CN113322198B

CN113322198B - Microbial agent based on xanthobacter oxamine and xanthobacter oryzae as well as preparation method and application of microbial agent

Info

Publication number: CN113322198B
Application number: CN202110493192.4A
Authority: CN
Inventors: 沈家葆; 李华一
Original assignee: Guangdong Lihao Biological Agriculture Co ltd
Current assignee: Guangdong Lihao Biological Agriculture Co ltd
Priority date: 2021-05-06
Filing date: 2021-05-06
Publication date: 2023-04-07
Anticipated expiration: 2041-05-06
Also published as: CN113322198A

Abstract

The invention belongs to the field of microorganisms, and particularly relates to a microbial agent based on xanthobacter oxamine and flavobacterium oryzae as well as a preparation method and application thereof. The microbial agent comprises mixed fermentation liquor of xanthobacter oxamine, xanthobacter oryzae and azospirillum brasilense, and the effective viable count of the three strains in the mixed fermentation liquor is not less than 7 multiplied by 10 ⁸ CFU/mL, the rate of mixed bacteria is not higher than 3%. The microbial agent provided by the invention can colonize in soil for a long time, has the effects of improving soil fertility and water retention, can secrete plant hormones, and can regulate and control plant growth by promoting the growth of plant roots.

Description

Microbial agent based on xanthobacter oxamine and xanthobacter oryzae as well as preparation method and application of microbial agent

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a microbial agent based on xanthobacter oxamine and flavobacterium oryzae as well as a preparation method and application thereof.

Background

With the increasing application of chemical fertilizers year by year, the adverse effect on the soil is gradually highlighted, the excessive application of the chemical fertilizers causes soil degradation, soil hardening, soil overnutrition, water and soil loss and soil salinization, so that the soil moisture retention and water retention capacity is reduced, the soil granular structure is damaged, the soil pH is out of control, the soil nitrogen fixation capacity is low and the like, the protection capacity of the soil on crops is reduced, the stress resistance of the crops is reduced, and finally the crop is pathogenic and yield reduction is caused, for example, the winter wheat planted in the southeast region of Henan, and the northern plain of Anhui is influenced by freezing damage in China in 2020 winter. Local agriculturists find that wheat seedlings are difficult to root into deep soil due to the hardened soil plough base layer, root systems are gathered in a ploughing stratum and are seriously influenced by cold air, and the overwintering seedlings of the wheat are frozen to death in a large area.

The root cause of the various soil problems is that the diversity of microorganisms in the soil is greatly reduced, and the problems are specifically represented by the loss of the diversity of beneficial bacteria in the soil and the reduction of the abundance of the beneficial bacteria in the soil. The agricultural microbial agent is a product which takes soil beneficial bacteria as a main active component. Can generally play a role in improving soil and increasing crop yield and solve various agricultural problems.

However, the existing agricultural microbial technology has a plurality of problems, and the important points are represented by three aspects of poor colonization ability of active bacteria, low efficacy and single strain formula.

In the prior art, for example, CN105820986B, CN104017750B, CN101709279B, CN102703363B and the like all adopt bacillus as main active bacteria, and the bacillus belongs to gram-positive bacteria. The prior art shows that most of strains capable of being effectively colonized in a root zone environment are gram-negative bacteria, so whether active ingredients can be effectively colonized or not needs to be considered in the prior art, and the prior art is difficult to colonize a plant root zone for a long time, so that the fertilizer efficiency is low and the duration is short. In addition, in the prior art such as CN103952349B, the effective active bacteria is only one strain of bacillus aryabhattai, the strain is a gram-positive bacterium and is difficult to work, and the single strain has only one aspect of its function, so that the problem of complex soil is difficult to solve.

The existing agricultural microbial agent products are mainly made of gram-positive bacteria in strain selection, and because the gram-positive bacteria are difficult to colonize and play roles in a rhizosphere environment, and a small amount of microbial agent products mainly made of gram-negative bacteria are difficult to colonize and play roles in a field due to single strain, low self-vitality and insufficient adaptability to field diversity, are difficult to produce and stably store.

In addition, the traditional planting technology usually depends on a large amount of chemical fertilizers, but the application of the large amount of chemical fertilizers into the field inevitably causes the reduction of the utilization rate of the fertilizers, the mineralization of inorganic fertilizers is chelated, the degradation of organic fertilizers is slow, and the existing microbial agents lack effective utilization measures for the potential fertility of the field and promote the rhizosphere absorption capacity of crops.

Disclosure of Invention

The invention aims to provide a microbial agent based on xanthobacter oxamium and xanthobacter oryzae, and a preparation method and application thereof.

Based on the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a microbial agent based on Xanthobacter oxamidum and Xanthobacter oryzae, which comprises mixed fermentation liquor of Xanthobacter oxamum (Xanthobacter aminoxidans), xanthobacter oryzae (Flavobacterium oryzae) and Azospirillum brasilens, wherein the effective viable count of the three strains in the mixed fermentation liquor is not less than 7 x 10 ⁸ CFU/mL, the rate of mixed bacteria is not higher than 3%.

According to the method, the flavobacterium amineoxide, the flavobacterium oryzae and the azospirillum brazilian are taken as composite microbial agents, the three microorganisms are mutually beneficial and symbiotic, and are promoted to mutually propagate, the azospirillum brazilian can increase the nitrogen element content in soil through the nitrogen fixation function, and the nitrogen element required by the growth of the flavobacterium amineoxide and the flavobacterium oryzae is supplied; the xanthobacter xylinum can consume metabolic waste generated by nitrogen fixation of the azospirillum brazilianum, so that the living efficacy of the azospirillum brazilianum is ensured; the flavobacterium oryzae produces extracellular polysaccharide to provide a micromolecular carbon source and protection for the flavobacterium oxamide and the azospirillum brasilense, and the three-bacterium compound solves the problems that the existing single strain is single in effect and difficult to colonize in soil for a long time to play the drug effect when being applied. In addition, the xanthobacter oxamine, the flavobacterium oryzae and the azospirillum brasilense are gram-negative bacteria, have strong colonization ability in a rhizosphere environment, can colonize in soil for a long time, and enrich the types of soil microorganisms.

In addition, the compound fertilizer has the capability of improving inorganic fertilizers of soil by phosphate solubilizing, sulfur solubilizing, amine oxide and the like and the capability of improving organic fertilizers by degrading long-chain aromatic hydrocarbons, benzene rings, carbon-containing heterocycles and the like by compounding the three bacteria; the water solubility of the inorganic fertilizer and the organic fertilizer after degradation is enhanced, the inorganic fertilizer and the organic fertilizer are convenient to absorb and utilize by plants, the potential fertility in the field is effectively utilized, and the growth of crops is promoted. In addition, the microbial agent provided by the application can also secrete phytohormones to regulate the growth of plants, enhance the absorption capacity of a mature root tip area by promoting the formation of plant hairy roots, and promote the growth of crop roots so as to promote the growth of plants.

In addition, the microbial agent can also be used for maintaining water and soil, reducing capillary effect and improving water retention capacity of soil by secreting a high molecular compound with a large number of sticky ends.

In a second aspect, the present invention provides a method for preparing the microbial agent, comprising the following steps:

(1) Inoculating the xanthobacter oxamine strain, the xanthobacter oryzae strain and the azospirillum brasilense strain into a co-culture solution, and performing mixed culture to obtain a co-culture solution;

(2) Carrying out amplification culture on the co-culture bacterial liquid in an improved fish peptone culture medium to prepare a mixed fermentation liquid; the effective viable count of the three strains in the mixed fermentation liquid is not less than 7 multiplied by 10 ⁸ CFU/mL, the rate of mixed bacteria is not higher than 3%.

Furthermore, the inoculation amount of the co-cultured bacterial liquid in the step (2) on the improved fish protein culture medium is 1.5-15 wt%.

Furthermore, the improved fish protein culture medium takes water as a medium and comprises 22.5 to 37.5g/L of fish meal, 7.5 to 12.5g/L of glucose and 0.75 to 1.25g/L of citric acid.

In a third aspect, the invention provides the use of the microbial agent in soil improvement.

The microbial agent provided by the invention can colonize in soil for a long time, enriches soil microbial species, and can enhance soil fertility by degrading organic matters and inorganic matters in soil into water-soluble organic fertilizers or inorganic fertilizers, so that plants can absorb and utilize the water-soluble organic fertilizers or inorganic fertilizers conveniently, and the growth of crops is promoted; in addition, the microorganism of the invention generates a macromolecular compound with a viscous end in the metabolic process, and has the functions of maintaining water and soil, reducing capillary effect and improving the soil moisture conservation and water retention capacity of soil.

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts the microbial agent formed by compounding the xanthobacter oxamine, the flavobacterium oryzae and the azospirillum brasilense, can colonize in soil for a long time, enrich the types of soil microorganisms, improve the effects of soil fertility and water retention and promote the growth of crops.

Drawings

FIG. 1 is a streaked plate of a colony of Xanthomonas amine oxide;

FIG. 2 is a microscopic image of a colony of Xanthomonas amino acid;

FIG. 3 is a flat-line drawing of the colony of Azospirillum brasilense;

FIG. 4 is a microscopic examination of Azospirillum brasilense colonies;

FIG. 5 is a streaked plate of a colony of Flavobacterium oryzae;

FIG. 6 is a photograph showing a colony of Flavobacterium oryzae by microscopic examination.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

Example 1

The embodiment provides a preparation method of a compound microbial agent, which comprises the following specific preparation processes:

(1) Culturing of a single strain

Three single strain freeze-dried powders of Xanthobacter oxamium (Xanthobacter aminoxidans) CGMCC1.3798 and Azospirillum brasilense strain CGMCC1.10379 and Flavobacterium oryzae (Flavobacterium oryzae) CGMCC 1.1584 which are obtained from commercial approaches are respectively put in corresponding slant culture media and cultured for 3 days at 28 ℃ to obtain Xanthobacter oxamium oxamatum colonies, azospirillum brasilense colonies and Flavobacterium oryzae colonies.

Wherein the formula of the slant culture medium for culturing the flavobacterium aminoxide is as follows: 10g of peptone, 3g of beef extract, 5g of sodium chloride, 10g of agar and 1L of distilled water.

The formula of the slant culture medium for culturing azospirillum brasilense is as follows: KH (Perkin Elmer) ₂ PO ₄ 0.2g、K ₂ HPO ₄ 0.8g、MgSO ₄ ·7H ₂ O 0.2g、CaSO ₄ ·2H ₂ O 0.1g、FeCl ₃ 0.0005g、Na ₂ MoO ₄ ·2H ₂ 0.0005g of O, 0.5g of yeast extract, 20g of mannitol, 15g of agar and 1L of distilled water.

The formula of the slant culture medium for culturing the flavobacterium oryzae comprises the following components: sucrose 15g, KH ₂ PO ₄ 0.2g、K ₂ HPO ₄ 0.8g、MgSO ₄ ·7H ₂ 0.2g of O, 0.2g of sodium chloride, 0.05g of calcium chloride, 0.025g of ferric sulfate, 0.0005g of sodium tungstate, 0.0005g of sodium molybdate, 0.0005g of manganese sulfate, 15g of agar and 1L of distilled water.

Respectively picking out an amine oxide flavobacterium colony, an azospirillum brasilense colony and a flavobacterium oryzae colony on the three slant culture media, uniformly mixing the colonies with a culture medium without agar, and performing streak plate culture and microscopic examination, wherein streaked plates and microscopic examination images of the amine oxide flavobacterium colony are respectively shown in a figure 1 and a figure 2; the streaking plate and the microscopic image of the Azospirillum brasilense colony are shown in FIG. 3 and FIG. 4, respectively; FIG. 5 and FIG. 6 show the streaked plates and the microscopic images of Flavobacterium oryzae colonies, respectively.

(2) Co-culture of three strains

And respectively selecting an amine oxide flavobacterium colony, an azospirillum brasilense colony and a flavobacterium oryzae colony on the three slant culture media to inoculate in a co-culture medium, wherein the inoculation proportion is that the strain weight/the volume of the co-culture medium is =0.3g/100mL. And (3) placing the inoculated co-culture solution into a constant-temperature oscillation box, setting the parameters of the constant-temperature oscillation box at 150r/min and 28 ℃, keeping out of the sun, and culturing for 3 days to obtain the co-culture solution.

Wherein, the formula of the co-culture solution is as follows: 5g of peptone, 5g of sucrose, 5g of mannitol, 3g of sodium chloride and KH ₂ PO ₄ 0.2g、K ₂ HPO ₄ 0.8g、MgSO ₄ ·7H ₂ 0.2g of O, 0.0005g of sodium tungstate, 0.0005g of sodium molybdate, 0.0005g of manganese sulfate, 0.0005g of ferric sulfate, 0.0005g of calcium chloride and 1L of distilled water.

(3) Amplification of Co-cultured strains

Inoculating the co-culture bacterial solution to modified fish peptone for culture in an inoculation amount of 1.5wt%In a fermentation tank of the substrate, performing spontaneous aerobic fermentation for 2 days in the fermentation tank to prepare mixed fermentation liquor, namely the composite microbial agent; the effective viable count of the three strains in the mixed fermentation liquor is not less than 7 multiplied by 10 ⁸ CFU/mL, the rate of mixed bacteria is not higher than 3%.

Wherein, the specific composition of the improved fish peptone culture medium is as follows: 30g of fish meal, 10g of glucose, 1g of citric acid and 1L of sterile water.

Example 2

This example will test the performance of the complex microbial inoculum prepared in the example by the following five aspects.

1. Hemolytic reaction of microbiome

The mixed fermentation liquid prepared in the example 1 is inoculated on a blood agar plate and cultured for 24 hours at 37 ℃, and no hemolytic ring is observed to be generated, which shows that the compound microbial agent prepared by the invention has good safety and can be used for preparing agricultural microbial agents.

The formula of the blood agar plate used in the test is as follows: 10g of peptone, 10g of beef extract powder, 5g of sodium chloride, 15g of agar and 1L of distilled water. The pH of the blood agar plate was 7.5.

The blood agar plate was prepared as follows: mixing the raw materials of the formula, sterilizing at high pressure, cooling to 60 ℃, adding 60mL of sterile defibered sheep blood, rotating and fully pouring the mixture into a flat plate to obtain a blood agar plate, wherein the thickness of the blood agar plate is 5mm.

2. Nitrogen fixation capacity determination of microbiome and components thereof

The method for respectively measuring the nitrogen fixation capacity of the microbiome by using an acetylene reduction method comprises the following specific implementation steps: adding 5mL LB agar into a test tube with the specification of 15mm x 150mm to prepare a slant culture medium, sealing the slant culture medium with a soft rubber plug for culture, standing for 24h until bacterial lawn grows out, extracting 5mL of air, injecting 5mL of acetylene, culturing for 48h, sucking 100 mu L of gas in the test tube, and measuring the ethylene production by using a gas chromatograph.

TABLE 1 mean nitrogen fixation capacity of microbial Agents

3. Effect of microbial Agents on soil Nitrogen content

Dividing soil with good soil conditions into two groups, namely an experimental group and a control group, wherein the experimental group adds the compound microbial agent into the soil according to the proportion of the compound microbial agent to the soil of 0.01g; and placing the treated soil of the test group and the control group in a constant temperature incubator with good ventilation environment for culture. And (3) culturing the two groups of soils in a 28 ℃ thermostat for 7 days, 14 days and 28 days, and detecting the percentage of total nitrogen content of the soils in the test groups and the reference map by using an automatic nitrogen determinator KA-1A according to national standard GB/T6423-94.

The detection results are shown in table 2, and on the 7 th day, the 14 th day and the 28 th day, the nitrogen content in the soil of the test group is improved, and the nitrogen content of the soil of the control group is reduced; the nitrogen content of the soil in the test group is gradually increased along with the extension of the culture time, and the nitrogen content of the soil in the control group is gradually decreased; the microbial agent has good fertility-increasing effect on soil.

TABLE 2 percent increase in nitrogen content in the soil of the test and control groups

4. Effect of microbial Agents on crop growth

A test field is set in a fresh district of Qingyuan city in Guangdong province and is divided into an experimental group and a control group, crops to be planted are wheat, peanut and rape, wheat seeds, peanut seeds and rape seeds in the experimental group are soaked in the compound microbial agent prepared in the embodiment diluted by 20 times for 1h before being planted, and the control group is soaked in clear water for 1h to serve as a control.

The overground parts of wheat, peanut and rape are harvested after 28 days of growth, the wheat, peanut and rape are placed in a constant-temperature drying box and dried to a constant temperature, and the growth promoting effect of the microbial agent on the overground parts of crops is analyzed by the dry weight increment of the overground parts of the crops of a test group relative to a control group.

The determination results are shown in table 3, and the dry weight average of the overground part of the wheat, the peanut and the rape treated by the compound microbial inoculum is increased compared with that of a control group, which shows that the compound microbial inoculum has a certain growth promotion effect on the wheat, the peanut and the rape.

TABLE 3 mean nitrogen fixation capacities of different microorganisms

Species of crop	Dry weight increment of aboveground parts of crops
		Wheat (Triticum aestivum L.) as one of raw materials	22％
Peanut	17％
		Rape seed	9％

5. Test of substitution effect of microbial agent on fertilizer

A test field is set in a fresh district of Qingyuan city in Guangdong province, the test field is respectively an experimental group and a control group, the planted crop is wheat, the fertilizing amount of the experimental group is 50% of that of the control group, but wheat seeds of the experimental group are treated by soaking the seeds in the compound microbial agent in the embodiment 1, the microbial agent diluted by 50 times is poured at intervals of 14 days, and the control group is poured with equal amount of water. The percentage of yield reduction of the experimental group relative to the control group was compared by harvesting the aerial parts on days 14, 28 and 42 of the experiment, respectively, and drying them in a constant temperature drying oven to a constant temperature.

As shown in Table 4, it can be seen that the reduction of the dry weight of the above-ground part tends to decrease with the increase of the treatment time of the microbial preparation of the present invention, and it is also shown that when the microbial preparation of the present invention is applied to crops instead of 50% of the fertilizer, the difference of the promotion effect of the sufficient amount of the fertilizer to the plants can be reduced by long-term application, indicating that the microbial preparation of the present invention can be applied instead of a part of the fertilizer.

Table 4 percent dry weight reduction of aerial parts of test groups

Example 3

In this example, different combinations of xanthobacter oxamine, azospirillum brasilense and flavobacterium oryzae were performed, and soil proliferation and colonization tests were performed on test strains of different combinations to analyze the interaction relationship among the three strains.

The test bacterial groups in the test are seven groups as follows: A. xanthobacter aminoxide; B. azospirillum brasilense; C. flavobacterium oryzae; D. xanthobacter oxamide + azospirillum brasilense; E. xanthobacter oxamide + flavobacterium oryzae; F. azospirillum brasilense + flavobacterium oryzae; G. xanthobacter oxamine + azospirillum brasilense + flavobacterium oryzae. The viable bacteria concentration of seven groups of tested bacteria is 1 × 10 ⁸ CFU/mL。

The specific test method is as follows: the seven groups of test bacterium groups are respectively added into soil, 10mL of test bacterium groups are added into each 1L of soil sample, the mixture is uniformly mixed and then placed in a constant temperature incubator, the temperature in the incubator is 25 ℃, and the humidity is 90%. Wherein, the soil sample used in the test is derived from humus collected from the mountain foot of Changbai mountain. And then taking the seven groups of test soil samples on the 7 th day, the 14 th day and the 21 st day respectively, and plating the test soil samples to detect the abundance of the strains.

As shown in Table 5, when Xanthomonas aminoxide or Azospirillum brasilense was applied alone to the soil, it was squeezed by indigenous flora and its abundance gradually decreased. When xanthobacter aminoxide was co-administered with azospirillum brasilense, an increase in its abundance was seen due to its synergistic effect. When the flavobacterium amineoxide, the azospirillum brasilense and the flavobacterium oryzae are applied together, the abundance rise speed is obviously increased; compared with the combination of any two strains, the microbial agent formed by combining the three strains of the xanthobacter xylinum, the azospirillum brasilense and the flavobacterium oryzae is more favorable for colonization and proliferation in soil, and the three strains of the xanthobacter xylinum, the azospirillum brasilense and the flavobacterium oryzae have good mutual growth promotion relationship.

TABLE 5 abundance of different test groups on days 7, 14 and 21

/>

Example 4

In this example, xanthobacter oxamine, azospirillum brasilense and flavobacterium oryzae were combined in different ways, and a soil moisture content retention test was performed using test bacterial groups of different combinations to analyze the water retention effect of the three strains on soil.

The test bacterial groups in the test are seven groups as follows: A. xanthobacter xylinum oxide; B. azospirillum brasilense; C. flavobacterium oryzae; D. xanthobacter oxamide + azospirillum brasilense; E. xanthobacter oxamide + flavobacterium oryzae; F. azospirillum brasilense + flavobacterium oryzae; G. xanthobacter oxamine + azospirillum brasilense + flavobacterium oryzae. The viable bacteria concentration of seven groups of tested bacteria is 1 × 10 ⁸ CFU/mL。

The specific test method is as follows: adding the seven groups of test bacterium groups into a soil sample, adding 10mL of test bacterium groups into each 1L of soil sample, uniformly mixing, placing into a constant-temperature drying oven, keeping the temperature in the constant-temperature drying oven at 30 ℃, keeping ventilation for a little, sampling and detecting on 7 th day, 14 th day and 21 st day, and detecting the moisture content in the soil through a moisture content detector. The soil sample used in the test is sourced from humus collected from the mountain foot of Changbai mountain, and the initial water content of the soil sample in the test is 70%.

The test results are shown in table 6, and on the 7 th day, the 14 th day and the 21 st day, the water content of the soil treated by the microbial agent compounded by the xanthobacter oxamine, the azospirillum brasilense and the xanthobacter oryzicola is higher than that of the soil treated by a single strain or the combination of any two strains, which indicates that the microbial agent compounded by the xanthobacter oxamine and the azospirillum oryzae has better soil moisture preservation capability. According to the table 5, the abundance of different tested bacteria in soil is combined, the water retention capacity of the microbial agent on the soil is presumed to have a positive correlation with the concentration of the bacteria contained in the soil, and relatively high-concentration microorganisms metabolize to generate relatively more polymers such as polyhydroxybutyrate and other macromolecular substances and have strong adsorption capacity on water, so that the soil moisture retention capacity of the soil is provided.

TABLE 6 Water content of soil samples treated with different test groups

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of the microbial agent based on the Xanthobacter oxaminum and the Flavobacterium oryzae in soil moisture conservation is characterized in that the microbial agent is mixed fermentation liquor of Xanthobacter oxaminum (Xanthobacter aminoxidans), flavobacterium oryzae (Flavobacterium oryzae) and Azospirillum brasilens, and the effective viable count of the three strains in the mixed fermentation liquor is not less than 7 x 10 ⁸ CFU/mL, the rate of mixed bacteria is not higher than 3%.

2. The use according to claim 1, wherein the microbial agent is prepared by a method comprising the steps of:

(2) Will co-cultureCarrying out enlarged culture on the bacterial liquid in an improved fish peptone culture medium to prepare a mixed fermentation liquid; the effective viable count of the three strains in the mixed fermentation liquid is not less than 7 multiplied by 10 ⁸ CFU/mL, the rate of mixed bacteria is not higher than 3%.

3. The use of claim 2, wherein the amount of the inoculum of the co-cultured broth in step (2) is 1.5-15 wt% on the modified fish protein medium.

4. The use of claim 2, wherein the modified fish protein culture medium is water, and comprises 22.5-37.5 g/L fish meal, 7.5-12.5 g/L glucose, and 0.75-1.25 g/L citric acid.