Preparation method and application of garlic planting soil improving bacterial agent

Abstract

The present invention provides a preparation method and application of a soil improving agent for garlic planting. The preparation method of the soil improving agent for garlic planting comprises: (1) selecting Bacillus amyloliquefaciens, Bacillus thuringiensis subspecies israeli and Streptomyces tenuiflavus; (2) fermenting Bacillus amyloliquefaciens and Bacillus thuringiensis subspecies israeli; (3) fermenting Streptomyces tenuiflavus. The selected strains and their formulas of the present invention can have the effects of increasing production, killing insects and antagonizing soil-borne diseases to a certain extent, alleviate the problem of soil flora imbalance caused by excessive fertilization and pesticide application in the garlic planting process, promote the increase of garlic production and income, increase farmers' income, break the bottleneck of agricultural development, and promote sustainable agricultural development.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, and in particular to a preparation method and application of a soil improving bacterial agent for garlic planting. Background Art

[0002] Garlic is an important economic crop. It is a kind of condiment and vegetable that is rich in nutritional value and has high medicinal value. As people gradually recognize the nutritional and medicinal value of garlic, the domestic market demand is large, and my country is the main producer and exporter of garlic. Garlic is grown on a large scale in Shandong, Jiangsu, Yunnan, Hebei, Henan and other places.

[0003] However, due to long-term continuous cropping and asexual reproduction, the varieties are mixed, chaotic, weak and degraded; the amount of chemical fertilizer input is too large, the amount of organic fertilizer used is small, and the nutrient ratio is unreasonable, resulting in a decrease in soil fertility; productivity is not high and the potential for fertilizer saving is huge; garlic planting yields have also begun to show a downward trend; soil continuous cropping obstacles have occurred, and the base number of pathogenic microorganisms of soil-borne diseases has increased year by year, leading to serious diseases and insect pests. Coupled with improper planting and management methods and other factors, the quality of garlic has continued to decline and its market competitiveness has declined.

[0004] Therefore, it is necessary to provide a preparation method and application of a garlic planting soil improving bacterial agent to solve the problems existing in the above-mentioned prior art. Summary of the invention

[0005] In view of this, the present invention provides a preparation method and application of a garlic planting soil improving microbial agent, aiming to alleviate the problem of soil flora imbalance caused by excessive fertilization and pesticide application during garlic planting, promote garlic production and income increase, increase farmers' income, break the bottleneck of agricultural development, and promote sustainable agricultural development.

[0006] In order to achieve the above technical effects, the present invention provides a preparation method and application of a garlic planting soil improving bacterial agent, which adopts the following technical scheme:

[0007] A method for preparing a soil improving microbial agent for garlic planting, comprising:

[0008] (1) Select Bacillus amyloliquefaciens, Bacillus thuringiensis subsp. israelensis and Streptomyces tenuiflavus;

[0009] (2) Fermentation of Bacillus amyloliquefaciens and Bacillus thuringiensis israeliensis: LB medium activation, culture at 35℃ for 24h; liquid LB medium, shaker culture, 180r / min, 35℃ for 24h; seed fermentation tank filled with fermentation medium, culture at 35℃ for 18h to obtain first-class seed culture liquid; 1 ton fermentation tank, temperature controlled at 35℃, dissolved oxygen controlled at 30%, rotation speed 150r / min, culture for 18h, blood cell counting plate microscopic examination of live bacteria count, Bacillus amyloliquefaciens subspecies israeliensis subspecies more than 5 billion CFU / mL, Bacillus thuringiensis subspecies israeliensis subspecies more than 2 billion CFU / mL, stop tanking;

[0010] (3) Fermentation of Streptomyces leucodermi: inoculate PYG solid culture medium and culture at 30°C for 48 h; pick a single colony and inoculate it into liquid PYG culture medium and culture it on a shaker at 200 r / min and 28°C for 48 h; culture it in a seed fermentation tank filled with fermentation medium at 35°C for 48 h; in a 1-ton fermentation tank, control the temperature at 35°C, the dissolved oxygen at 20%, the rotation speed at 100 r / min, culture for 48 h, and stop tanking when the live bacterial count of Streptomyces leucodermi is above 5 billion CFU / mL.

[0011] Furthermore, the strains in (1) were purchased from the China General Microbiological Culture Collection Center (CGMCC), among which the accession number of Bacillus amyloliquefaciens is CGMCC 1.803, the accession number of Bacillus thuringiensis subsp. israelensis is CGMCC1.1754, and the accession number of Streptomyces tenuifolius is CGMCC 4.5123.

[0012] Further, in (2), the fermentation medium is prepared as follows: 30-40 parts of soluble corn starch, 40-50 parts of molasses, 10-20 parts of soybean meal, 1.0-2.0 parts of KH2PO4, 0.5-1 parts of MgSO4·7H2O, 0.2 parts of MnSO4·H2O and 5.0 parts of CaCO3, mixed well, pH 7.2, and sterilized at 121°C for 30 min.

[0013] Furthermore, in (3), the fermentation medium is prepared as follows: 20-30 parts of starch, 10-20 parts of soybean meal, 5-10 parts of bran, 2-5 parts of ammonium sulfate, 2-5 parts of magnesium sulfate, 2-5 parts of dipotassium hydrogen phosphate and 5 parts of calcium carbonate, which are mixed evenly, adjusted to volume with water, pH 7.5, and sterilized at 121°C for 40 min.

[0014] An application of the microbial agent prepared by the above preparation method is to use 20L of the microbial agent per mu, and apply it with water during the two best irrigation periods of garlic, namely, water for strengthening seedlings and water for moss growth, and use it once respectively.

[0015] Further, as an application of a garlic yield-increasing microbial agent, the preparation of the garlic yield-increasing microbial agent is as follows: 50 parts of Bacillus amyloliquefaciens, 10 parts of Bacillus thuringiensis subsp. israelensis, 20 parts of Streptomyces tenuiflavos, 5 parts of zinc sulfate, 5 parts of magnesium sulfate and 5 parts of potassium humate, adjusting the pH to 6.0, and mixing evenly to obtain the product.

[0016] Furthermore, as an application of a garlic maggot control bacterial agent, the preparation of the garlic maggot control bacterial agent is as follows: 40 parts of Bacillus thuringiensis subspecies israelensis, 10 parts of Bacillus amyloliquefaciens and 10 parts of Streptomyces tenuiflavos, adjusting the pH to 6.0, and mixing evenly.

[0017] Furthermore, as an application of a soil improving agent for garlic continuous cropping obstacles, the soil improving agent for garlic continuous cropping obstacles is prepared as follows: 30 parts of Bacillus amyloliquefaciens, 30 parts of Streptomyces tenuifolius, 10 parts of Bacillus thuringiensis subspecies israelensis, 5 parts of zinc sulfate, 5 parts of magnesium sulfate and 5 parts of potassium humate, the pH is adjusted to 6.0, and the mixture is mixed evenly.

[0018] The above technical solution of the present invention includes at least the following beneficial effects:

[0019] The strains and formulations selected by the present invention can have the effects of increasing production, killing insects and antagonizing soil-borne diseases to a certain extent, alleviate the problem of soil flora imbalance caused by excessive fertilization and pesticide application during garlic planting, promote the increase of garlic production and income, increase farmers' income, break the bottleneck of agricultural development, and promote sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the promoting effect of the bacterial agent on indigenous beneficial microorganisms in Example 2;

[0021] Figure 2 This is a comparison chart of the effect of the bacterial agent on the growth of garlic roots in Example 2, wherein A is the control plot and B is the test plot;

[0022] Figure 3 This is a comparison diagram of the garlic root conditions of user 6 in Example 3, where C is the control site and D is the test site;

[0023] Figure 4 This is a statistical result diagram of the diversity of soil-borne diseases in Example 4. DETAILED DESCRIPTION

[0024] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the following will be combined with the appended drawings of the embodiments of the present invention. Figure 1-4, the technical scheme of the embodiment of the present invention is clearly and completely described. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0025] The invention provides a preparation method and application of a garlic planting soil improving bacterial agent. The bacterial strains are all purchased from China General Microbiological Culture Collection Center (CGMCC), wherein the preservation number of Bacillus licheniformis is CGMCC 1.803, the preservation number of Bacillus thuringiensis subsp. israelensis is CGMCC 1.1754, and the preservation number of Streptomyces microflavus is CGMCC 4.5123.

[0026] The specific implementation is as follows:

[0027] Example 1

[0028] A method for preparing a soil improving microbial agent for garlic planting, comprising:

[0029] (1) Fermentation production of Bacillus

[0030] The fermentation process of Bacillus amyloliquefaciens and Bacillus thuringiensis subsp. israelensis was respectively operated according to the following steps: taken out from the -80°C refrigerator, inoculated with LB medium for activation, and cultured at 35°C for 24 hours; picked a single colony and inoculated it into liquid LB medium, cultured in a shaking table at 180r / min and 35°C for 24 hours; transferred to a seed fermentation tank filled with fermentation medium, cultured at 35°C for 18 hours, and obtained a primary seed culture solution.

[0031] Transfer 3% of the first-level seed culture solution inoculated into a 1-ton fermentation tank with fermentation medium and control the temperature at 35°C. Control the dissolved oxygen at 30%, the rotation speed at 150 r / min, and culture for 18 h. Count the live bacteria under a blood cell count plate after staining. If the number of Bacillus amyloliquefaciens is above 5 billion CFU / mL and the number of Bacillus thuringiensis is above 2 billion CFU / mL, stop tanking.

[0032] The preparation process of the culture medium is as follows:

[0033] LB medium (g / L): 5% yeast extract, 10% tryptone, 10% NaCl, dilute to 1.0L with distilled water, pH 7.0-7.2. Add 15.0% agar to the solid medium; pH 7.2, sterilize at 121℃ for 30min;

[0034] The fermentation medium formula of Bacillus amyloliquefaciens and Bacillus thuringiensis subsp. israelensis is: 30% soluble corn starch, 40% molasses, 20% soybean meal (100 mesh fermentation grade), 1.0% KH2PO4, 0.5% MgSO4·7H2O, 0.2% MnSO4·H2O and 5.0% CaCO3; pH 7.2, sterilization at 121℃ for 30min.

[0035] (2) Fermentation production of Streptomyces leucoflavus

[0036] The Streptomyces tenuifolius strain was inoculated into a PYG solid culture medium and cultured at 30°C for 48 hours; a single colony was picked and inoculated into a liquid PYG culture medium and cultured in a shaking incubator at 200 r / min and 28°C for 48 hours; the colony was transferred to a seed fermentation tank filled with a fermentation medium and cultured at 35°C for 48 hours to obtain a primary seed culture solution.

[0037] The first-level seed culture liquid was inoculated into a 1-ton fermentation tank with a 10% inoculation rate, and the temperature was controlled at 35°C; the dissolved oxygen was controlled at 20%, the rotation speed was 100r / min, and the culture was carried out for about 48 hours. When the live bacterial count of Streptomyces tenuifolius was above 5 billion CFU / mL, the fermentation was terminated and the tank was removed.

[0038] The preparation process of the culture medium is as follows:

[0039] PYG medium (g / L): tryptone 10, yeast extract 10, glucose 5;

[0040] Fermentation medium of Streptomyces tenuifolius (g / L): starch 30, soybean meal 10 (100 mesh fermentation grade), bran 10 kg (100 mesh fermentation grade), ammonium sulfate 5, magnesium sulfate 5, dipotassium hydrogen phosphate 5, calcium carbonate 5, make up to 1000 kg with water; pH 7.5, sterilize at 121℃ for 40min.

[0041] Example 2

[0042] The application of microbial agents as garlic yield-increasing agents; that is, the application of microbial agents to increase yield in medium-level garlic cultivation.

[0043] Four households with medium garlic planting levels in Qi County in previous years were selected as yield-increasing test households, and each household selected 1 mu of test plot and control plot, and the garlic seeds and fertilization were basically the same; 20L of bacterial agent was added to the test field, and the use time and frequency were once in early April and early May, and irrigated with water. The production method described in Example 1 uses the preferred bacterial agent and formula weight parts: 50 parts of Bacillus amyloliquefaciens, 10 parts of Bacillus thuringiensis subspecies israelensis, 20 parts of Streptomyces tenuifolius, 5 parts of zinc sulfate, 5 parts of magnesium sulfate and 5 parts of potassium humate, adjust the pH to 6.0, and after mixing evenly, obtain the garlic yield-increasing bacterial agent.

[0044]

[0045] Table 1.

[0046] Note: a. The yield is calculated based on 70% of the fresh weight; b. The grade rate is the proportion of garlic larger than 5 cm.

[0047] The yield measurement method is to take samples at five points, randomly sample 100 garlics, and calculate 25,000 heads per mu; the field of the microbial agent is affected by many factors, and the yield increase effect is shown in Table 1, with an increase range of 5%-21%, and an average increase of 11.25%. At the same time, the rate of high-quality products in the experimental field is higher than that in the control field. Figure 1 The figure shows the promoting effect of the microbial agent on the beneficial indigenous microorganisms. The Bacillus and Pseudomonas in the applied microbial agent can effectively stimulate the effects of the indigenous microorganisms related to the nutrient cycle.

[0048] like Figure 2 The figure below is a comparison chart of the effects of the microbial agent on the growth of garlic roots. The microbial agent effectively promoted the growth of garlic root fibrous roots, which were more developed than the control and the garlic heads were fuller.

[0049] Example 3

[0050] The application of microbial agents as a control agent for garlic maggots; that is, the effect of microbial agents on garlic maggots in the roots of garlic.

[0051] A test plot in Qi County where insect pests were relatively serious in previous years was selected, and 3 households were selected as insecticide test households. Each household selected 1 mu of test plot and control plot, and the garlic seeds and fertilizers were basically the same; 20L of microbial agent was added to the test field, and the application time and frequency were once in early April and early May, and irrigated with water. The production method described in Example 1 uses the preferred microbial agent and formula weight parts: 40 parts of Bacillus thuringiensis subspecies israelensis, 10 parts of Bacillus amyloliquefaciens and 10 parts of Streptomyces tenuifolius, adjust the pH to 6.0, and mix evenly to obtain garlic maggot insecticide;

[0052]

[0053] Table 2.

[0054] The insecticidal effect of the fungicide is shown in Table 2. In the control plot of User 5, 80% of the garlic roots were infested with maggots when the pesticide thiazophos was used for root irrigation, while only 10% of the garlic roots were infested with maggots in the test plot that only used the fungicide. However, the test field used 20 kg less compound fertilizer, resulting in a lower yield than the control plot. In the control plot of User 7, 60% of the garlic roots were infested with maggots, but none were found in the test plot.

[0055] User 6 is the household with the lowest yield and the most serious insect pests in previous years. He reported that the insecticide effect was obvious, earthworms appeared, and there were signs of improvement. Figure 3As shown; and the control garlic stems were smaller than those in the test and died earlier; soil diversity analysis also found that user 6 had a more serious insect pest, with the proportion of Fusarium in the fungal flora being 8.19%, which was absolutely dominant in population competition, and Fusarium was the pathogen of garlic root rot, which was consistent with literature reports. Diseases are often accompanied by insect pests, resulting in crop yield reduction. The microbial agent of the present invention has a significant effect in killing garlic maggots at the root of garlic, and also has a certain yield-increasing effect, which will provide an alternative option for reducing the use of pesticides in the future.

[0056] Example 4

[0057] The application of microbial agents as soil improvement agents for garlic continuous cropping obstacles; that is, the application of microbial agents in the soil where garlic is planted for many years in continuous cropping.

[0058] A plot of land in Qi County where garlic has been planted for more than 10-15 years was selected, and 3 households were selected as insecticide test households. Each household selected 1 mu of test land and control land, and the garlic seeds and fertilizers were basically the same; 20L of bacterial agent was added to the test field, and the application time and frequency were once in early April and early May, and irrigated with water. The production method described in Example 1 uses the preferred bacterial agent and formula weight parts: 30 parts of Bacillus amyloliquefaciens, 30 parts of Streptomyces tenuifolius, 10 parts of Bacillus thuringiensis subspecies israelensis, 5 parts of zinc sulfate, 5 parts of magnesium sulfate and 5 parts of potassium humate, and the pH is adjusted to 6.0. After mixing evenly, the soil improvement bacterial agent for garlic continuous cropping obstacles is obtained.

[0059]

[0060] Table 3.

[0061] As shown in Table 3, after the use of soil improvement agents for continuous cropping obstacles, the garlic yield increased by 10%-28%, with an average increase of 16%;

[0062] like Figure 4 As shown in the figure, it is a statistical result of soil-borne diseases diversity. It can be seen that through the comparison of the abundance of the top 50 pathogens in the soil samples of 3 households, it is found that the microbial agent can effectively inhibit the expansion of pathogens, especially from the results of 3 test households, it is shown that the microbial agent can effectively inhibit the dominant soil pathogen Fusarium that causes root rot. It shows that the microbial agent of the present invention can effectively alleviate the obstacles of continuous cropping and inhibit the soil-borne pathogenic microorganisms caused by it.

[0063] The strains and their formulations selected by the present invention can have the effects of increasing production, killing insects and antagonizing soil-borne diseases to a certain extent, alleviate the problems of soil flora imbalance and pests caused by excessive fertilization and pesticide application during garlic planting, and promote the increase of garlic production and income. However, the above is only a specific embodiment of the present invention, and the scope of implementation of the present invention cannot be limited by this. Therefore, the replacement of equivalent components, or equivalent changes and modifications made according to the scope of protection of the patent of the present invention, should still fall within the scope of the claims of the present invention.

[0064] The above are preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a soil improving agent for garlic planting, characterized in that: include: (1) Select Bacillus amyloliquefaciens, Bacillus thuringiensis subsp. israelensis and Streptomyces tenuiflavus; (2) Fermentation of Bacillus amyloliquefaciens and Bacillus thuringiensis israeliensis: LB medium activation, culture at 35°C for 24 hours; liquid LB medium, shaker culture, 180r / min, 35°C for 24 hours; seed fermentation tank filled with fermentation medium, culture at 35°C for 18 hours to obtain first-class seed culture liquid; 1 ton fermentation tank, temperature controlled at 35°C, dissolved oxygen controlled at 30%, rotation speed 150r / min, culture for 18 hours, blood cell counting plate microscopic examination of live bacteria count, Bacillus amyloliquefaciens subspecies israeliensis subspecies more than 5 billion CFU / mL, Bacillus thuringiensis subspecies israeliensis subspecies more than 2 billion CFU / mL, stop tanking; (3) Fermentation of Streptomyces leucodermi: inoculate PYG solid culture medium and culture at 30°C for 48h; pick a single colony and inoculate it into liquid PYG culture medium and culture it on a shaker at 200r / min and 28°C for 48h; culture it in a seed fermentation tank filled with fermentation medium at 35°C for 48h; in a 1-ton fermentation tank, control the temperature at 35°C, the dissolved oxygen at 20%, the rotation speed at 100r / min, culture it for 48h, and stop the fermentation when the live bacterial count of Streptomyces leucodermi is above 5 billion CFU / mL; The accession number of the Bacillus amyloliquefaciens is CGMCC 1.803, the accession number of the Bacillus thuringiensis subspecies israelensis is CGMCC 1.1754, and the accession number of the Streptomyces tenuiflavos is CGMCC 4.5123. The above strains were purchased from China General Microbiological Culture Collection Center (CGMCC).

2. The method for preparing the garlic planting soil improving microbial agent according to claim 1, characterized in that: (2) The fermentation medium is prepared as follows: 30-40 parts of soluble corn starch, 40-50 parts of molasses, 10-20 parts of soybean meal, 1.0-2.0 parts of KH2PO4, 0.5-1 parts of MgSO4·7H2O, 0.2 parts of MnSO4·H2O and 5.0 parts of CaCO3, which are mixed evenly, pH 7.2, and sterilized at 121°C for 30 min.

3. The preparation method of the garlic planting soil improving bacterial agent according to claim 1, characterized in that, (3) The fermentation medium is prepared as follows: 20-30 parts of starch, 10-20 parts of soybean meal, 5-10 parts of bran, 2-5 parts of ammonium sulfate, 2-5 parts of magnesium sulfate, 2-5 parts of dipotassium hydrogen phosphate and 5 parts of calcium carbonate. The mixture is evenly mixed, adjusted to volume with water, pH 7.5, and sterilized at 121°C for 40 min.

4. An application of the bacterial agent prepared by the preparation method according to any one of claims 1 to 3, characterized in that: Use 20L of bacterial agent per mu, and apply it with water during the two best irrigation periods for garlic: water for strong seedlings and water for moss growth, and use it once each time.

5. The use of the bacterial agent according to claim 4, characterized in that: As an application of a garlic yield-increasing microbial agent, the garlic yield-increasing microbial agent is prepared by: 50 parts of Bacillus amyloliquefaciens, 10 parts of Bacillus thuringiensis subspecies israelensis, 20 parts of Streptomyces tenuiflavos, 5 parts of zinc sulfate, 5 parts of magnesium sulfate and 5 parts of potassium humate, adjusting the pH to 6.0, and mixing evenly to obtain the agent.

6. The use of the bacterial agent according to claim 4, characterized in that: As an application of a garlic maggot control bacterial agent, the preparation of the garlic maggot control bacterial agent is as follows: 40 parts of Bacillus thuringiensis subspecies israelensis, 10 parts of Bacillus amyloliquefaciens and 10 parts of Streptomyces tenuiflavos, adjusting the pH to 6.0, and mixing evenly to obtain the agent.

7. The use of the bacterial agent according to claim 4, characterized in that: As an application of a soil improving bacterial agent for garlic continuous cropping obstacles, the soil improving bacterial agent for garlic continuous cropping obstacles is prepared by: 30 parts of Bacillus amyloliquefaciens, 30 parts of Streptomyces tenuiflavus, 10 parts of Bacillus thuringiensis subspecies israelensis, 5 parts of zinc sulfate, 5 parts of magnesium sulfate and 5 parts of potassium humate, adjusting the pH to 6.0, and mixing evenly to obtain the agent.

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