CN114560735A

CN114560735A - Biogas slurry bacterial fertilizer and preparation method and application thereof

Info

Publication number: CN114560735A
Application number: CN202210167026.XA
Authority: CN
Inventors: 慕康国; 刘艳丽; 李乃荟; 赵娜娜; 陈清
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-02-23
Filing date: 2022-02-23
Publication date: 2022-05-31
Anticipated expiration: 2042-02-23
Also published as: CN114560735B

Abstract

The invention relates to the technical field of compound fertilizer production, and particularly discloses a biogas slurry bacterial fertilizer and a preparation method and application thereof. The biogas slurry bacterial fertilizer comprises bacillus amyloliquefaciens, concentrated biogas slurry, active substances and a protective agent; the active substances are itaconic acid and polyglutamic acid, and the protective agent is trehalose. The biogas slurry bacterial fertilizer can obviously promote the germination and growth of seeds, obviously improve the inhibition effect on pathogenic bacteria, reduce the number of viable bacteria after being stored for one month and still have ideal bacteriostasis rate.

Description

Biogas slurry bacterial fertilizer and preparation method and application thereof

Technical Field

The invention relates to the technical field of compound fertilizer production, in particular to a biogas slurry bacterial fertilizer and a preparation method and application thereof.

Background

The direct discharge of biogas slurry can cause many environmental problems, such as eutrophication of water bodies and damage to animals and plants in the water bodies, so the development, utilization and research of biogas slurry resources are concerned. The biogas slurry contains rich nutrient elements, vitamins, amino acids, bioactive substances and the like, and is a good organic liquid fertilizer. A large number of researches show that the biogas slurry can replace part of chemical fertilizers, can effectively improve soil fertility after being applied for a long time, improves the contents of nitrogen, phosphorus, potassium and the like in the soil, promotes the growth and development of crops, increases the crop yield, and can also alleviate or prevent and control crop diseases and insect pests to a certain extent. But the biogas slurry has low nutrient content, generally more than 95 percent of the nutrient content is water, the fertilizer efficiency is not high when the biogas slurry is directly used, the transportation cost is high, and the commercial utilization is difficult. In recent years, the concentration technology can remove redundant moisture in the biogas slurry, so that the storage and transportation cost is reduced, the nutrient content can be greatly improved, and the fertilizer value of the biogas slurry is improved.

The phenomenon of soil-borne diseases caused by continuous cropping obstacles is common, and environmental pollution and food safety problems caused by the use of chemical pesticides have attracted people's attention and concern. The beneficial microorganism is a method for effectively preventing and controlling soil-borne diseases. The biocontrol bacteria play an important role in preventing and controlling soil-borne diseases, and the number of soil-borne pathogenic bacteria can be reduced by inducing plant system resistance, secreting bacteriostatic substances, competing with pathogenic bacteria for resources and the like. In addition, the biocontrol bacterium can secrete some plant growth hormones, and has a stimulation effect on plant growth. The bacillus is widely distributed in nature, has the advantages of various varieties, strong stress resistance, high temperature tolerance, good biocontrol effect and the like, and becomes an ideal biocontrol bacterium resource for preventing and treating soil-borne diseases.

The bacillus is compounded with the biogas slurry, so that the organic fertilizer and microbial fertilizer have the advantages of both organic fertilizers and microbial fertilizers, contain a large amount of organic matters (amino acids, proteins, sugar, fat and the like) and certain nitrogen, phosphorus and potassium nutrients, also contain a large amount of biocontrol microbes and metabolites thereof, and can effectively enhance the soil fertility, improve the soil environment, improve the crop quality and enhance the disease resistance of plants. The combination of the two can not only avoid the environmental problem caused by biogas slurry discharge, but also realize the value-added utilization of the biogas slurry, and have higher environmental protection benefit, economic benefit and social benefit.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide the biogas slurry fertilizer which can promote the growth of seeds, effectively inhibit pathogenic bacteria and has long lasting period.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a biogas slurry bacterial fertilizer, which comprises: bacillus amyloliquefaciens, concentrated biogas slurry, active substances and a protective agent; the active substances are itaconic acid and polyglutamic acid, and the protective agent is trehalose.

According to the invention, bacillus amyloliquefaciens, concentrated biogas slurry, specific active substances (with growth promoting and soil improving properties) and a protective agent (with a microbial protection effect) are compounded, so that the biogas slurry bacterial fertilizer with good effects of promoting plant growth and continuously inhibiting pathogenic bacteria is obtained.

The microbial fertilizer has the nutrient balance of the organic fertilizer, the quick acting performance of the fertilizer and the promoting effect of microorganisms, and is considered to be the best choice for replacing the traditional fertilizer. The essential feature of microbial fertilizers is that they contain microorganisms with specific functions, the vital activities of which are the main factors of microbial fertilizers over other fertilizers. After the microbial fertilizer is applied to soil, active substances generated by functional microorganisms can dissolve part of indissolvable nutrients in the soil, so that richer nutrition is provided for plants, and meanwhile, bacteriostatic substances generated by the functional microorganisms can reduce crop diseases and promote the growth of crops. Therefore, the number of functional microorganisms and the survival time of the functional microorganisms in the microbial fertilizer are important points for the efficacy of the fertilizer.

The bacillus is added into the concentrated biogas slurry to prepare the fertilizer, and the survival condition of the bacillus in the biogas slurry needs to be considered, so that the bacillus can have higher survival rate. The growth and reproduction of bacillus are influenced by various factors such as pH value, salt concentration, nutrients and the like. When the pH is too high or too low, the growth of the bacillus can be inhibited, the salt concentration is favorable for the growth of the bacillus within a certain range, but the life activity of the bacillus is inhibited due to the too high salt concentration, and the growth, the reproduction, the cell metabolism and the like tend to slow. The concentrated biogas slurry has high nutrient content and may influence the growth of bacillus. In addition, the number of viable bacteria in commercial bio-organic fertilizers on the market at present is remarkably reduced along with the increase of storage time. Although some protective agents are usually added in order to improve the survival rate of functional bacteria. The protective agent can play a role in buffering when the pH, osmotic pressure and temperature in the environment change, protect thalli and prolong the storage time of the microbial fertilizer. However, how to find a bacterial fertilizer formula which can effectively match the microbial inoculum and the biogas slurry and realize good overall comprehensive effect still needs to be researched and researched repeatedly.

The invention researches the influence of the concentrated biogas slurry and the bacillus after compounding on the activity of the bacillus, and researches the influence of EC and pH changes on the activity of the bacillus caused by adding different nutrient element factors under the condition that the concentrated biogas slurry is used as a fertilizer carrier. And further, the maximum survival rate of the bacillus in the biogas liquid fertilizer is ensured by optimizing the bacillus protective agent, and the method has important significance for improving the commodity value and the application of the biogas liquid bacterial fertilizer.

In the biogas slurry bacterial fertilizer, the bacillus amyloliquefaciens has the capability of producing siderophores, the IAA production capability is 7-40mg/kg, the phosphorus solubility is 40-90mg/kg, the inhibition effect on fusarium oxysporum is 40-50%, and the viable count is 1 multiplied by 10 when the NaCl concentration is 60g/L⁹-1.5×10⁹CFU/mL。

Preferably, the bacillus amyloliquefaciens has the capability of producing siderophores, the IAA production capability is 7.5mg/kg, the phosphorus solubility is 68mg/kg, the inhibition effect on fusarium oxysporum is 47.8 percent, and the viable count is 1.3 multiplied by 10 percent when the NaCl concentration is 60g/L⁹CFU/mL, maximum viable count at pH 7 of 2.9X 10⁹CFU/mL can further ensure the growth promoting effect and the lasting antibacterial effect of the bacterial fertilizer, effectively enhance the soil fertility, improve the soil environment, improve the crop quality and enhance the disease resistance of plants.

In the present invention, the ability of bacteria to produce IAA is determined by colorimetric methods.

The phosphorus solubility of the microorganism is measured by a colorimetric method after the microorganism is cultured in a relevant liquid culture medium for 30-50 h.

The siderophore production capacity is determined by using detection liquid.

The antibacterial property is measured by adopting a flat plate opposing method:

specifically, the pathogenic bacteria are inoculated on a PDA culture medium, the PDA culture medium is placed in an incubator at the temperature of 20-30 ℃ for culture for 3-7 d, and a puncher is used for preparing a bacterial cake for later use. Inoculating the pathogenic bacteria cake in the center of a corresponding culture medium, symmetrically inoculating 2 separated and purified bacillus strains at a certain position away from the center, performing dark culture at 20-30 ℃ for 3-10 d (according to the growth condition of bacterial colonies), measuring the diameter of the pathogenic bacteria colony, and calculating the bacteriostasis rate.

And (3) determining stress resistance: (1) determination of salt resistance: adding a certain amount of NaCl into a corresponding culture medium, setting a certain mass concentration gradient for the NaCl, inoculating the bacillus into the culture medium with different salt concentrations in a certain volume in an inoculation amount of 5-20 g/L after high-temperature sterilization, performing shake culture for 20-30 h at 20-30 ℃ under the condition of 150-200 r/min, and determining the viable count by adopting a dilution coating plate method. (2) And (3) acid and alkali resistance determination: adding a certain amount of HCl or NaOH into a corresponding culture medium to adjust the pH, setting the pH of the culture medium with a certain concentration gradient, inoculating bacillus into NA liquid culture media with a certain volume and different pH values in an inoculation amount of 5-20 g/L after high-temperature and high-pressure sterilization, performing shake culture for 20-30 hours at 20-30 ℃ under the condition of 150-200 r/min, and determining the number of viable bacteria by using a dilution coating plate method.

The biogas slurry bacterial fertilizer comprises 10-30g/L of itaconic acid and 60-80g/L of polyglutamic acid.

The biogas slurry bacterial manure comprises 30-50mg/L trehalose.

Preferably, the biogas slurry bacterial fertilizer comprises 15g/L of itaconic acid, 80g/L of polyglutamic acid and 50mg/L of trehalose, so that the viable count of bacillus amyloliquefaciens in the bacterial fertilizer can be better improved, the activity time and the survival rate of the bacillus amyloliquefaciens in the bacterial fertilizer can be maintained, and the overall action function and the activity preservation time of the biogas slurry bacterial fertilizer can be prolonged.

In the invention, the mass-to-volume ratio of the bacillus amyloliquefaciens to the concentrated biogas slurry is (30-70): 1g/L, preferably 50: 1g/L to give consideration to economy and action effects.

In the biogas slurry bacterial fertilizer, the concentrated biogas slurry is livestock and poultry manure biogas slurry subjected to filtration and impurity removal, and the physicochemical properties of the livestock and poultry manure biogas slurry are as follows: pH, 9.2; EC, 32.00 mS/cm; TN, 5.00 g/L; TP: 2.40 g/L; TK: 5.60 g/L; organic matter, 1.90 g/L; 4.50g/L of humic acid; ca, 864 mg/L; mg, 25 Mg/L; s, 352 mg/L; fe, 104 mg/L; mn, 16 mg/L; zn, 84 mg/L; cu, 11mg/L, so that the added bacillus amyloliquefaciens has higher activity and longer acting time in bacterial manure.

As a preferred embodiment, the concentrated biogas slurry is prepared by firstly pre-treating chicken manure biogas slurry before a membrane to remove a large amount of suspended matters in the chicken manure biogas slurry, and then filtering the pre-treated biogas slurry through an inorganic ceramic ultrafiltration membrane, so that redundant moisture and impurities in the biogas slurry are removed, micro organic matters in the biogas slurry are intercepted, and the nutrient content and the fertilizer value of the biogas slurry are improved. The concentrated biogas slurry is a tan or dark brown liquid in appearance.

The invention also provides a method for preparing the biogas slurry fertilizer, which comprises the step of mixing the bacillus amyloliquefaciens, the concentrated biogas slurry, the active substances and the protective agent.

The invention also provides application of the biogas slurry fertilizer or the method in preparation of products for promoting plant growth and/or inhibiting pathogenic bacteria.

Preferably, the pathogenic bacteria are Fusarium oxysporum f.sp.cucumerinum, Fusarium oxysporum f.sp.lycopersici, and Fusarium solani;

and/or the plant is cucumber, wheat or Chinese cabbage.

The invention has the beneficial effects that:

the bacillus and concentrated biogas slurry compounded organic bacterial fertilizer has a synergistic effect on seed growth, and can remarkably promote seed germination and growth. Compared with the single use of the biogas liquid fertilizer, the biogas liquid fertilizer containing the bacillus can obviously improve the inhibition effect on pathogenic bacteria. In addition, the number of viable bacteria in the biogas slurry fertilizer containing the bacillus is reduced a little after the biogas slurry fertilizer is stored for one month, and the bacteriostasis rate of the biogas slurry fertilizer to soil-borne pathogenic fungi is reduced by about 5 percent compared with the prior use. In conclusion, the bacillus and concentrated biogas slurry compounded organic bacterial fertilizer has good growth promoting and disease inhibiting effects on crops, and the bacillus and concentrated biogas slurry compounded organic bacterial fertilizer have a synergistic effect.

Drawings

Fig. 1 is a statistical result of viable count change in a biogas slurry bacterial manure storage process, wherein the significance difference between different treatments is completed through Duncan test, and different lower case letters indicate that the significance difference exists between the treatments, and the significance P is less than 0.05.

Fig. 2 shows the effect of different treated fertilizers on cucumber seed germination, wherein (a) shows the result of comparison of germination vigor between different treatments, (b) shows the result of comparison of germination percentage between different treatments, (c) shows the result of comparison of bud length between different treatments, (d) shows the result of comparison of root length between different treatments, (e) shows the result of comparison of germination index between different treatments, and (f) shows the result of comparison of vigor index between different treatments, wherein the significant difference between different treatments is determined by Duncan test. Different letters indicate significant differences between treatments at the same dilution (P < 0.05).

Fig. 3 shows the effect of different treated fertilizers on wheat seed germination, wherein (a) shows the result of comparison of germination vigor between different treatments, (b) shows the result of comparison of germination percentage between different treatments, (c) shows the result of comparison of bud length between different treatments, (d) shows the result of comparison of root length between different treatments, (e) shows the result of comparison of germination index between different treatments, and (f) shows the result of comparison of vigor index between different treatments, wherein the significant difference between different treatments is determined by Duncan test. Different letters indicate significant differences between treatments at the same dilution (P < 0.05).

Fig. 4 shows the effect of different treated fertilizers on the germination of Chinese cabbage seeds, wherein (a) shows the result of comparing the germination potential between different treatments, (b) shows the result of comparing the germination percentage between different treatments, (c) shows the result of comparing the bud length between different treatments, (d) shows the result of comparing the root length between different treatments, (e) shows the result of comparing the germination index between different treatments, and (f) shows the result of comparing the vitality index between different treatments, wherein the significant difference between different treatments is determined by Duncan test. Different letters indicate significant differences between treatments at the same dilution (P < 0.05).

FIG. 5 is a graph showing the effect of different treatment fertilizers on the specialized growth of Fusarium oxysporum cucumbers.

FIG. 6 is a graph showing the effect of different treatments on the growth of Fusarium oxysporum tomato specialization.

FIG. 7 is a graph showing the effect of different treatments on the growth of Fusarium solani.

In fig. 2 to 7, CK is blank; BS is the original concentrated biogas slurry; BSF is a biogas slurry fertilizer without bacillus; b3 is Bacillus amyloliquefaciens B3 bacterial liquid; FA is the biogas slurry bacterial manure (ready for use) of example 1; FB is the biogas slurry bacterial manure of example 1 (stored for one month).

FIG. 8 shows the effect of different active substances on the viable count of Bacillus amyloliquefaciens, wherein CK represents that no material is added into the concentrated biogas slurry; YK represents addition of itaconic acid; NM indicates addition of citric acid; SL represents the addition of sorbitol; JG represents addition of polyglutamic acid; YS represents addition of itaconic acid + sorbitol; YJ represents addition of itaconic acid + polyglutamic acid; NS means addition of citric acid + sorbitol; NJ represents addition of citric acid + polyglutamic acid; YNSJ indicates addition of itaconic acid + citric acid + sorbitol + polyglutamic acid. Significant differences between different treatments were accomplished by Duncan test. Different letters indicate that the significance difference exists between different treatments at the same time, and the significance P is less than 0.05.

FIG. 9 shows the effect of different protective agents on the viable count of Bacillus amyloliquefaciens, wherein CK represents that no material is added to the concentrated biogas slurry; HZ represents the addition of trehalose; BJ represents the addition of p-hydroxybenzoic acid; TW represents addition of tween 20; GY represents addition of glycerol. Significant differences between different treatments were accomplished by Duncan test. Different letters indicate that the significance difference exists between different treatments at the same time, and the significance P is less than 0.05.

FIG. 10 shows the effect of adding protective agents and nutrients under optimal conditions on the viable count of Bacillus amyloliquefaciens, wherein CK represents that no material is added to the concentrated biogas slurry; YJ denotes addition of itaconic acid + polyglutamic acid; HZ represents the addition of trehalose; YJH denotes addition of itaconic acid + polyglutamic acid + trehalose; FB means addition of potassium nitrate based on YJH treatment. Significant differences between different treatments were accomplished by Duncan test. Different letters indicate that the significance difference exists between different treatments, and the significance P is less than 0.05.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The bacillus amyloliquefaciens B3 bacterial fertilizer used in the detailed description of the invention was purchased from a neutral organism.

Example 1

The embodiment provides a biogas slurry bacterial fertilizer and a preparation method thereof.

Raw materials:

1. strain: the bacillus amyloliquefaciens B3 bacterial fertilizer has the following properties:

(1) IAA production capacity: 7.5mg/kg (measured by colorimetry);

(2) phosphorus solubility: 68mg/kg (measured by colorimetry);

(3) has the capability of producing siderophores;

(4) the inhibition effect on fusarium oxysporum reaches 47.8 percent;

(5) when the NaCl concentration is 60g/L, the viable count is 1.3 multiplied by 10⁹CFU/mL；

(6) The highest viable count is 2.9 × 10 when pH is 7⁹CFU/mL。

2. Concentrating biogas slurry: the method comprises the following steps of pretreating chicken manure biogas slurry from Shandong people and biotechnology limited company before membrane treatment, and filtering and removing impurities by an ultrafiltration membrane to obtain the chicken manure concentrated biogas slurry, wherein the appearance color of the chicken manure concentrated biogas slurry is brown, and the physicochemical properties of the chicken manure concentrated biogas slurry are as follows: pH, 9.2; EC, 32.00 mS/cm; TN, 5.00 g/L; TP, 2.40 g/L; TK, 5.60 g/L; organic matter, 1.90 g/L; 4.50g/L of humic acid; ca, 864 mg/L; mg, 25 Mg/L; s, 352 mg/L; fe, 104 mg/L; mn, 16 mg/L; zn, 84 mg/L; cu, 11 mg/L.

3. Active substance: itaconic acid, polyglutamic acid.

4. A protective agent: trehalose.

The preparation method comprises the following steps: mixing 50g of bacillus amyloliquefaciens B3 with 1L of concentrated biogas slurry, and adding active substances and a protective agent to ensure that the concentration of itaconic acid, polyglutamic acid and trehalose in the biogas slurry bacterial manure is 15g/L, 80g/L and 50 mg/L.

Experimental example 1 changes in viable count during biogas slurry bacterial manure storage

In the experimental example, the change of the viable count of the biogas slurry bacterial fertilizer obtained in the example 1 in the storage process is measured. The storage mode is as follows: and placing the biogas slurry bacterial fertilizer in a sterilized centrifugal tube, and storing the biogas slurry bacterial fertilizer at room temperature in a dark place. The results are shown in FIG. 1.

The viable count measuring method comprises the following steps: diluting the biogas slurry bacterial manure prepared in the example 1 into diluent with different gradients, sucking a proper amount of the diluent by using a sterile gun head, coating the diluent on corresponding culture medium flat plates with different gradients, uniformly coating the three flat plates with different gradients, putting the flat plates into an incubator at 26 ℃ for culturing for 3 days, and counting.

As shown in figure 1, the number of bacillus amyloliquefaciens B3 in the biogas slurry bacterial manure is reduced along with the increase of the storage time. Wherein the reduction trend is most obvious in 0-30 days, and the viable count in the biogas slurry bacterial manure is 3.0 multiplied by 10 in the 30 th day⁹CFU/mL, survival 48.4%. The number of viable bacteria is basically kept stable within 30-90 days, and no obvious difference exists. The number of viable bacteria of Bacillus amyloliquefaciens B3 at day 90 was 2.8 × 10⁹CFU/mL, meets the regulation of the number of viable bacteria in the composite microbial fertilizer (5 multiplied by 10)⁷CFU/ml)。

Experimental example 2 influence of biogas slurry bacterial manure on germination effect of cucumber, wheat and cabbage seeds

The experimental example determines the influence of the biogas liquid bacterial manure prepared in example 1 on the germination effect of cucumber, wheat and cabbage seeds.

The experimental method is as follows:

test seeds:

wheat seeds (alternate 987), cabbage seeds (luyi) and cucumber seeds (jin yan 4) were purchased from china agri-academy of sciences and hongfeng vegetable research ltd.

The experiment was set up with 16 treatments:

the original Biogas Slurry (BS) described in example 1 was diluted 250 times, 500 times and 1000 times; the prepared biogas liquid fertilizer (BSF, which is different from the BSF prepared in the example 1 only in that no bacillus amyloliquefaciens B3 is added) is diluted by 250 times, 500 times and 1000 times; 50g/L of Bacillus amyloliquefaciens B3 was added to 100mL of sterile water (B3) to dilute 250-fold, 500-fold and 1000-fold; the biogas slurry bacterial manure (FA) prepared in the embodiment 1 is diluted by 250 times, 500 times and 1000 times; the biogas slurry bacterial manure (FB) prepared in example 1 after being stored for one month is diluted by 250 times, 500 times and 1000 times; sterile water (CK) treatment served as control.

The test process comprises the following steps:

selecting seeds with plump seeds and basically consistent sizes, soaking and sterilizing the seeds in 1% NaClO solution for 10min, continuously stirring the seeds, then washing the seeds for 3 times by using deionized water, and placing the seeds in a shade place to be dried to reach the initial water content. Then, 10mL of the treatment solution diluted by different times was added to each of the 9 cm-diameter petri dishes. And (3) putting a layer of sterile qualitative filter paper in each culture dish, uniformly placing 20 seeds, repeating each treatment for 9 times, adding a dish cover for moisturizing, placing the culture dish in an incubator at the temperature of 25 ℃ for dark culture, recording the germination number of the seeds at each fixed point until the control germination rate does not change, and recording the root length, the bud length, the germination rate, the germination index, the germination vigor and the vitality index of the seeds.

Measurement indexes are as follows:

according to the crop seed test protocol, germination was counted as 1/2 with radicle length exceeding the length of the seed, and the number of germination of the seed was recorded per d (test end standard: no change in the number of germination of the control group of 2d consecutively).

The germination rate, germination index, germination vigor and vigor index were obtained by calculation in the following manner.

Wherein GP represents Germination Potential (Germination Potential); n2 is the germination number of the 2d seed; n is the total number of the tested seeds.

Wherein GR represents Germination Rate (Germination Rate); n is_tT d th seed germination number, t is 4 when testing cucumber and Chinese cabbage seeds, t is 6 when testing wheat seeds; n is the total number of the tested seeds.

Wherein GI represents Germination Index (Germination Index); n is_tT d seed germination number, Dt is corresponding seed germination days, and t is the same as calculated germination rate.

VI＝GI×S

Wherein VI represents the viability Index (Vigor Index); GI is germination index; s is the average root length.

Root length (Root length) and Bud length (Bud length) were measured directly with an accuracy of 1mm meter.

The results are shown in FIGS. 2 to 4. As can be seen from FIG. 2, the germination vigor and germination index of 250-fold and 500-fold diluted biogas slurry bacterial manure (FA and FB) in example 1 are significantly lower than those of the control treatment, and the treatment has an inhibiting effect on germination of cucumber seeds. However, the biogas liquid bacterial manure (FA and FB) of example 1 treated by 1000 times dilution has greatly improved germination vigor and germination index of seeds. Wherein, the germination potentials of FA and FB diluted by 1000 times are respectively 87.8% and 87.2%, and the two are not significantly different and are respectively improved by 15.3% and 14.6% compared with a control. Most treatments have a different degree of promotion of both shoot and root length in cucumber seeds compared to controls. The biogas slurry bacterial manure (FA and FB) diluted by 1000 times in the embodiment 1 has obvious promotion effects on the bud length and the root length of cucumber seeds: the bud length of the cucumber seed after being diluted by 1000 times can respectively reach 48.9mm and 47.0mm, and the bud length of the cucumber seed have no significant difference, and are respectively improved by 56.3 percent and 50.1 percent compared with a control; the root length of the cucumber seeds after being diluted by 1000 times can reach 52.7mm and 53.0mm respectively, and the root length are not significantly different and are improved by 72.3 percent and 73.3 percent respectively compared with a control. The root length and the bud length of the cucumber seeds treated by the 1000-time diluted liquid of the biogas slurry bacterial fertilizer (FA and FB) in the embodiment 1 are obviously higher than those treated by the 1000-time diluted liquid of the biogas slurry bacterial fertilizer and the bacillus amyloliquefaciens B3 which are not contained in bacillus and are diluted 1000 times. Compared with the singly used diluted 1000 times biogas slurry fertilizer, the treatment root length of the 1000 times biogas slurry bacterial fertilizer (FA and FB) of the embodiment 1 is respectively increased by 12.1 percent and 12.8 percent; compared with 1000-time dilution of the bacillus amyloliquefaciens B3 bacterial liquid, the dilution is improved by 18 percent.

Therefore, the biogas slurry bacterial manure in the embodiment 1 has a synergistic effect on the growth of cucumber seeds. In addition, the number of viable bacteria in the biogas slurry bacterial manure in the embodiment 1 is reduced after the biogas slurry bacterial manure is stored for one month, but the germination and growth of cucumber seeds are not obviously different.

As can be seen from FIG. 3, the germination vigor and germination index of the 250-fold and 500-fold diluted biogas slurry bacterial manure (FA and FB) in example 1 are significantly lower than those of other treatments, and the biogas slurry bacterial manure has an inhibiting effect on the germination of wheat seeds. But the germination vigor and the germination index of the biogas liquid bacterial manure (FA and FB) seeds treated by 1000 times of dilution in the embodiment 1 are greatly improved. Wherein, the germination potentials of FA and FB diluted by 1000 times are respectively 87.2% and 86.4%, and the two are not significantly different and are respectively improved by 22.7% and 21.6% compared with a control. Most treatments have different degrees of promotion of shoot and root length in wheat seeds compared to controls. The biogas liquid bacterial manure (FA and FB) in the embodiment 1 has a remarkable effect of promoting the roots of the wheat seeds, the root lengths of the wheat seeds after being diluted 1000 times can reach 118.9mm and 117.1mm respectively, and the two are not significantly different and are respectively increased by 47.5% and 45.2% compared with a control. The root length and activity index of the wheat seeds treated by the 1000-time diluted liquid of the biogas liquid bacterial fertilizer (FA and FB) in the embodiment 1 are obviously higher than those treated by the 1000-time diluted liquid of the biogas liquid bacterial fertilizer without bacillus and the bacillus amyloliquefaciens B3 bacterial liquid. Compared with the singly used diluted 1000 times biogas slurry fertilizer, the treatment root length of the 1000 times biogas slurry bacterial fertilizer (FA and FB) of the embodiment 1 is respectively improved by 7.1 percent and 5.4 percent; compared with 1000-time dilution of the bacillus amyloliquefaciens B3 bacterial liquid, the yield is respectively improved by 27.3 percent and 25.3 percent.

Therefore, the biogas liquid bacterial manure in the embodiment 1 has a synergistic effect on wheat seeds. In addition, the number of viable bacteria in the biogas slurry bacterial manure in the example 1 is reduced after the biogas slurry bacterial manure is stored for one month, but no significant difference exists in germination and growth of wheat seeds.

As can be seen from FIG. 4, the germination vigor and germination index of 250-fold and 500-fold diluted biogas slurry bacterial manure (FA and FB) in example 1 are significantly lower than those of other treatments, and the treatment has an inhibiting effect on the germination of white cabbage seeds. But the germination vigor and germination index of the biogas slurry bacterial manure (FA and FB) seeds of example 1 treated by 1000 times of dilution are greatly improved. Wherein, the germination potentials of FA and FB diluted by 1000 times are respectively 62.2% and 61.9%, and the two are not significantly different and are respectively improved by 14.3% and 13.7% compared with a control. Most of the treatments have different degrees of promotion effects on the bud length and the root length of the Chinese cabbage seeds compared with the control. The biogas slurry bacterial manure (FA and FB) in the embodiment 1 has a remarkable effect of promoting roots of Chinese cabbage seeds, the root lengths of the Chinese cabbage seeds diluted 1000 times can reach 78.3mm and 78.9mm respectively, and the two are not significantly different and are improved by 50.6% and 51.7% respectively compared with a control. The root length and activity index of the Chinese cabbage seeds treated by the 1000-time diluted liquid of the biogas slurry bacterial fertilizer (FA and FB) in the embodiment 1 are obviously higher than those treated by the 1000-time diluted liquid of the 1000-time biogas slurry bacterial fertilizer without bacillus and the bacillus amyloliquefaciens B3 bacterial liquid. Compared with the singly used diluted 1000 times biogas slurry fertilizer, the treatment root length of the 1000 times biogas slurry bacterial fertilizer (FA and FB) of the embodiment 1 is respectively improved by 6.2 percent and 7.1 percent; compared with 1000-time dilution of the bacillus amyloliquefaciens B3 bacterial liquid, the yield is respectively improved by 50.6 percent and 51.7 percent.

Therefore, the biogas slurry bacterial manure in the embodiment 1 has a synergistic effect on the growth of the seeds of the Chinese cabbage. In addition, the number of viable bacteria is reduced after the biogas slurry bacterial manure in the embodiment 1 is stored for one month, but no obvious difference exists in germination and growth of the Chinese cabbage seeds.

Experimental example 3 inhibitory effects of biogas slurry bacterial manure on Fusarium oxysporum cucumber specialization type, Fusarium oxysporum tomato specialization type and Fusarium solani

In the experimental example, the inhibition effects of the biogas slurry bacterial manure prepared in the example 1 on fusarium oxysporum cucumber specialization type, fusarium oxysporum tomato specialization type and fusarium solani are measured.

The experimental method is as follows:

pathogenic bacteria to be tested:

fusarium oxysporum f.sp.Cucumerinum, Fusarium oxysporum f.sp.lycopersici, and Fusarium solani (Fusarium solani) are provided by the Chinese academy of agricultural sciences. The activated PDA culture medium is ready for use.

The experiment was set up with 16 treatments:

the original Biogas Slurry (BS) described in example 1 was diluted 1000 times, 5000 times and 10000 times; the prepared biogas liquid fertilizer (BSF, which is different from the BSF prepared in the example 1 only in that no bacillus amyloliquefaciens B3 is added for dilution by 1000 times, 5000 times and 10000 times; 50g/L of Bacillus amyloliquefaciens B3 is added into 100mL of sterile water (B3) to be diluted by 1000 times, 5000 times and 10000 times; the biogas slurry bacterial manure (FA) prepared in the embodiment 1 is diluted 1000 times, 5000 times and 10000 times; the biogas slurry bacterial manure (FB) prepared in example 1 after being stored for one month is diluted by 1000 times, 5000 times and 10000 times; sterile water (CK) treatment served as control.

The test process comprises the following steps:

sucking 100 mu L of treatment solution diluted by different times, uniformly coating the treatment solution on the surface of a PDA culture medium, repeating each treatment for 9 times, taking a bacterial cake with the diameter of 5mm from a cultured pathogenic bacteria strain by using a puncher, reversely buckling the bacterial cake in the center of a plate, placing 1 bacterial cake in each culture dish, putting the plate inoculated with the pathogenic bacteria into an incubator at 26 ℃ for 3-10 days in the dark (according to the growth condition of the bacterial colony), measuring the diameter of the bacterial colony by using a cross method, and calculating the inhibition rate of the growth of the bacterial colony (AI).

Measurement indexes are as follows:

the measurement results are shown in FIGS. 5 to 7.

The inhibitory effect of different treatments and different dilution factors on the specialized growth of fusarium oxysporum cucumbers is shown in fig. 5. In the concentration range set in the test, the colony diameter of pathogenic bacteria increases along with the increase of dilution times of all treatments, namely the growth inhibition effect on the pathogenic bacteria is weakened. The reason may be that the number of beneficial microorganisms decreases after dilution and the competitive action against pathogenic bacteria is reduced. The bacillus amyloliquefaciens B3 bacterial liquid and the biogas slurry bacterial fertilizer (FA, FB) in the embodiment 1 have strong inhibition effect on pathogenic bacteria because of containing a large amount of bacillus amyloliquefaciens B3, wherein the inhibition rates of 1000-time diluent on the pathogenic bacteria are respectively 87.5%, 87.4% and 83.2%, and the inhibition rates of 10000-time diluent on the pathogenic bacteria are respectively 65.3%, 68.5% and 65.3%. Compared with the biogas liquid fertilizer (without bacillus) used alone, the biogas liquid bacterial fertilizer in the embodiment 1 can obviously improve the inhibition effect on pathogenic bacteria. Therefore, the bacteriostatic effect of the biogas liquid fertilizer can be improved by adding the bacillus amyloliquefaciens B3. In addition, the number of live bacteria in the biogas slurry fertilizer containing the bacillus amyloliquefaciens B3 (the biogas slurry bacterial fertilizer in the embodiment 1) after being stored for one month is reduced, compared with the existing biogas slurry fertilizer containing the bacillus amyloliquefaciens B3, the growth inhibition effect on pathogenic bacteria is reduced, and the bacteriostasis rate is reduced by 4.8%.

The inhibition effect of different treatments and different dilution times on the specialized growth of fusarium oxysporum tomatoes is shown in fig. 6, and in the concentration range set in the test, the colony diameter of pathogenic bacteria is increased along with the increase of the dilution times in all the treatments, so that the inhibition effect on the growth of the pathogenic bacteria is weakened. The bacillus amyloliquefaciens B3 bacterial liquid and the biogas liquid bacterial manure (FA, FB) in the embodiment 1 have strong inhibition effect on fusarium oxysporum tomato specialization because a large amount of bacillus amyloliquefaciens B3 is contained, 10000 times of diluent still has higher inhibition effect on pathogenic bacteria, the inhibition rates are 71.3%, 75.8% and 72.6%, and the inhibition rate of the biogas liquid bacterial manure prepared in the embodiment 1 by using the conventional FA is obviously higher than that of other treatments. Wherein, the biogas liquid fertilizer containing the bacillus amyloliquefaciens B3 (the biogas liquid bacterial fertilizer of the example 1) stored for one month has the bacteriostasis rate reduced by only 3.2 percent compared with the prior use. Compared with the single use of biogas liquid fertilizer (without bacillus), the 10000-fold diluted biogas liquid fertilizer containing the bacillus amyloliquefaciens B3 stored for one month has the advantage that the inhibition effect on pathogenic bacteria is improved by 52.1 percent. Therefore, the bacteriostatic effect of the biogas liquid fertilizer can be improved by adding the bacillus amyloliquefaciens B3.

The inhibition effect of different treatments and different dilution times on the growth of fusarium solani is shown in fig. 7, and in the concentration range set in the test, the colony diameter of fusarium solani is increased along with the increase of the dilution times in all the treatments, so that the inhibition effect on the growth of pathogenic bacteria is weakened. The bacillus amyloliquefaciens B3 bacterial liquid and the biogas slurry bacterial manure (FA, FB) in the embodiment 1 have strong inhibition effect on fusarium solani due to the large amount of bacillus amyloliquefaciens B3, and the inhibition rates of 1000-time diluent on the fusarium solani are respectively 87.5%, 88.8% and 86.5%. 10000 times of diluent still has higher inhibition effect on fusarium solani, and the inhibition rate is respectively reduced by 10.5%, 4.8% and 8.2% compared with 1000 times of diluent. Compared with the biogas liquid fertilizer (without bacillus) singly used, the biogas liquid fertilizer (biogas liquid bacterial fertilizer in example 1) containing the bacillus amyloliquefaciens B3 can obviously improve the inhibition effect on fusarium solani. Wherein, compared with the single use of biogas liquid fertilizer (without bacillus), the 10000 times of the biogas liquid fertilizer diluent containing the bacillus amyloliquefaciens B3 stored for one month has the inhibition effect on fusarium solani improved by 50.7 percent, and the inhibition rate is only reduced by 6.0 percent compared with the prior use.

In conclusion, the number of bacillus in the biogas slurry bacterial fertilizer is reduced along with the increase of the storage time. The number of viable bacteria of Bacillus is 2.8 × 10 by 90 days⁹CFU/mL, meets the regulation of the number of viable bacteria in the composite microbial fertilizer (5 multiplied by 10)⁷CFU/ml)。

The biogas slurry bacterial fertilizer diluted by 250 times has an inhibiting effect on the growth of the seeds of the cucumbers, the wheat and the cabbages, the 1000 times of the diluent is beneficial to promoting the germination and the growth of the seeds of the cucumbers, the wheat and the cabbages, and compared with a control, the 1000 times of the diluent is improved in 6 germination characteristic indexes of the seeds to different degrees.

The biogas slurry bacterial fertilizer can obviously improve the inhibition effect on pathogenic bacteria, has extremely strong inhibition effect on the growth of soil-borne pathogenic fungi, and still has very high inhibition effect on 10000 times of diluent. The number of viable bacteria in the biogas slurry bacterial manure stored for one month is reduced, but the inhibition rates of the biogas slurry bacterial manure.

EXAMPLE 4 investigation of active substances and protective Agents

1. Investigation of active substances

Itaconic acid and citric acid belong to organic acids, and the addition of the organic acids to concentrated biogas slurry can reduce the pH. In addition, the itaconic acid and the citric acid can effectively activate phosphorus in soil and promote nutrient dissolution and crop growth. Sorbitol and polyglutamic acid can reduce EC of concentrated biogas slurry through the action of chelating ions. Sorbitol belongs to sugar alcohol micromolecule organic matter, and can effectively improve the growth of crops, improve the quality, promote nutrient absorption and the like. Polyglutamic acid is a high molecular polymer and has the effects of saving fertilizer, increasing yield, improving quality, improving soil physicochemical property and the like.

However, the study of this example shows that the addition of a specific active substance to the biogas slurry bacterial manure of the present invention is more suitable for the overall effect.

Specifically, in this experimental example, the number of viable bacteria of bacillus amyloliquefaciens B3 was examined to examine the active substance.

The experiment is totally provided with 10 treatments, specifically, each treatment material is respectively added into the concentrated biogas slurry described in example 1 to reach corresponding target concentration, then the bacillus amyloliquefaciens B3 is inoculated into the concentrated biogas slurry in an amount of 50g/L, and then the viable count of the bacillus amyloliquefaciens is measured according to the method of the experiment example 1. In each treatment: CK represents that no material (original biogas slurry) is added into the concentrated biogas slurry; YK denotes addition of itaconic acid; NM indicates addition of citric acid; SL represents the addition of sorbitol; JG represents addition of polyglutamic acid; YS represents addition of itaconic acid + sorbitol; YJ denotes addition of itaconic acid + polyglutamic acid; NS indicates addition of citric acid + sorbitol; NJ represents addition of citric acid + polyglutamic acid; YNSJ indicates addition of itaconic acid + citric acid + sorbitol + polyglutamic acid. The amounts of the substances added are shown in Table 1. In Table 1, pH and EC are the physical and chemical indexes of the biogas slurry after each treatment substance is added.

TABLE 1

The results of the test for the effect of different treatments on B3 are shown in FIG. 8. Itaconic acid and citric acid can obviously improve the degradation of the concentrated biogas slurryViable count of bacillus amyloliquefaciens B3. Wherein, after the itaconic acid is added, the viable count of the bacillus amyloliquefaciens B3 is increased by 35.8 percent compared with the control at the 30 th day, the viable count of the bacillus amyloliquefaciens B3 is increased by 24.3 percent compared with the control at the 30 th day, and the difference of the viable counts is not obvious. Both sorbitol and polyglutamic acid can obviously improve the viable count of the bacillus amyloliquefaciens B3 in the concentrated biogas slurry. Wherein, the viable count of the Bacillus amyloliquefaciens B3 can be increased by 47.2% compared with the control at 30d after the polyglutamic acid is added, and the viable count of the Bacillus amyloliquefaciens B3 is only increased by 10.5% compared with the control at 30d after the sorbitol is added. The number of viable bacteria of bacillus amyloliquefaciens B3 in the concentrated biogas slurry after the polyglutamic acid is added is obviously higher than that of sorbitol. After the itaconic acid and the polyglutamic acid are added, the number of viable bacteria of the bacillus amyloliquefaciens B3 in the concentrated biogas slurry at the 30 th day is the maximum and is 2.8 multiplied by 10⁹CFU/mL, an increase in survival rate of 52.1% compared to control. Therefore, the biogas slurry bacterial manure system finally selects itaconic acid and polyglutamic acid as active substance components.

2. Investigation of protective Agents

In this example, the protective agent was investigated by examining the number of viable bacteria of Bacillus amyloliquefaciens B3.

The experiment is totally provided with 5 treatments, specifically, each treatment material is respectively added into the concentrated biogas slurry described in example 1 to reach corresponding target concentration, then, the bacillus amyloliquefaciens B3 is inoculated into the concentrated biogas slurry in an amount of 50g/L, and then, the viable count of the bacillus amyloliquefaciens is measured according to the method of the experiment example 1. In each treatment: CK represents that no material (original biogas slurry) is added into the concentrated biogas slurry; HZ represents the addition of trehalose; BJ represents the addition of p-hydroxybenzoic acid; TW represents addition of tween 20; GY stands for addition of glycerol. The amounts of the substances added are shown in Table 2. In Table 2, pH and EC are the physical and chemical indexes of the biogas slurry after each treatment substance is added.

TABLE 2

The test results are shown in FIG. 9, and it can be seen from FIG. 9 that after the protective agent is added, all the protective agent is decomposed in the concentrated biogas slurryThe viable count of the bacillus amyloliquefaciens B3 has certain improvement effect. Wherein, the viable count of the bacillus amyloliquefaciens B3 in the concentrated biogas slurry can be obviously improved after 50g/L of trehalose is added, and the viable count of the 30 th viable count can reach 3.2 multiplied by 10⁹CFU/mL, compared with the control viable count increased by 1.04 times. Therefore, trehalose is selected as a protective agent in the biogas slurry bacterial manure system.

3. Investigation of addition of active substances and protective Agents

Through the tests, the number of the viable bacteria of the bacillus amyloliquefaciens B3 is the largest after a proper amount of itaconic acid and polyglutamic acid are added into the biogas slurry bacterial fertilizer system, and the number of the viable bacteria of the bacillus amyloliquefaciens B3 is the largest after trehalose is added into the protective agent, so that the bacillus amyloliquefaciens B3 and the protective agent are simultaneously added into concentrated biogas slurry to measure the change condition of the viable bacteria. Meanwhile, nutrients are added on the basis, and the change of the viable count is measured.

Experiments are carried out for 5 treatments in total, specifically, each treatment material is respectively added into the concentrated biogas slurry recorded in the example 1 to reach corresponding target concentration, then the bacillus amyloliquefaciens B3 is inoculated into the concentrated biogas slurry in the amount of 50g/L, and the viable count of the bacillus amyloliquefaciens is measured according to the method of the experiment example 1. In each treatment: CK represents that no material (original biogas slurry) is added into the concentrated biogas slurry; YJ denotes addition of itaconic acid + polyglutamic acid; HZ represents the addition of trehalose; YJH denotes addition of itaconic acid + polyglutamic acid + trehalose; FB means addition of itaconic acid + polyglutamic acid + trehalose + potassium nitrate. The amounts of the substances added are shown in Table 3. In Table 3, pH and EC are the physical and chemical indexes of the biogas slurry after each treatment substance is added.

TABLE 3

The test results are shown in FIG. 10, and it can be seen from FIG. 10 that the viable count of Bacillus amyloliquefaciens B3 added with only the active substance at 30d is 2.7X 10⁹CFU/mL, the viable count of trehalose added only 50g/L as a protective agent was 3.2X 10⁹CFU/mL. Active substances and trehalose as protective agent are added simultaneously, and the viable count can reach 3.5 multiplied by 10⁹CFU/mL, active and protective addition compared to controlThe number of treated viable bacteria is increased by 1.2 times. Therefore, the active substances and the protective agent trehalose which are researched and determined by the invention are added simultaneously, so that the viable count of the bacillus amyloliquefaciens B3 can be effectively increased.

On the basis of adding active substances and trehalose which are determined by the research of the invention, necessary nutrients are added, and the nutrient content of the liquid fertilizer is improved. As can be seen from FIG. 10, the addition of nutrients made by Bacillus amyloliquefaciens B3 was very beneficial, but not optimal. The number of viable bacteria at 30d is 3.0 × 10⁹CFU/mL, the survival rate is 48.4%.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The biogas slurry bacterial fertilizer is characterized by comprising: bacillus amyloliquefaciens, concentrated biogas slurry, active substances and a protective agent; the active substances are itaconic acid and polyglutamic acid, and the protective agent is trehalose.

2. The biogas slurry bacterial fertilizer as claimed in claim 1, wherein the bacillus amyloliquefaciens has an siderophore production capacity, an IAA production capacity of 7-40mg/kg, a phosphorus solubility of 40-90mg/kg, an inhibition effect on fusarium oxysporum of 40-50%, and a viable count of 1 x 10 at a NaCl concentration of 60g/L⁹-1.5×10⁹CFU/mL。

3. The biogas slurry bacterial fertilizer as claimed in claim 2, wherein the bacillus amyloliquefaciens has an siderophore production capacity, an IAA production capacity of 7.5mg/kg, a phosphorus solubility of 68mg/kg, an inhibiting effect on fusarium oxysporum of 47.8%, and a viable count of 1.3 x 10 at a NaCl concentration of 60g/L⁹CFU/mL。

4. The biogas slurry bacterial fertilizer according to any one of claims 1 to 3, comprising 10 to 30g/L of itaconic acid and 60 to 80g/L of polyglutamic acid.

5. The biogas slurry bacterial fertilizer according to any one of claims 1 to 4, comprising 30 to 50mg/L trehalose.

6. The biogas slurry bacterial fertilizer according to claim 5, which comprises 15g/L of itaconic acid, 80g/L of polyglutamic acid and 50mg/L of trehalose;

and/or the mass-volume ratio of the bacillus amyloliquefaciens to the concentrated biogas slurry is (30-70): 1g/L, preferably 50: 1 g/L.

7. The biogas slurry bacterial fertilizer as claimed in any one of claims 1 to 6, wherein the concentrated biogas slurry is livestock and poultry manure biogas slurry after filtration and impurity removal, and the physicochemical properties of the livestock and poultry manure biogas slurry are as follows: pH, 9.2; EC, 32.00 mS/cm; TN, 5.00 g/L; TP: 2.40 g/L; TK: 5.60 g/L; organic matter, 1.90 g/L; 4.50g/L of humic acid; ca, 864 mg/L; mg, 25 Mg/L; s, 352 mg/L; fe, 104 mg/L; mn, 16 mg/L; zn, 84 mg/L; cu, 11 mg/L.

8. A method for preparing the biogas slurry fertilizer according to any one of claims 1 to 7, which comprises the step of mixing the bacillus amyloliquefaciens, the concentrated biogas slurry, the active substance and the protective agent.

9. Use of a biogas slurry fertiliser as claimed in any one of claims 1 to 7 or a method as claimed in claim 8 in the manufacture of a product for promoting plant growth and/or inhibiting pathogenic bacteria.

10. Use according to claim 9, wherein the pathogenic bacteria are Fusarium oxysporum f.sp.cumerinum f.sp.cucumerinum, Fusarium oxysporum f.sp.lycopersici and Fusarium solani (Fusarium solani);

and/or the plant is cucumber, wheat or Chinese cabbage.