CN102965319B - Microbial agent and bio-organic fertilizer special for winter jujube and preparation methods thereof - Google Patents

Abstract

The invention discloses a bacterial liquid of microbial composition, which comprises rhizosphere growth-promoting Bacillus subtilis DZ1, Saccharomyces cerevisiae and Lactobacillus casei. The invention also discloses a microbial agent specially used for winter jujube and a preparation method thereof. The microbial agent is prepared by mixing the bacterial liquid of the microbial composition and a stabilizing auxiliary agent at a weight ratio of 100:4-7. The present invention also discloses a bio-organic fertilizer specially used for jujube, which is formed by mixing microbial composition bacterial liquid and organic fertilizer, the organic fertilizer: microbial composition bacterial liquid = 10:1~2 (W/V). Test proves, when the winter jujube seedlings are inoculated with the microbial bacterial agent of the present invention than the single inoculation of three kinds of bacterial strain fermented liquid or stabilizing aids, the winter jujube root weight, ground diameter and seedling height all significantly increase; Compared with the bio-fertilizer or organic fertilizer made by inoculating the rhizosphere growth-promoting Bacillus subtilis DZ1 into the organic fertilizer, the yield is increased by 10-20%, and the quality is obviously improved.

Description

Special microbial agent for winter jujube, bio-organic fertilizer and preparation method thereof

technical field

The invention relates to a special microbial agent for jujube, biological organic fertilizer and a preparation method thereof.

Background technique

The production management process of winter jujube is very complicated, and fertilization is one of the very important links. The fertilization effect is not only affected by the soil, the nutritional characteristics of the plant itself and the amount of fertilizer applied, but also related to the type of fertilizer. At present, the fertilizers used in winter jujube are mainly compound fertilizers composed of chemical fertilizers and organic fertilizers based on farmyard manure. The combined application of compound fertilizers and organic fertilizers improves the yield and quality of winter jujubes. Because the root system of winter jujube is underdeveloped, the growth is slow, the number of capillary roots is small, and the nutrient absorption capacity is poor, a large amount of compound fertilizer is used, and the leaching of fertilizer nutrients is serious, resulting in eutrophication of surface water and pollution of groundwater; Although fertilizers can improve the physical and chemical properties of soil, the organic matter in them varies from good to bad, and contains germs and eggs of diseased insects, which can easily cause soil pollution. Microbial fertilizers can not only improve the soil, improve the ecological environment of the soil flora, but also effectively improve the absorption and utilization of soil nutrients by plants. Therefore, research on finding new fertilizers suitable for jujube, especially microbial fertilizers, to reduce the amount of chemical fertilizers and improve the effect of organic fertilizers has attracted much attention.

There are few domestic research reports on the microbial fertilizer of winter jujube. CN102643144A discloses a special active biomedicine fertilizer for winter jujube, which is mainly used for spraying on the leaves of winter jujube. It has dual functions of disease prevention and fattening. The special active biomedical fertilizer for winter jujube disclosed in this patent is a foliar fertilizer, which has no effect on improving soil and rhizosphere micro-ecological environment.

Studies have shown that the interaction between functional microorganisms and crops is often unstable, and this instability stems from the impact of different environmental conditions on the growth of microorganisms, thereby restricting their effects. This requires that the functional microorganisms in microbial fertilizers should be more targeted. At present, there are few domestic research reports on the use of microbial fertilizers in the production of winter jujube, and there is no research report on the special microbial compound bacterial agent for winter jujube.

For winter jujube, it is an urgent need for the production of winter jujube to develop special microbial compound bacteria agent and special bio-organic fertilizer to steadily and continuously improve the soil micro-ecological environment, so as to increase the yield and quality of winter jujube.

The present inventor has screened out a rhizosphere growth-promoting Bacillus subtilis DZ1 of Dongzao rhizosphere DZ1 from the rhizosphere soil samples of Dongzao. For this strain and its application, the inventor has applied for a national invention patent "A Strain of Dongzao Rhizosphere Growth-Promoting Bacillus subtilis and Its Application", the patent application number is 201110376966.1. The patent discloses the application of Dongzao rhizosphere growth-promoting Bacillus subtilis DZ1: cultivate the strain to obtain a bacterial suspension, which is used as a liquid fertilizer; inoculate the bacterial suspension into organic fertilizer to make Dongzao bio-organic fertilizer. These applications are only preliminary, specifically as follows: (1) The microbial fertilizer produced by the above application only contains Bacillus subtilis DZ1, which promotes growth in the rhizosphere of winter jujube, and needs to add a variety of specific microbial populations to achieve the microbial cluster growth-promoting effect; ( 2) The fermentation conditions of Dongzao rhizosphere growth-promoting Bacillus subtilis DZ1 need to be further optimized to realize industrial production; (3) The bacterial suspension is directly used as liquid fertilizer, and its shelf life is short, so appropriate stabilizers and protective agents need to be added to Protect the survival of live bacteria and prolong the shelf life.

Contents of the invention

The purpose of the present invention is to provide a microbial composition bacterial liquid, in which the bacterial strains are not antagonistic but synergistic with each other, and are used to prepare special microbial bacterial agents and bio-organic fertilizers for winter jujube.

Another object of the present invention is to provide a special microbial bacterial agent for winter jujube containing the bacterial liquid of the microbial composition and a preparation method thereof.

Another object of the present invention is to provide a special bio-organic fertilizer for winter jujube containing the bacterial liquid of the microbial composition and a preparation method thereof.

To achieve the above object, the present invention provides the following technical solutions:

A microbial composition bacterial liquid, including rhizosphere growth-promoting Bacillus subtilis DZ1, Saccharomyces cerevisiae and Lactobacillus casei. The test showed that there was no antagonistic effect among the three strains.

The CFU ratio of the rhizosphere-promoting Bacillus subtilis DZ1, the Saccharomyces cerevisiae, and the Lactobacillus casei is: 10:1-2:2-3, and the number of viable bacteria in the microbial composition liquid is >3 ×10 ⁸ cfu/ml.

The present invention additionally provides a microbial agent specially used for winter jujube, which is formed by mixing the above-mentioned microbial composition bacterial solution and a stabilizing auxiliary agent at a weight ratio of 100:4-7, and the rhizosphere growth-promoting Bacillus subtilis of winter jujube The cfu ratio of the liquid culture solution of DZ1, Saccharomyces cerevisiae and Lactobacillus casei is: 10:1-2:2-3, and the number of viable bacteria in the bacterial liquid of the microbial composition is >3×10 ⁸ cfu/ml.

The microbial bacterial agent after mixing the bacterial liquid of the microbial composition and the stabilizing auxiliary agent contains a total effective number of viable bacteria >2.5×10 ⁸ cfu/ml.

The stabilizing auxiliary agent is composed of the following components by weight: 3-5 parts of calcium carbonate, 0.2-0.5 parts of xanthan gum, 0.2-0.3 parts of hydrophilic white carbon black, 0.3-0.6 parts of sodium chloride parts, 0.3-0.6 parts of sodium carboxymethylcellulose.

The preparation method of the microbial bacterial agent specially used for winter jujube, the steps are as follows:

(1) The rhizosphere growth-promoting Bacillus subtilis DZ1, Saccharomyces cerevisiae and Lactobacillus casei were respectively cultured to obtain a fermentation broth;

(2) The fermented liquid is mixed according to the cfu ratio of 10:1-2:2-3 to obtain a mixed bacterial liquid, and the mixed bacterial liquid is mixed with a stabilizing auxiliary agent to obtain the product.

The cultivation method of the rhizosphere growth-promoting Bacillus subtilis DZ1 of the winter jujube is (the following are percentages by weight):

(1) Test tube seed culture

The formula of the seed medium is, based on the total weight of the seed medium: 0.3% beef extract, 1% peptone, 0.5% sodium chloride, pH7.0-7.2, and the balance is water;

The rhizosphere growth-promoting Bacillus subtilis DZ1 of winter jujube is inoculated on the seed medium, and cultured at a constant temperature of 30-35° C. for 36 hours to obtain test-tube species.

(2) Liquid fermentation culture

The formula of the liquid fermentation medium is, based on the total amount of the liquid fermentation medium: 1% soluble starch, 1% yeast extract, 0.15% K ₂ HPO ₄ , 0.02% MgSO ₄ 7H ₂ O, 0.01% CaCl ₂ , and The amount is water, pH6.5-7.5.

Inoculate the test tube seeds into 500ml Erlenmeyer flask liquid culture medium, the inoculum amount is 10% (volume ratio), 200ml per bottle, cultivate in a shaker at 30-35°C, with a rotation speed of 180r/min, and cultivate for 12 hours. The bacterial content of the growth-promoting Bacillus subtilis DZ1 can reach 40×10 ⁸ cfu/ml.

The present invention also provides a bio-organic fertilizer specially used for winter jujube, which is formed by mixing the liquid culture solution of winter jujube rhizosphere growth-promoting Bacillus subtilis DZ1, Saccharomyces cerevisiae, and Lactobacillus casei with organic fertilizer. The organic fertilizer: microorganism Composition bacteria solution=10:1~2 (W/V).

The bio-organic fertilizer contains a total effective number of viable bacteria ≥ 0.25×10 ⁸ cfu/g.

The production of the organic fertilizer: mix chicken manure and fungus chaff at a weight ratio of 9:1, then inoculate the decomposed bacteria agent of organic materials, carry out aerobic fermentation, and pulverize through a 1-2mm sieve after decomposing and air-drying, with a water content of ≤20%. Organic matter content ≥ 30%, total nutrient N+P ₂ O ₅ +K ₂ O ≥ 5%. Chicken manure, fungus chaff and organic material decomposing bacteria agents can all be purchased from the market.

The Saccharomyces cerevisiae and Lactobacillus casei used in the present invention are derived from the General Microbiology Center (CGMCC) of China Microbial Culture Collection Management Committee. is 2.0666. They can be purchased through commercial channels, and their culture and growth conditions are carried out according to the instructions provided by the strain preservation unit.

Compared with the prior art, the present invention has the following effects and advantages:

(1) The present invention uses Dongzao rhizosphere growth-promoting Bacillus subtilis DZ1 as the main component, and optimizes the combination of other functional bacteria to realize the cluster growth-promoting effect of multiple strains on Dongzao, so that the microbial fertilizer product has a stronger effect on Dongzao pertinence and effectiveness.

(2) The present invention provides an optimized cultivation method for rhizosphere growth-promoting Bacillus subtilis DZ1 of winter jujube. Without increasing the production cost, the bacterial content in the fermentation broth reaches 40×10 ⁸ cfu/ml, and the production efficiency is significantly improved .

(3) The addition of the stabilizer in the microbial agent of the present invention ensures the activity and stability of the functional bacteria.

(4) Tests have shown that when Dongzao seedlings are inoculated with the microbial agent of the present invention, the Dongzao rhizosphere growth-promoting Bacillus subtilis DZ1 fermented liquid, Saccharomyces cerevisiae fermented liquid, Lactobacillus casei fermented liquid or stabilizing aids when inoculated alone, Dongzao jujube Root weight, ground diameter and seedling height all significantly increase; Winter jujube fruit tree uses bio-organic fertilizer of the present invention and uses winter jujube rhizosphere growth-promoting Bacillus subtilis DZ1 to be inoculated into the biological fertilizer or the organic fertilizer that make in organic fertilizer, yield Increased by 10-20%, the quality improved significantly.

Description of drawings

Fig. 1 is a process flow diagram of the present invention;

Fig. 2 is the result figure of the effective viable bacteria number test of the special microbial bacterial agent for winter jujube of the present invention.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

Embodiment 1 is specially used in the preparation of the microbial bacterial agent of winter jujube, bio-organic fertilizer

The rhizosphere growth-promoting Bacillus subtilis DZ1 fermentation broth of Dongzao rhizosphere was prepared according to the aforementioned cultivation method of the rhizosphere-promoting Bacillus subtilis DZ1.

Prepare the fermentation liquid of Saccharomyces cerevisiae and Lactobacillus casei according to the culture method provided by the strain preservation unit.

The obtained fermented liquid is uniformly mixed according to the quantity ratio of the number of living bacteria contained: the ratio of cfu of Dongzao rhizosphere growth-promoting Bacillus subtilis DZ1, said Saccharomyces cerevisiae, and said Lactobacillus casei=10:1.5:2.5. The bacterial liquid of the microbial composition contains a total number of viable bacteria of 3.36×10 ⁸ cfu/ml.

The stabilizing aid is composed of the following components by weight: 3.5 parts of calcium carbonate, 0.35 parts of xanthan gum, 0.2 parts of hydrophilic white carbon black, 0.5 parts of sodium chloride, and 0.5 parts of sodium carboxymethylcellulose. The bacterial liquid of the microbial composition and the stabilizing agent are mixed at a weight ratio of 100:5.05. After mixing, a special microbial bacterial agent for winter jujube is prepared, and the total number of viable bacteria is 3.10×10 ⁸ cfu/ml.

Mix chicken manure and fungus chaff according to the weight ratio of 9:1, then inoculate the decomposed bacteria agent of organic materials, carry out aerobic fermentation, pulverize and pass through a 2mm sieve after decomposing and air-drying, and obtain water content ≤ 20%, organic matter content ≥ 30%, total Organic fertilizer with nutrients N+P ₂ O ₅ +K ₂ O≥5%.

According to the ratio of organic fertilizer: microbial composition bacterial solution = 10:1, fully absorb and mix to make a special bio-organic fertilizer for winter jujube. The number of live bacteria in the fertilizer is 0.28×10 ⁸ cfu/g.

Embodiment 2 is specially used in the microbial bacterial agent of winter jujube

The rhizosphere growth-promoting Bacillus subtilis DZ1 fermentation broth of Dongzao rhizosphere was prepared according to the aforementioned cultivation method of the rhizosphere-promoting Bacillus subtilis DZ1.

Prepare the fermentation liquid of Saccharomyces cerevisiae and Lactobacillus casei according to the culture method provided by the strain preservation unit.

The obtained fermented liquid is uniformly mixed according to the quantity ratio of the number of living bacteria contained: the ratio of the cfu of Dongzao rhizosphere growth-promoting Bacillus subtilis DZ1, the Saccharomyces cerevisiae, and the Lactobacillus casei is 10:1.3:2.3. The bacterial liquid of the microbial composition contains 3.64×10 ⁸ cfu/ml of total viable bacteria.

The stabilizing aid is composed of the following components by weight: 4.0 parts of calcium carbonate, 0.30 parts of xanthan gum, 0.2 parts of hydrophilic white carbon black, 0.6 parts of sodium chloride, and 0.4 parts of sodium carboxymethylcellulose. The bacterial liquid of the microbial composition and the stabilizing auxiliary agent are mixed at a weight ratio of 100:5.5. After mixing, a special microbial bacterial agent for winter jujube is prepared, and the total number of viable bacteria is 3.41×10 ⁸ cfu/ml.

Example 3 Application test of special microbial agent for winter jujube

The experiment was arranged in the experimental nursery of Shandong Academy of Forestry. Choose the year-old jujube seedlings with consistent growth, plant them in a plastic pot with a height of 20cm and a diameter of 30cm, get the special microbial inoculum for jujube made in Example 1 and dilute 10 times with tap water, get 100ml of the diluted inoculum (total effective live bacteria 40×10 ⁸ cfu) were evenly watered around the roots of winter jujube seedlings, and measured the dry weight of the aboveground part, the dry weight of the underground part, and the total dry weight (experimental group) after 6 months of growth, and watered with 100ml of clean water (control group 1), Only water 100ml of the diluted Dongzao rhizosphere growth-promoting Bacillus subtilis DZ1 fermentation broth (total effective viable count 40×10 ⁸ cfu) (control group 2), and only water the diluted Saccharomyces cerevisiae fermentation broth (total effective viable count 40×10 ⁸ cfu) 100ml (control group, 3), and only watered 100ml of diluted Lactobacillus casei fermentation broth (total effective number of viable bacteria 40×10 ⁸ cfu) (control group 4) for control. The results are shown in Table 1.

It can be seen from Table 1 that the dry weight of aboveground part, dry weight of underground part, and total dry weight of winter jujube seedlings treated in the experimental group were all higher than those in the control group, and the differences reached a significant level. It shows that the special microbial agent for winter jujube can obviously promote the growth of winter jujube.

Table 1 The growth-promoting effect of microbial bacterial agents on winter jujube seedlings

Example 4 field test of special bio-organic fertilizer for winter jujube

The field test of Dongzao bio-organic fertilizer was arranged in the Dongzao Demonstration Garden of Binzhou Forestry Bureau, and the age of the tree was 8 years. Apply the special bio-organic fertilizer for winter jujube (experimental group) prepared in Example 1, and the organic fertilizer (control group 1) prepared in example 1, and use the organic fertilizer prepared in example 1 to absorb the rhizosphere of winter jujube and promote the growth of subtilized grass Bio-organic fertilizer (0.28×10 ⁸ cfu/g) (control group 2) made from Bacillus DZ1 fermentation broth was used as a control. It was applied twice in 2011: 5kg of test fertilizer or control fertilizer was applied to each tree at the end of June, and 10kg of test fertilizer or control fertilizer was applied to each tree at the beginning of September. The yield and quality of Dongzao were measured on October 9, 2012, and the results are shown in Table 2.

It can be seen from the test results that the fruit quality and yield of winter jujube special bio-organic fertilizer were higher than those of the control group, and the difference reached a significant level.

Table 2 Effects of bio-organic fertilizers on fruit yield and quality of Dongzao

The effective viable count test of embodiment 5 winter jujube special microbial agent

Using the microbial bacterial agent specially used for winter jujube in Example 2, and only adding calcium carbonate (control group 1) in the stabilizing auxiliary agent, only adding xanthan gum in the stabilizing auxiliary agent (control group 2), adding only Hydrophilic white carbon black in the stabilizing agent (control group 3), only adding sodium chloride in the stabilizing agent (control group 4), only adding carboxymethyl cellulose sodium in the stabilizing agent ( Control group 5) and without adding any stabilizing agent (control group 6) were compared, and the number of viable bacteria in the bacterial agent was measured after natural storage for 90 days, and the results are shown in Figure 2. It can be seen that the number of effective viable bacteria in the microbial inoculum of winter jujube is significantly greater than that of the experimental treatment with the addition of some stabilizing adjuvants.

Claims (7)

1. a microbial composite bacterium liquid that is exclusively used in the winter jujube, it is characterized in that, comprise winter jujube rhizosphere growth-promoting subtilis DZ1, S. cervisiae, lactobacillus johnsonii, wherein, the ratio of the cfu of described winter jujube rhizosphere growth-promoting subtilis DZ1, described S. cervisiae, described lactobacillus johnsonii is: 10:1～2:2～3, microbial composite bacterium liquid is containing viable count>3 * 10 ⁸cfu/ml; Described winter jujube rhizosphere growth-promoting subtilis DZ1 Classification And Nomenclature is bacillus sp., on October 11st, 2011, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its deposit number is CGMCC No.5318.

2. a microbiobacterial agent that is exclusively used in the winter jujube, it is characterized in that, to be mixed by weight 100:4～7 by the liquid medium of winter jujube rhizosphere growth-promoting subtilis DZ1, S. cervisiae, lactobacillus johnsonii and steady effect auxiliary agent, the ratio of the cfu of the liquid medium of described winter jujube rhizosphere growth-promoting subtilis DZ1, S. cervisiae, lactobacillus johnsonii is: 10:1～2:2～3, microbial composite bacterium liquid is containing viable count>3 * 10 ⁸cfu/ml;

Described steady effect auxiliary agent is comprised of the component of following weight proportion: 3～5 parts, calcium carbonate, 0.2～0.5 part of xanthan gum, 0.2～0.3 part of wetting ability White Carbon black, 0.3～0.6 part, sodium-chlor, Xylo-Mucine 0.3-0.6 part.

3. the microbiobacterial agent that is exclusively used in the winter jujube according to claim 2, is characterized in that, described microbiobacterial agent is containing total living bacteria count>=2.5 * 10 ⁸cfu/ml.

4. the preparation method who is exclusively used in the microbiobacterial agent of winter jujube claimed in claim 2, is characterized in that, step is as follows:

(1) get winter jujube rhizosphere growth-promoting subtilis DZ1, S. cervisiae, lactobacillus johnsonii and cultivated respectively to obtain fermented liquid;

(2) by fermented liquid, by the ratio of the cfu of winter jujube rhizosphere growth-promoting subtilis DZ1, S. cervisiae, lactobacillus johnsonii, be: 10:1～2:2～3 are mixed to get mixed bacteria liquid, and mixed bacteria liquid is mixed with steady effect auxiliary agent, obtain.

5. the preparation method who is exclusively used in the microbiobacterial agent of winter jujube claimed in claim 4, is characterized in that, the cultural method of described winter jujube rhizosphere growth-promoting subtilis DZ1 is below to be weight percentage:

(1) test tube seed culture

The seed culture based formulas is, in the gross weight of seed culture medium: extractum carnis 0.3%, peptone 1%, sodium-chlor 0.5%, pH7.0-7.2, surplus is water;

Winter jujube rhizosphere growth-promoting subtilis DZ1 is inoculated on seed culture medium, and 30～35 ℃ of constant temperature culture 36 hours, obtain the test tube kind;

(2) liquid fermentation and culture

The liquid fermentation medium formula is, in the total amount of liquid fermentation medium: Zulkovsky starch 1%, yeast extract paste 1%, K ₂hPO ₄0.15%, MgSO ₄7H ₂o0.02%, CaCl ₂0.01%, surplus is water, pH6.5-7.5;

The test tube seed is inoculated in 500ml triangular flask liquid nutrient medium, and inoculum size is 10% by volume, every bottled 200ml, in 30～35 ℃ of shaking tables, cultivate, rotating speed 180r/min, cultivate 12 hours, and in fermented liquid, winter jujube rhizosphere growth-promoting subtilis DZ1 bacteria containing amount can reach 40 * 10 ⁸cfu/ml.

6. a biological organic fertilizer that is exclusively used in the winter jujube, it is characterized in that, that liquid medium and fertilizer by winter jujube rhizosphere growth-promoting subtilis DZ1, S. cervisiae, lactobacillus johnsonii mixes, described fertilizer: microbial composite bacterium liquid=10:1～2(W/V); Described biological organic fertilizer is containing total living bacteria count>=0.25 * 10 ⁸cfu/g.

7. the biological organic fertilizer that is exclusively used in the winter jujube according to claim 6, it is characterized in that, the making of described fertilizer: chicken manure and bacterium chaff are mixed by weight 9:1, inoculate the organic material composting microbial inoculum, carry out aerobic fermentation, become thoroughly decomposed after air-dry and pulverized 1～2mm sieve, water content≤20%, organic content>=30%, total nutrient N+P ₂o ₅+ K ₂o>=5%.

Images