG strains were inoculated onto MRS agar plates, and cultured to form different colonies. S2, inoculating colonies of Lactobacillus plantarum P9 and Lactobacillus casei ZHANG into a first MRS liquid culture medium and culturing for 24-48 hours; S3. Take 2-5% of the culture solution and inoculate it into the second MRS liquid medium for expansion culture. When the pH value of the culture solution is stable and the bacterial growth reaches the exponential phase, use a high-speed centrifuge at 4-8 rpm for 1-15 minutes to precipitate the bacteria. S4. Taking out the bacterial precipitate, washing it with physiological saline for multiple times, concentrating the washed Lactobacillus plantarum P9 and Lactobacillus casei ZHANG to obtain the microbial composite agent.

[0020] The above method comprises the following steps in actual operation: 1.1 Inoculation Purified Lactobacillus plantarum P9 and Lactobacillus casei ZHANG strains were selected and inoculated onto MRS agar plates, respectively, and cultured for 48 hours in a 37°C incubator until typical colonies were formed.

[0021] 1.2 Bacteria Activation A single colony was picked from the MRS plate and inoculated into MRS liquid culture medium for 24-48 hours. Microscopic observation confirmed that Lactobacillus plantarum P9 and Lactobacillus casei ZHANG were growing well in the liquid culture medium and had entered the logarithmic growth phase.

[0022] 1.3 Expansion culture Take a small amount of activated culture (usually 2-5% of the inoculum) and inoculate it into a larger volume of MRS liquid medium, usually 2-5 mL of culture volume, and continue to culture at 37°C for 48 hours.

[0023] 1.4 Temperature and pH Control Lactobacillus plantarum P9 and Lactobacillus casei ZHANG prefer to grow at 37°C. Maintain this temperature during the culture process. Use a pH self-regulating fermenter and regularly monitor the pH value of the culture medium (the pH should drop to 4.5-5.5). When the pH value is low, add an appropriate amount of sodium hydroxide solution to adjust it.

[0024] 1.5 Centrifugal Collection When the pH value of the culture solution is stable and the bacterial growth reaches the exponential phase, use a high-speed centrifuge to centrifuge at 4-8 rpm for 1-15 minutes to precipitate the bacteria.

[0025] 1.6 Washing Remove the bacterial pellet and wash it twice with physiological saline to remove residual substances in the culture medium. Centrifuge again after each wash until the bacterial purity reaches the required level.

[0026] 1.7 Bacterial Concentration The washed Lactobacillus plantarum P9 and Lactobacillus casei ZHANG bacteria can be concentrated and adjusted to a bacterial solution concentration suitable for use.

[0027] The ratio of viable bacteria of Lactobacillus plantarum P9 to Lactobacillus casei ZHANG in the microbial composite agent prepared by the present invention is (1-3):1, preferably 2:1. The number of viable bacteria per milliliter of the microbial composite agent suspension is determined by plate count method (e.g., tenfold dilution method). The bacterial concentration generally needs to reach 1×10 9 CFU / mL or higher.

[0028] Example 2 Application test of microbial composite agent on Wuyuanhuang persimmon In this example, the microbial composite agent prepared in Example 1 was tested.

[0029] 2.1 Trial location Location: Wuyuan County, Bayannur City, Inner Mongolia Soil type: Sandy loam, pH 6.8, rich in organic matter Climate conditions: Typical temperate continental climate, with annual rainfall of 180-200mm, suitable for persimmon growth 2.2 Experimental design The experiment adopted a randomized block design (RCBD) with 4 treatment groups and 3 replicates for each treatment.

[0030] Treatment 1 (control group): conventional fertilization without microbial preparations Treatment 2: Reduce chemical fertilizer by 1% and use a microbial compound agent Treatment 3: Reduce chemical fertilizer by 2% and use a microbial compound agent Treatment 4: Use only compound microbial agents and no chemical fertilizers 2.3 Test indicators Growth indicators: plant height, leaf area, root growth Yield indicators: single plant yield, single fruit weight, total yield Fruit quality: sugar content, vitamin C content, fruit firmness, skin glossiness Disease resistance: Incidence and resistance of major persimmon diseases (e.g. anthracnose, late blight) Soil nutrients: nitrogen, phosphorus, potassium content, soil microbial diversity 3. Experimental Data 3.1 Growth indicators Plant height: Control group: average plant height 11.5cm Treatment 2: Average plant height 12.6 cm Treatment 3: Average plant height 11.7 cm Treatment 4: average plant height 13.4 cm Leaf area (cm²): Place the leaf on graph paper, trace the leaf's outline, and count the number of squares within the outline. For any incomplete squares, estimate and merge them to form a whole square. Finally, calculate the leaf area based on the graph paper's specifications (e.g., each square is 1 cm²).

[0031] Control group: average leaf area 17cm² Treatment 2: Average leaf area 19 cm² Treatment 3: Average leaf area 17.5 cm² Treatment 4: average leaf area 22cm² 3.2 Production indicators Yield per plant (kg): Control group: average single plant yield 5.3kg Treatment 2: Average yield per plant 5.8kg Treatment 3: Average yield per plant 5.6kg Treatment 4: Average yield per plant 6.4kg Single fruit weight (g): Control group: average single fruit weight 152g Treatment 2: Average single fruit weight 153g Treatment 3: Average single fruit weight 144g Treatment 4: Average single fruit weight 164g Total production (tons / hectare): Control group: 3.1 tons / hectare Treatment 2: 3.4 tons / hectare Treatment 3: 3.2 tons / hectare Treatment 4: 3.7 tons / hectare 3.3 Fruit quality Sugar content (Brix): using high performance liquid chromatography (HPLC) Control group: 11.5% Treatment 2: 11.3% Treatment 3: 11.8% Treatment 4: 12.3% Vitamin C content (mg / 1g): using high performance liquid chromatography (HPLC) Control group: 8.5 mg Treatment 2: 9.5mg Treatment 3: 8.8 mg Treatment 4: 10.1 mg Fruit firmness (N): using electronic universal testing machine Control group: 13N Process 2: 14N Processing three: 12.6N Processing 4: 16N Peel glossiness (glossiness score, 1-5 points): Control group: 3.1 points Process 2: 3.5 points Processing 3: 3.2 points Processing 4: 4.4 points 3.4 Disease resistance Disease incidence rate (%): Disease incidence rate = number of diseased plants / number of tested plants * 100% Control group: anthracnose incidence was 15%, late blight incidence was 12%, and morbidity rate was 27%; Treatment 2: anthracnose incidence was 8%, late blight incidence was 7%, and morbidity was 15%; Treatment 3: anthracnose incidence was 1%, late blight incidence was 9%, and morbidity was 10%; Treatment 4: The anthracnose incidence was 5%, the late blight incidence was 4%, and the disease rate was 9%.

[0032] 3.5 Soil nutrients Nitrogen (N), phosphorus (P), potassium (K) content: Control group: nitrogen content 11.6 mg / kg, phosphorus content 46 mg / kg, potassium content 14 mg / kg Microbial preparation group: nitrogen content 12.5 mg / kg, phosphorus content 51 mg / kg, potassium content 15 mg / kg Soil microbial diversity: Control group: microbial species are single and the number is small Microbial preparation group: significantly increased soil microbial diversity and the number was significantly increased 4. Experimental Conclusion Compared with the control group, 1% fertilizer reduction + microbial preparation group, and 2% fertilizer reduction + microbial preparation group, the plant height of the microbial composite agent group increased by 16.5%, 5.8%, and 12.7%; the leaf area index increased by 22.7%, 13.6%, and 20.5%; the single plant yield increased by 17.2%, 9.4%, and 12.5%; the total yield increased by 16.2%, 5.4%, and 13.5%; the sugar content increased by 6.5%, 8.1%, and 4.1%; the vitamin C content increased by 6.5%, 8.1%, and 4.1%; and the incidence rate decreased by 66.7%, 40%, and 10%.

[0033] The group using the microbial compound maintained a high yield even with reduced fertilizer use. The compound also increased the sugar and vitamin C content of the persimmons, enhanced the firmness and glossiness of the fruit, and improved their disease resistance.

[0034] Soil improvement: The use of microbial preparations can effectively improve soil microbial communities, increase soil fertility, and reduce dependence on chemical fertilizers.

[0035] In summary, the microbial compound agent showed significant advantages in increasing the yield of Wuyuan yellow persimmon, improving fruit quality, enhancing disease resistance and promoting soil health, providing strong support for the sustainable development of persimmon cultivation in Wuyuan area.

[0036] The above is merely the preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions within the technical scope disclosed in this application shall be included in the scope of protection of this application. Therefore, the scope of protection of this application shall be based on the scope of protection of the claims.

Claims

1. A microbial composite agent, characterized in that: The microbial composite agent comprises Lactobacillus plantarum P9 and Lactobacillus casei ZHANG, and the ratio of the viable bacteria of Lactobacillus plantarum P9 to Lactobacillus casei ZHANG is (1-3):

1.

2. The microbial composite agent according to claim 1, characterized in that: The deposit number of the Lactobacillus plantarum P9 is CGMCC NO.16662; the deposit number of the Lactobacillus casei ZHANG is CGMCC NO.5469.

3. The microbial composite agent according to claim 1, characterized in that: The ratio of the viable bacteria of the Lactobacillus plantarum P9 to the Lactobacillus casei ZHANG is 2:

1.

4. The microbial composite agent according to claim 1, characterized in that: The bacterial concentration in the microbial composite agent is not less than 1×10 9 CFU / mL.

5. The microbial composite agent according to claim 1, characterized in that: The microbial composite agent is stored in a freeze-dried protective agent, and the components of the freeze-dried protective agent are: 12-18% trehalose, 2-3% skim milk powder, 0.5-0.8 mmol / L glutathione, 0.05-0.1% nano-silicon dioxide and phosphate.

6. A method for preparing the microbial composite agent according to any one of claims 1 to 5, characterized in that: The following steps are involved: Purified Lactobacillus plantarum P9 and Lactobacillus casei ZHANG were inoculated onto MRS agar plates, and different colonies were formed after cultivation. Inoculating colonies of Lactobacillus plantarum P9 and Lactobacillus casei ZHANG into a first MRS liquid culture medium and culturing for 24-48 hours; Take 2-5% of the culture medium and inoculate it into the second MRS liquid medium for expansion culture. When the pH value of the culture medium is stable and the bacterial growth reaches the exponential phase, use a high-speed centrifuge at 4-8 rpm for 1-15 minutes to precipitate the bacteria. The bacterial precipitate is taken out and washed with physiological saline for multiple times. The washed Lactobacillus plantarum P9 and Lactobacillus casei ZHANG are concentrated to obtain the microbial composite agent.

7. The method for preparing the microbial composite agent according to claim 6, characterized in that: The first MRS liquid culture medium and the second MRS liquid culture medium include the following components by mass: 1 g / L peptone, 2 g / L glucose, 2 g / L bile salt, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, and 2 g / L magnesium sulfate. The volume is adjusted to 1000 mL with distilled water and the pH is adjusted to 6.4-6.

6.

8. Use of the microbial composite agent according to any one of claims 1 to 5 in improving the quality and yield of Wuyuan persimmon.

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