Complex microbial inoculant and application thereof in lettuce planting

Abstract

The invention discloses a complex microbial inoculant and application thereof in lettuce planting, the complex microbial inoculant comprises lactobacillus plantarum P8 and lactobacillus plantarum P9, the preservation number of the lactobacillus plantarum P8 is CGMCC (China General Microbiological Culture Collection Center) NO.5468, and the preservation number of the lactobacillus plantarum P9 is CGMCC NO.16662. The invention further discloses a preparation method of the complex microbial inoculant. By applying the complex microbial inoculant in the lettuce planting process, the purposes of increasing the yield, improving the quality and inhibiting plant diseases and insect pests to increase economic benefits can be achieved; compared with a control group, the lettuce using the complex microbial inoculant has the advantages that the seedling survival rate is high, the seedling morbidity is effectively reduced, the yield is relatively effectively increased, and the yield increasing proportion is 34.86%.

Description

Technical Field

[0001] The invention belongs to the field of agricultural microbial applications, and particularly relates to a combined preparation of two specific plant lactobacilli and a multifunctional application thereof in lettuce crop cultivation. Background Art

[0002] As a green leafy vegetable with high economic value, lettuce is susceptible to infection by a variety of pathogens during its cultivation process. Among the many diseases, rot caused by Pseudomonas is particularly prominent. This disease is characterized by rapid onset and difficulty in prevention and control. In the early stage of the disease, translucent infiltrated spots appear on the edges of the leaves. As the disease progresses, the spots gradually turn dark green and are accompanied by tissue necrosis. During the heading stage, the inside of the leaf head may turn brown and rot. Current prevention and control methods mostly rely on chemical agents, but there are risks of increased resistance of pathogens and excessive pesticide residues. Once this disease occurs, it is very difficult to treat, and the conventional method is spraying, which is not only ineffective but also causes pesticide pollution. Therefore, it is necessary to provide a more scientific prevention and control method. Summary of the Invention

[0003] The present invention aims to provide a composite microbial agent and its application in lettuce planting. By applying the composite microbial agent during the lettuce planting process, the yield can be increased, pests and diseases can be suppressed, and the economic benefits can be increased.

[0004] The present invention innovatively proposes to construct a synergistic bacterial community using Lactobacillus plantarum P8 (CGMCC NO.5468) and P9 (CGMCC NO.16662). The Lactobacillus plantarum P8 has a preservation number of CGMCC NO.5468, and the Lactobacillus plantarum P9 has a preservation number of CGMCC NO.16662. The composition exhibits triple efficacy in lettuce cultivation:

[0005] 1) Promote nutrient absorption and increase biomass accumulation through root colonization;

[0006] 2) Produce antimicrobial metabolites to inhibit the growth of soil-borne pathogens;

[0007] 3) Activate plant system resistance and enhance its own immunity.

[0008] The technical solutions provided by the present invention are as follows:

[0009] A first aspect of the present invention provides a composite bacterial agent, comprising Lactobacillus plantarum P8 and Lactobacillus plantarum P9.

[0010] Furthermore, the effective viable bacteria count of the composite bacterial agent is ≥2×10 8 CFU.

[0011] Furthermore, the ratio of the viable bacteria count of the Lactobacillus plantarum P8 to the Lactobacillus plantarum P9 is 4:1.

[0012] A second aspect of the present invention provides a use of the composite bacterial agent as described above in increasing lettuce yield.

[0013] The third aspect of the present invention provides a use of the composite bacterial agent as described above in inhibiting gray mold, soft rot and rot of lettuce.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides a composite microbial agent and its application in lettuce cultivation. By applying the composite microbial agent during the lettuce cultivation process, the purpose of increasing yield, improving quality, and inhibiting pests and diseases can be achieved, thereby increasing economic benefits. Compared with the control group, the lettuce using the composite microbial agent has a high survival rate of seedlings, an effectively reduced incidence rate of seedlings, and a relatively effective increase in yield, with an increase rate of 34.86%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Comparison diagram of lettuce treated with and without the composite microbial agent in the embodiment of the present invention, wherein a is a real picture of lettuce treated with the composite microbial agent, and b is a real picture of lettuce not treated with the composite microbial agent;

[0017] Figure 2 Figure 2 is a diagram showing the growth status of lettuce with and without the composite microbial agent in an embodiment of the present invention, wherein the left side shows the growth status of lettuce with the composite microbial agent, and the right side shows the growth status of lettuce without the composite microbial agent. DETAILED DESCRIPTION

[0018] 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.

[0019] 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.

[0020] Example 1 Preparation of composite bacterial agent

[0021] The present invention provides a composite bacterial agent, comprising Lactobacillus plantarum P8 and Lactobacillus plantarum P9, wherein the ratio of viable bacteria of Lactobacillus plantarum P8 to Lactobacillus plantarum P9 is 4:1. Lactobacillus plantarum P8 was deposited with the China General Microorganism Center of the China National Culture Collection Administration on November 18, 2011, with a deposit number of CGMCC No. 5468. Lactobacillus plantarum P9 was deposited with the China General Microorganism Center of the China National Culture Collection Administration on October 31, 2018, with a deposit number of CGMCC No. 16662.

[0022] The preparation of the composite bacterial agent comprises the following steps:

[0023] 1.1 Inoculation

[0024] Purified Lactobacillus plantarum P8 and Lactobacillus plantarum P9 were selected and inoculated onto MRS agar plates, respectively, and cultured for 48 hours in a 37° C. incubator until typical colonies were formed.

[0025] 1.2 Bacteria Activation

[0026] 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 P8 and Lactobacillus plantarum P9 were growing well in the liquid culture medium and had entered the logarithmic growth phase.

[0027] 1.3 Expansion Culture

[0028] A small amount of activated culture fluid (usually 2-5% of the inoculation volume) is inoculated into a larger volume of MRS liquid culture medium, usually 2-5 mL of culture volume, and culture is continued at 37°C for 48 hours.

[0029] 1.4 Temperature and pH Control

[0030] Lactobacillus plantarum P8 and Lactobacillus plantarum P9 prefer to grow at a temperature of 37°C. This temperature should be maintained during the culture process, and the pH value of the culture medium should be monitored regularly (the pH should be reduced to 4.5-5.5). When the pH value is low, it can be adjusted by adding an appropriate amount of sodium hydroxide solution.

[0031] 1.5 Differential Centrifugation Enrichment

[0032] 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.

[0033] 1.6 Washing

[0034] 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.

[0035] 1.7 Bacterial Concentration

[0036] The washed Lactobacillus plantarum P8 and Lactobacillus plantarum P9 cells can be concentrated and adjusted to a bacterial solution concentration suitable for use.

[0037] The ratio of the viable bacteria of plantarum Lactobacillus P8 to the viable bacteria of plantarum Lactobacillus P9 in the composite microbial agent prepared by the present invention is 4:1. The number of viable bacteria per milliliter of the microbial composite microbial agent suspension is determined by plate count method (e.g., ten-fold dilution method). The bacterial concentration usually needs to reach 2×10 8 CFU / mL or higher.

[0038] Example 2 Application test of composite microbial agent in lettuce planting

[0039] In this example, the composite bacterial agent prepared in Example 1 was tested.

[0040] 2.1 Test Materials

[0041] Lettuce, compound microbial agent (the number of effective live bacteria in the compound microbial agent is ≥2×10 8 CFU).

[0042] 2.2 Test Location

[0043] At the Haofeng Agricultural Planting Base in Wangbojigou Village, Zhangbei County, Zhangjiakou City, one planting area with an area of 12 mu was selected for the regional experiment, and the adjacent areas with the same farming practices were used as the control group.

[0044]

[0045] Note: 1. The application group and the control group were managed with the same agricultural practices;

[0046] 2. The application group and the control group were selected from planting areas with the same planting and equipment hardware facilities, and the temperature and humidity were consistent.

[0047] 2.3 Test methods

[0048] Directions:

[0049] When watering, the compound bacterial agent was evenly poured onto the roots along with water according to the watering time of each acre of land. After the seedlings emerged, watered once (2 liters / time in early June) and once (2 liters / time in late June). The irrigated areas were compared in early July.

[0050] 2.4 Test Results

[0051] Seedling transplant survival rate: The planting amount of lettuce per mu is 4800 / mu, and 7 seeds are planted per meter in ridge planting. According to the actual situation of the plot, 5-meter ridges of the application field and the control field are randomly selected 5-7 days after transplanting to calculate the lettuce survival rate (as shown in Table 1). Ten groups of data are taken from each group to finally calculate the average survival rate.

[0052] Table 1

[0053]

[0054]

[0055] Result analysis: The survival rate of seedlings was measured by randomly sampling plots. The survival rate of seedlings in the control group was 97.14%, and the survival rate of seedlings in the application group was 99.14%. The application of compound microbial agent can improve the survival rate of lettuce seedlings.

[0056] Seedling incidence rate: The same method as the seedling survival rate was used to calculate the incidence rate of lettuce (gray mold, soft rot, and rot) by randomly selecting 5-meter ridges from the application field and the control field according to the actual situation of the plot. As shown in Table 2, 10 groups of data were taken from each group to finally calculate the average incidence rate.

[0057] Table 2

[0058]

[0059] Result analysis: The seedling morbidity data were measured by randomly sampling plots. The seedling morbidity of the control group was 25.71%, and the seedling morbidity of the application group was 10%. Figure 1 It can be seen that the application of compound bacterial agents can effectively reduce the incidence of lettuce seedlings.

[0060] The yield comparison is shown in Table 3.

[0061] Table 3

[0062]

[0063] Result analysis: Figure 2 As shown, compared to the control group, the lettuce treated with the composite inoculant had a higher survival rate, significantly reduced seedling disease incidence, and significantly increased yield by 34.86%. Due to the long-term continuous cropping of lettuce, the plot had severe outbreaks of gray mold, soft rot, and rot, leading to reduced yields. However, the experimental group achieved a yield of 1,750 kg per mu, compared to only 1,140 kg in the control group, a yield increase of 610 kg per mu. This demonstrates that the composite inoculant can effectively reduce losses caused by common lettuce diseases, improve the yield of lettuce, and increase production and ensure a stable income.

[0064] Observation indicators:

[0065] 1. Colonization survival rate: 99.14% in the experimental group (97.14% in the control group)

[0066] 2. Disease inhibition rate: The comprehensive prevention effect on gray mold, soft rot, etc. is up to 61.2%

[0067] 3. Yield increase: Average yield per mu increased by 34.86% (1750kg vs 1140kg)

[0068] 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 composite bacterial agent, characterized in that: The composite bacterial agent comprises Lactobacillus plantarum P8 and Lactobacillus plantarum P9, the preservation number of Lactobacillus plantarum P8 is CGMCC NO.5468, and the preservation number of Lactobacillus plantarum P9 is CGMCC NO.16662.

2. The composite bacterial agent according to claim 1, characterized in that: The effective viable bacteria count of the composite bacterial agent is ≥2×10 8 CFU.

3. The microbial composite agent according to claim 1, characterized in that: The ratio of the viable bacteria of the Lactobacillus plantarum P8 to the Lactobacillus plantarum P9 is 4:

1.

4. Use of the composite bacterial agent according to any one of claims 1 to 3 in increasing lettuce yield.

5. Use of the composite bacterial agent according to any one of claims 1 to 3 in inhibiting gray mold, soft rot and rot of lettuce.

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