CN113980877B

CN113980877B - Compound microbial agent and preparation method thereof

Info

Publication number: CN113980877B
Application number: CN202111615832.0A
Authority: CN
Inventors: 曹鸿一; 梁锏文; 孙玉婷; 康品; 刘仁燕; 王科晶; 王琳
Original assignee: Moon Guangzhou Biotech Co ltd
Current assignee: Moon Guangzhou Biotech Co ltd
Priority date: 2021-12-28
Filing date: 2021-12-28
Publication date: 2022-07-01
Anticipated expiration: 2041-12-28
Also published as: CN113980877A

Abstract

The invention provides a compound microbial agent which is characterized by comprising Burkholderia (A), (B) and (C)Burkholderia sp.M928), Bacillus pumilus (B.pumilus: (B.pumilus) ((B.pumilus))Bacillus pumilus M101) and Bacillus subtilis, wherein the Burkholderia is isolated from rhizosphere soil of rubber tree in Yunnan province. The compound microbial agent has the main functions of effectively improving the soil environment for crop growth, being widely applied to preventing and treating common plant diseases and insect pests such as nematode, rhizoctonia and the like, improving the capability of crop to absorb nutrition, enhancing the disease resistance of crops, promoting growth and improving yield and quality.

Description

Compound microbial agent and preparation method thereof

Technical Field

The invention relates to the field of microorganisms, and in particular relates to a compound microbial agent and a preparation method thereof.

Background

The growth process of crops is accompanied by plant diseases and insect pests, and the problems of yield and quality are also encountered.

In the 20 th century, chemical agents are used for solving the problems, but the defects of the chemical agents are slowly discovered, and although the problems can be solved for a while, the problems of drug resistance, pesticide residues and soil caused by long-term use are often difficult to reverse and even threaten the physical health of people.

In recent years, people have focused on the biological field, and natural products such as plants, animals and bacteria are used to solve the problems, so that some results are obtained, and the eosin is seen, and microorganisms including bacteria become important sources of medicines and pesticides.

A large number of researches and practices show that the microbial agent can effectively improve soil, promote ecological restoration of the soil and improve the disease and insect resistance of crops. Among these microorganisms, those of interest include Bacillus subtilis, Paenibacillus mucilaginosus, Bacillus licheniformis, Bacillus megaterium, Bacillus amyloliquefaciens, Saccharomyces cerevisiae, Brevibacillus laterosporus, Streptomyces microflavus, and Lactobacillus plantarum, and recently, the research on a new bacterium, i.e., Burkholderia, has been increasing.

The order Burkholderia belongs to the beta-Proteobacteria of the phylum Proteobacteria among bacteria, and is the second major group of bacteria from which natural products are derived following actinomycetes. Burkholderia can exist in various ecoenvironments such as water and soil, and can secrete various secondary metabolites including various extracellular enzymes having proteolytic activity and hemolytic activity. Through research, people find that part of the burkholderia is plant growth-promoting bacteria, can be planted at the roots and rhizomes of plants, and accretes with plant hosts, and is widely concerned and applied in the field of plant microorganism combined repair. However, the effect exhibited by using Burkholderia varies depending on the species of Burkholderia, the test conditions, and the host plant.

In order to pursue efficient and convenient use, people are also exploring and developing a compounded microbial agent. However, the bacteria often have different survival conditions, and the obtained microbial inoculum has a low number of effective bacteria, so the effect is not ideal.

Disclosure of Invention

According to the invention, microorganisms with similar survival conditions are screened out through screening and proportioning and compounded to obtain the microbial agent, and the microbial agent can effectively improve the soil environment for crop growth, improve the nutrient absorption capacity of crops, enhance the disease resistance of crops, promote growth and improve yield and quality.

The inventor screens and separates in the early stage to obtain a burkholderia strain ()Burkholderia sp.M928) which is separated from rhizosphere soil of rubber trees in Yunnan province and has the preservation number of GDMCC No:61156 said Burkholderia has the following features:

(1) better control plant diseases and insect pests, such as plant parasitic nematodes, damping off and the like;

(2) improving the soil environment for crop growth;

(3) effectively promotes the growth of the root system of the plant and improves the yield and the quality of the crop.

Based on the burkholderia, the inventor screens various microorganisms and formulas to finally form a compound microbial agent, the compound microbial agent can better retain the characteristics of various groups of microorganisms, has better effects of killing nematodes and promoting growth compared with a single microbial agent, can simultaneously generate control effects on various germs such as rhizoctonia solani, citrus canker, ralstonia solanacearum, fusarium wilt and the like, and has broad-spectrum bacteriostasis.

The specific technical scheme is as follows:

the microbial agent mainly comprises microbial fermentation liquor, potassium fulvate, water and diammonium phosphate, and is characterized in that the microbial fermentation liquor is prepared by compounding fermentation liquor of more than two strains of Burkholderia, Bacillus pumilus and Bacillus subtilis.

The burkholderia is separated from rhizosphere soil of rubber trees in Yunnan province, samples are collected from the rhizosphere soil of the rubber trees in Yunnan province, and the separation process is as follows:

weighing 4 g of soil sample, adding into sterilized 36 mL of Tween water with the concentration of 0.1% (v/v), and performing vortex oscillation for 10 min to obtain a 10-1 concentration diluent. The soil liquid is diluted to the concentrations of 10-4, 10-5 and 10-6 by Tween water in a gradient manner, then the soil liquid is coated on an LB solid culture medium and cultured in a constant temperature incubator at 30 ℃, a single colony is selected after 1 day, the single colony is continuously inoculated on the LB solid culture medium and is inversely cultured in the constant temperature incubator at 30 ℃ for 1 day, then the colony morphology is observed, and the thallus morphology is observed by an optical microscope (1000X).

LB solid medium: 10g of tryptone, 5g of yeast powder, 10g of NaCl, 15 g of agar powder, 1L of water supplement, and high-pressure steam sterilization at 121 ℃ for 20 minutes.

The strain is gram-negative bacteria, the bacterial colony is opaque milk white, the shape is circular, the bacterial colony is convex upwards, and the edge is wet.

Has been preserved in Guangdong province microorganism culture collection center in 21.8.2020, the preservation address is No. 59 building and five buildings of Michelia Tokyo No. 100, Guangzhou city, and the preservation number is GDMCC No: 61156.

the Bacillus pumilus strain: (A), (B)Bacillus pumilusM101) was isolated from water from Guangzhou city, the strain was gram-positive, the colony was opaque, milky white and round in shape. The strain is rod-shaped, round-ended, single or short-chained and about 2.0 microns long. The spores can move, the diameter of the spores is 1.0-1.2 multiplied by 1.5-2.0 microns, and the spores are oval. Has been preserved in Guangdong province microorganism culture collection center in 22.10.2021, the preservation address is No. 59 building and five buildings of the Mieli Zhou 100 college in Guangzhou city, the preservation number is GDMCC No: 61962.

the Bacillus subtilis can be purchased from conventional commercial sources.

The fermentation can be carried out by a person skilled in the art according to methods customary in the art to obtain the desired microbial fermentation broth.

The microorganism fermentation liquor is prepared from burkholderia, bacillus pumilus and bacillus subtilis fermentation liquor according to the mass ratio of 1: 1: 1 is prepared by compounding.

Preferably, the microorganism fermentation liquid is prepared from Burkholderia and Bacillus pumilus fermentation liquids according to a mass ratio of 1: 1 is prepared by compounding.

The number of effective viable bacteria in the Burkholderia fermentation liquid and the Bacillus pumilus fermentation liquid is more than or equal to 90 hundred million/mL.

Further, the microbial agent comprises the following main components in percentage by weight: 40-80 parts of microbial fermentation liquor, 40-90 parts of potassium fulvate, 10-50 parts of water and 2-5 parts of diammonium phosphate.

Further, the microbial agent comprises the following main components in percentage by weight: 50 parts of microbial fermentation liquor, 80 parts of potassium fulvate, 20 parts of water and 3 parts of diammonium phosphate.

Further, the main preparation method of the microbial agent comprises the following steps:

(1) preparing microbial fermentation liquor: separating microbial strains, activating, fermenting, and mixing in proportion.

(2) Weighing potassium fulvate, microbial fermentation liquor, water and diammonium phosphate, wherein diammonium phosphate and water are heated and dissolved, then the mixture is placed to normal temperature, and the microbial fermentation liquor and the potassium fulvate are added and stirred uniformly.

(3) And (3) spray-drying the mixed solution obtained in the step (2), and subpackaging the obtained powder to obtain the microbial agent.

The microbial agent can be used for preventing and treating plant diseases and insect pests, and has good prevention and treatment effects on diseases including but not limited to root rot, stego rosea, citrus canker, root-knot nematode, yellow dragon disease, cucumber damping off, potato scab, neck rot, gray mold, scab, anthracnose, damping off, bacterial wilt and the like;

the microbial agent also has the function of promoting the growth of crops, especially eggplants, tomatoes and cucumbers.

The invention has the beneficial effects that:

the microorganisms and the auxiliary materials used in the microbial agent disclosed by the invention can meet the growth conditions of all microorganisms and ensure the number of effective bacteria in the microbial agent, thereby ensuring the effect of the microbial agent: effectively improve the soil environment, promote the plant growth, enhance the plant disease and pest resistance, provide technical support for reducing chemical fertilizers and pesticides and producing green agricultural products, and have large-area application and popularization prospects.

Drawings

FIG. 1 shows the control effect of the microbial inoculum of the invention on the root-knot nematode of cucumber in pot experiments, which is a comparison of the effect on the root growth of cucumber.

FIG. 2 shows the control effect of the microbial inoculum of the invention on the root-knot nematode of cucumber in pot experiments, which is a comparison of the effect on the growth of cucumber plants.

Detailed Description

It should be noted that, due to the limitation of the test site, the invention only preferably selects part of the test scheme and representative crop development potting and plot tests to verify the disease prevention and growth promotion effects of the compound microbial agent, but this does not negate the effectiveness of other test schemes covered by the invention except for the preferred test scheme.

Example 1 preparation of microbial Agents

(1) Taking Burkholderia, bacillus pumilus and bacillus subtilis, activating, fermenting and counting, wherein the number of effective viable bacteria in each fermentation liquid is more than or equal to 90 hundred million/mL, and the effective viable bacteria in each fermentation liquid are calculated according to the mass ratio of 1: 1: 1 mixing to obtain the microbial fermentation liquor.

(2) Taking 80 parts of potassium fulvate, 50 parts of microbial fermentation liquor, 20 parts of water and 3 parts of diammonium phosphate, heating and dissolving diammonium phosphate and water, then placing the mixture to normal temperature, adding the microbial fermentation liquor and the potassium fulvate, and uniformly stirring the mixture.

Example 2 preparation of microbial Agents

(1) Taking Burkholderia and bacillus pumilus, activating, fermenting and counting, wherein the number of effective viable bacteria in each fermentation liquid is more than or equal to 90 hundred million/mL, and the effective viable bacteria are obtained according to the mass ratio of 1: 1 mixing to obtain the microbial fermentation liquor.

Example 3 preparation of microbial Agents

(2) Taking 40 parts of potassium fulvate, 40 parts of microbial fermentation liquor, 10 parts of water and 2 parts of diammonium phosphate, heating and dissolving diammonium phosphate and water, then placing the solution to normal temperature, adding the microbial fermentation liquor and the potassium fulvate, and uniformly stirring the solution.

Example 4 preparation of microbial Agents

(2) Taking 90 parts of potassium fulvate, 80 parts of microbial fermentation liquor, 50 parts of water and 5 parts of diammonium phosphate, heating and dissolving diammonium phosphate and water, then placing the mixture to normal temperature, adding the microbial fermentation liquor and the potassium fulvate, and uniformly stirring the mixture.

Example 5 preparation of microbial Agents

(2) Taking 60 parts of potassium fulvate, 60 parts of microbial fermentation liquor, 25 parts of water and 3 parts of diammonium phosphate, heating and dissolving diammonium phosphate and water, then placing the solution to normal temperature, adding the microbial fermentation liquor and the potassium fulvate, and uniformly stirring the solution.

Example 6 lethal Effect of microbial Agents on root-knot nematodes

(1) The microbial agents obtained in examples 1 and 2 were diluted with 100 times the amount of water to give microbial dilutions 1 and 2, respectively. The Burkholderia fermentation broth and Bacillus pumilus fermentation broth used in example 1 or 2 were diluted with 100 times of water to give positive control dilutions 1 and 2, respectively.

(2) And (4) grouping. Six experimental groups are set in the experiment, namely a treatment group 1 group, a treatment group 2 group, a negative control group 1 group, a positive control group 1 group, a 2 group and a 3 group.

Wherein, the treatment liquids added in the treatment groups 1 and 2 are microbial diluents 1 and 2 respectively; the treatment fluid added in the negative control group is sterile water; the treatment solutions added in the positive control group 1 and the positive control group 2 are positive control diluent 1 and positive control diluent 2 respectively, and the treatment solution added in the positive control group 3 is 1.8% avermectin missible oil 1500 times of diluent.

5 wells (samples) were made for each experimental group and each experiment was repeated 3 times.

(3) To a 24-well cell culture plate, 450. mu.L of the treatment solution and 50. mu.L of the nematode suspension (100 nematode second-instar larvae) were added and allowed to stand at room temperature. After the nematodes in each treatment are stimulated by 1 mol/mL NaOH solution at 24h, dead insects are dead and live insects are alive when the nematodes are bent and wriggled. The number of dead insects at 24h was recorded and the corrected mortality was calculated.

TABLE 1 nematicidal effect of each experimental group

The 24h lethal effect of each treatment solution on root-knot nematodes is shown in table 1. The results show that the mortality rate of the Burkholderia fermentation broth and the Bacillus pumilus fermentation broth treatment group for the root-knot nematode for 24h is 94.57 percent and 92.50 percent respectively, and is higher than 87 percent of the mortality rate of the Bacillus brevis in the patent CN102283253B for the root-knot nematode for 24 h. In addition, the microbial agent treatment group compounded by the two fermentation liquids has better nematicidal effect (the death rate after 24h correction is 100%) as that of the abamectin control group. The compounded microbial agent has more stable and efficient nematode killing effect than that before compounding.

Example 7 prevention and treatment effects of microbial Agents on cucumber root-knot nematodes in potting experiments

(1) Based on the findings of example 6, the combination of burkholderia and bacillus pumilus has stable and efficient nematicidal effect, and the microbial preparation obtained in example 2 is directly taken and diluted with 100 times of water to obtain the microbial dilution 1. Purchasing a similar product sold in the market, namely the paecilomyces neologineus, and adding 300 times of water to dilute the product to obtain a microbial diluent 2.

(2) Grouping: four experimental groups are set, namely a positive control group 1 and a positive control group 2, a microbial agent treatment group and a blank control group. The treatment solutions used in the experimental groups were as follows: the positive control group 1 uses abamectin (diluted by 1500 times), the positive control group 2 uses a microorganism diluent 2, the microorganism agent treatment group uses the microorganism diluent 1, and the blank control group uses sterile water for treatment.

10 cucumber seedlings were set per group and repeated 3 times.

(3) Sowing the cucumber seeds (variety: Yuexiu No. 3) in sterilized substrate soil for seedling. When the first true leaf grows out, transplanting the seedlings into a plastic pot (7 cm x 10 cm) filled with sterilized matrix soil, and inoculating 15 mL of corresponding treatment solution into the seedling soil of each experimental group when the cucumber seedlings grow into two true leaves. And (3) inoculating second-instar larvae of the root-knot nematodes after 1 day, inoculating 300 larvae per plant, normally managing at room temperature, detecting the number of root knots after 30 days, and calculating the control effect on the root-knot nematodes. The root conditions and aerial parts of each group were compared.

The control effect of each experimental group on cucumber root-knot nematode is shown in table 2 and figure 1, and the growth promoting effect on cucumber seedlings is shown in figure 2.

TABLE 2 number of root knots treated differently and controlling effect

As can be seen from Table 2 and FIG. 1, the control effect of the microbial agent treatment group is 83.13% in the aspect of controlling root-knot nematodes, which is slightly lower than 89.44% of the control effect of the positive control group 1 treated by chemical agents, but is obviously higher than 66.86% of the control effect of the positive control group 2 treated by similar products on the market.

In addition, as can be seen from fig. 2, the avermectin chemical and paecilomyces lilacinus agent used in the positive control groups 1 and 2 do not have an obvious promoting effect on the growth of cucumber seedlings, and the growth advantage of the cucumber seedlings treated by the microbial agent is extremely obvious (shown in the aspects of plant height, leaf surface opening degree, root system development degree and the like), which may be related to the toxicity of the chemical itself on the plant seedlings, the planting ability of the strain itself, functional characteristics and other factors. Therefore, under the condition of equivalent nematicidal effect, the compounded microbial agent has better growth promoting effect than the traditional chemical agents and/or similar products sold in the market.

Example 8 cell experiment of growth promoting effect of microbial inoculum on tomato and eggplant

Based on the growth promoting effect of the microbial agent discovered in example 7 on potted cucumber seedlings, a cell amplification experiment is further carried out to verify the reliability and stability of the growth promoting effect.

According to the existing plot experimental land, two plants, namely tomato and eggplant, are selected to carry out the plot experiment, wherein the microbial agent obtained in the example 1 is selected for the tomato plot, and the microbial agent obtained in the example 2 is selected for the eggplant plot. The specific experimental protocol and experimental results are as follows:

first, cell experiment of growth promoting effect of microbial agent on tomato

(1) Preparing: the microbial preparation obtained in example 1 was diluted with 200 times the amount of water and used as a microbial preparation diluent.

(2) Grouping: the experiment is divided into three groups, including a microbial agent treatment group, a positive control group and a blank control group. The irrigation liquid for each group of irrigation is respectively as follows: the microorganism bacterial agent treatment group uses microorganism bacterial agent diluent, and the positive control group uses commercially available brand^®Microbial inoculum (Bacillus amyloliquefaciens), blank control group was treated with clear water.

Random block arrangement, 1 action 1 cell, 20 plants from head to tail of each cell were taken for testing, 1 cell was treated each, and the test was repeated 3 times.

(3) Seedling culture: and sowing tomato seeds in a seedling raising plate, and planting after 3 true leaves grow. After the tomato seedlings are transplanted for 2 weeks, carrying out first irrigation, carrying out second irrigation after 14 days, and carrying out third irrigation at the early stage of flowering. In each irrigation, 250 mL of microbial agent diluent is taken for the microbial agent treatment group for irrigation, the same amount of clear water is taken for the blank control group, and the positive control group is applied according to the recommended dose.

(4) And (3) observation and recording: and (3) observing the flowering condition in the flowering period, investigating the plant height of each group 20 d after the application of the pesticide, randomly picking 100 fruits per cell during harvesting, weighing the weight of each fruit and the transverse diameter of each fruit, measuring the quality, and recording the total yield of the cells. The results are shown in tables 3 and 4.

TABLE 3 Effect of different treatments on tomato plant height and fruit set percentage

As can be seen from Table 3, compared with the blank control group and the positive control group, the plant height and the fruit setting rate of the tomato plants treated by applying the compound microbial agent are significantly increased, and the growth-promoting long-acting fruits are embodied in the whole growth cycle of the plants, which shows that the microbial agent can effectively promote the growth of the plants and the whole growth vigor of the plants is good. Better plant growth usually means more desirability of fruit yield and quality.

TABLE 4 Effect of different treatments on tomato yield and quality

Table 4 shows that the average yield of the tomato plot treated by the compound microbial agent is 201.10 kg, which is 26.35kg and 9.72kg higher than the average yield of the blank control group and the positive control group, respectively, and the weight and the fruit diameter of the single tomato are more ideal. The compound microbial agent can achieve the same or even better growth promoting and yield increasing effects than the same type of products sold in the market, and the fruit quality is more ideal.

Cell experiment of growth promoting effect of microbial agent on eggplant plants

The experiment adopts the same experimental scheme as the cell experiment of the tomato growth promoting effect, and the difference is as follows: (1) the seeds are replaced by eggplant seeds from tomato seeds; (2) the microbial inoculum treatment group used the microbial inoculum obtained in example 2, the positive control group used staphylaxin 17-17-17 (commercially available fertilizer with root strengthening effect) for corresponding replacement, and the others were consistent.

The plant heights and root fresh weights of different treatment groups are observed and determined in the eggplant picking period, and the result comparison is shown in a table 5.

TABLE 5 Effect of different treatments on eggplant plants

Compared with the positive control group, the eggplant plant applied with the compound microbial inoculum is stably increased (47.10 cm), and the eggplant plant is increased by 4.74cm (11.19%) compared with the positive control group (42.36 cm). Regular observation shows that the growth promoting effect is reflected in the whole growth stage of flowering and fruiting of plants, which shows that the microbial inoculum can effectively promote the growth of the plants and the overall growth vigor of the plants is good. In addition, after the picking period is finished, the root system weight of the plants is weighed, and the average root system weight of the eggplant plants to which the microbial agent is applied is 170g, is equivalent to (168 g) the average root system weight of the eggplant plants of a positive control group with a strong root system effect, and is obviously higher than the average root system weight (103 g) of a blank control group, so that the microbial agent has the capability of promoting the root system development.

Therefore, the microbial agent provided by the invention can obviously promote the development of crop roots and can also improve the overall growth situation and fruit yield of plants, thereby achieving the purposes of improving the lodging resistance of the plants and ensuring the yield and quality of crops.

Example 9 prevention and treatment effects of microbial Agents on cucumber damping-off in Pot culture experiments

The irrigation liquid for each group of irrigation is respectively as follows: the treatment groups 1 and 2 are microbial dilutions 1 and 2, respectively; the negative control group was sterile water; the positive control group 1 and the positive control group 2 are positive control diluent 1 and positive control diluent 2 respectively, and the positive control group 3 is 1000-time thiophanate methyl diluent. Each group was set with 6 cucumber seedlings and repeated 3 times.

(3) Seeds of Yuexiu No. 3 cucumber were sown in plastic pots (7 cm. about.7 cm. about.10 cm) filled with sterilized matrix soil, 3 plants per pot. When the first true leaf grows out (14 days), after the root system of the plant is damaged by a gardening small flower shovel, 100 mL of irrigation liquid of the corresponding group is irrigated in each pot. After 24h, the pathogenic bacterium Rhizoctonia solani (A) is inoculatedRhizoctonia solani) After 1 week, the 2 nd inoculation was performed. And 5 days after the 2 nd inoculation, cleaning soil at the root of the cucumber seedling, observing and recording the disease level of the plant, and measuring physiological indexes such as fresh weight, leaf area, dry weight and the like. And calculating disease index and relative prevention effect, the results are shown in table 6.

Grading index of disease grade:

stage 0: the cucumber seedling stem base has no disease spot

Level 1: small disease spots exist at the base of the stem of the cucumber seedling, and the proportion of the disease spots in the stem circumference is less than 1/4

Stage 2: the disease spots at the base of the cucumber seedling stem are large and account for 1/4-1/2 of the stem circumference ratio

And 3, level: the ratio of the disease spots at the base of the cucumber seedling is more than 1/2, but the whole stem circumference is not damaged

And 4, stage 4: the root and stem of cucumber seedling have the symptoms of dent or rupture, rot and browning and the like

The disease index calculation formula is as follows:

the relative prevention effect calculation formula is as follows:

TABLE 6 index of disease and prevention and treatment effect

From the results shown in table 6, it is understood that the control effect of the burkholderia monocytogenes alone is only 66.46%, while the control effect of the bacillus pumilus alone or after the burkholderia monocytogenes and the bacillus pumilus are compounded (example 2) is respectively improved to 76.24% and 74.85%, and the control effect of the microbial agent compounded in the manner of example 1 is improved to 80.44%, which is equivalent to the control effect of the thiophanate methyl product having various crop disease control effects (81.83%). The microbial agent has better control effect on cucumber rhizoctonia solani caused by rhizoctonia solani.

Example 10 in vitro prevention and treatment test of microbial Agents against Citrus canker

(1) The microbial preparation obtained in example 2 was diluted with 100 times the amount of water to prepare a microbial preparation diluent.

(2) Will be provided withCulturing the citrus canker pathogen to be tested on a culture dish with the diameter of 90mm for 48h at 28 ℃ by using a culture medium, scraping a ring of bacterial lawn by using a sterile inoculating ring, culturing the bacterial lawn in 50ml of beef extract peptone sterile liquid culture medium for 24h, measuring the OD value of the bacterial liquid by using an ultraviolet visible spectrophotometer, converting the OD value into the bacterial concentration by using a standard curve, and diluting the bacterial liquid to about 10 by using the beef extract peptone liquid culture medium⁸cfu/ml pathogen bacterial suspension. The test fungicide was diluted 500 times with sterile water for use.

1ml of microbial agent diluent and 1ml of 10-concentration microbial agent are mixed by a trace adjustable pipette⁸Sequentially adding cfu/ml of pathogenic bacteria suspension into a 20ml glass bottle filled with 8ml of beef extract peptone liquid culture medium to serve as a microbial agent treatment group; adding equal amount of sterile water as blank control group; adding 500 times of equivalent 27.12% basic copper sulfate solution as positive control group, culturing in constant temperature oscillator at 120rpm and 28 deg.C for 24 hr, and measuring OD value of bacterial liquid with ultraviolet-visible spectrophotometer, wherein each treatment is repeated for 3 times.

The calculation formula of the inhibition rate of the bactericide on citrus canker germs is as follows:

(3) according to the related phenomena of the concentration of the medicament and the inhibition rate, two medicament concentrations corresponding to the inhibition rates of about 20 percent and 90 percent are firstly found out, the common ratio is calculated, and five equal ratio concentrations with different dosages are arranged between the inhibition rates of 20 percent and 90 percent for virulence determination. According to the bacteria inhibition rate and the corresponding bactericide concentration, the effective inhibition concentration of the bactericide on the citrus canker pathogenic bacteria is calculated by using a bioassay method in DPS7.05 statistical software, namely EC50, and the result is shown in Table 7.

TABLE 7 biocontrol results for citrus canker pathogens with different treatments

As can be seen from Table 7, the microbial agent provided by the invention has 85.13% of inhibition rate on the citrus canker pathogen, and the capability of resisting the citrus canker pathogen is not weaker than that of basic copper sulfate.

EXAMPLE 11 potted biocontrol effects of microbial Agents on L.solani R.s

(1) The microbial preparation obtained in example 2 was diluted with 100 times the amount of water to prepare a treatment group drug.

(2) And (4) grouping. Divided into three groups including a treatment group, a positive control group, a negative control group and a blank control group. Wherein the blank control group and the negative control group are treated by clear water; the positive control group used 1000-fold dilutions of neomycin.

(3) R.s bacterial suspension preparation: l. solani R.s: (Ralstonia solanacearum) Activated from glycerin tube to TTC plate (formulation: 5g of glucose, 10g of peptone, 1 g of hydrolyzed fibronectin and 15 g of agar, adding water to 1L, sterilizing at 121 ℃ for 20min, cooling to 60 ℃, adding TTC to a final concentration of 0.005% (W/V)), culturing for 2 d, selecting high-activity ralstonia solanacearum colonies, and inoculating the ralstonia solanacearum colonies to an SPA liquid medium (formula: sucrose 20 g, bacteriological peptone 5g, K₂HPO₄ 0. 5 g，MgSO₄0.25 g, adding water to 1L, adjusting pH to 7.0-7.2, sterilizing at 121 deg.C for 20 min), culturing on shaking table for 8-16 h, and then according to OD₆₀₀The absorbance value of R.s is diluted to 10⁷CFU/mL, R.s bacterial suspension was prepared for use.

(4) And (3) tomato seedling treatment: taking tomato seedlings growing for 20 days, gently shaking off soil on the surfaces of the roots, soaking the roots in the treatment solutions for 10 min, immediately planting the tomato seedlings in sterilized soil, treating 1 tomato seedling in each pot, treating 6 tomato seedlings in each pot, and repeating 5 treatment steps.

The potted plant is placed in an environment with the temperature of 25-28 ℃ for seedling revival for 7 days, and then a gardening small shovel is inserted into soil around the root, so that the root of the tomato is damaged to cause a wound. Then, each seedling of the negative control group, the positive control group and the microbial inoculum treatment group is filled with 50mL 10⁷cfu/mL R.s bacterial suspension, blank control group filled with equal amount of clear water.

(5) Observation and results: when the first sign of symptoms (tomato seedlings have a withered symptom) begins (generally, the tomato seedlings begin to get ill at the 5 th day after root irrigation), the tomato seedling state is observed and recorded once a day (the ill condition is recorded according to the grading standard) to the 10 th to 13 th days (determined according to the actual ill condition), the number of plants of each disease treatment grade is recorded, and the disease index and the prevention and treatment effect are calculated, and the results are shown in a table 8.

The disease grades of the plants are divided into the following 5 grades:

level 0: no symptoms

Stage 1: wilting leaves in one piece

And 2, stage: 2-3 leaf wilting

And 3, level: withering of the whole plant

4, level: death of the plant

As can be seen from Table 8, the disease index of the tomato seedlings treated by the microbial inoculum is reduced to 25.39%, the pathogen prevention and treatment effect reaches 41.86%, and the disease index and the pathogen prevention and treatment effect of the positive control group treated by the new phytomycin diluent are 27.60% and 36.80%, respectively. The compounded microbial agent has a similar or even better germ control effect with the common products sold in the market for controlling plant diseases caused by the ralstonia solanacearum.

TABLE 8 biocontrol effects of various treatment groups on L.solani

Example 12 microbial Agents on wilt of winter melon: (Fusarium oxysporum) The field biocontrol effect

(1) The microbial preparation was prepared as described in example 2, and diluted with 100 times the amount of water to prepare microbial preparation diluent 1. The Bacillus pumilus fermentation broth used in example 1 or 2 was diluted with 100 times the amount of water to obtain microorganism dilution 2.

(2) And (4) grouping. Divided into 5 groups including treatment 1 group, treatment 2 group, positive control 1 group, positive control 2 group, negative control group and blank control group. Wherein the blank control group and the negative control group are treated by clear water; treatment 1 group was treated with microbial inoculum; treating the group 2 by using a bacillus pumilus fermentation diluent; the positive control group 1 was treated with Liangdun (fludioxonil and metalaxyl-M as the main ingredients) according to the instruction; positive control 2 groups were performed using commercially available microbial inoculum products^®(Bacillus amyloliquefaciens) was treated as per the instructions.

(3) The experimental method comprises the following steps: white gourd variety guan yan No. 2 small white gourd; and (5) planting in a greenhouse. The experiment was repeated 3 times for each 10 seedlings treated. Watering 250 mL of treatment agent for each wax gourd seedling in field planting, and after 3 days of field planting, beginning to water at a concentration of 1 × 10⁶cfu/mL wax gourd Fusarium oxysporum (C.A.)Fusarium oxysporum) Spore suspension, 50mL of each melon seedling is watered. And then, irrigating the roots with the treatment agent for 1 time at intervals of 10-15 days according to the weather conditions, and applying the treatment agent for 3 times in total. And (5) performing cultivation management by adopting a conventional method. After 2 times of irrigation for 3 weeks, the incidence rate of the blight of the winter melon was investigated.

(4) Observation and results: see table 9.

TABLE 9 biocontrol effect of strains on blight of winter melon

As can be seen from Table 9, the Bacillus pumilus diluent used in the present invention has better biocontrol effect on the blight of the melon (the morbidity is 8.23% and 14.47% respectively) than the commercial Bacillus amyloliquefaciens product, and the compounded microbial agent can further reduce the morbidity of the blight of the melon (6.15%). And the observation shows that the growth vigor of the wax gourd plants treated by the compound microbial agent is better, and the expansion area of leaves, the thickness of vines, the flowering quantity and the like are obviously superior to those of other treatment groups.

Claims

1. The application of a microbial agent in controlling plant diseases, wherein the microbial agent comprises microbial fermentation liquor, potassium fulvate, water and diammonium phosphate, and is characterized in that the microbial fermentation liquor comprises fermentation liquor of Burkholderia GDMCC No:61156 and Bacillus pumilus GDMCC No:61962, and the plant diseases are cucumber Fusarium wilt, citrus ulcer, cucumber Rhizoctonia solani or tomato bacterial wilt, wherein the cucumber Rhizoctonia solani is caused by Rhizoctonia solani, the citrus ulcer is caused by Xanthomonas axonodis pv. citri pathogenic bacteria, the tomato bacterial wilt is caused by Ralstonia solani, and the cucumber Fusarium wilt is caused by Fusarium oxysporum; the effective viable count in various bacterial strain fermentation liquor used by the microbial fermentation liquor is more than or equal to 90 hundred million/mL.

2. The application of a microbial agent in preventing and treating plant diseases comprises microbial fermentation liquor, potassium fulvate, water and diammonium phosphate, and is characterized in that the microbial fermentation liquor is compounded by fermentation liquor of Burkholderia GDMCC No:61156, Bacillus pumilus GDMCC No:61962 and Bacillus subtilis, and the plant diseases are cucumber Fusarium wilt, citrus ulcer, cucumber Rhizoctonia solani or tomato bacterial wilt, wherein the cucumber Rhizoctonia solani is caused by Rhizoctonia solani, the citrus ulcer is caused by Xanthomonas anodis pv. citri pathogenic bacteria, the tomato bacterial wilt is caused by Ralstonia solani, and the cucumber Fusarium wilt is caused by Fusarium oxysporum; the effective viable count in the fermentation liquor of various bacterial strains used by the microbial fermentation liquor is more than or equal to 90 hundred million/mL.

3. The application of the microbial agent in controlling plant diseases according to claim 1 or 2, wherein the microbial agent is prepared by proportionally mixing fermentation liquor of various strains used by the microbial fermentation liquor according to a mass ratio.

4. The use of the microbial agent according to claim 1 or 2 for controlling plant diseases, wherein the microbial agent comprises the following components in parts by weight: 40-80 parts of microbial fermentation liquor, 40-90 parts of potassium fulvate, 10-50 parts of water and 2-5 parts of diammonium phosphate.

5. The application of the microbial agent in controlling plant diseases according to claim 4, wherein the microbial agent comprises the following components in parts by weight: 50 parts of microbial fermentation liquor, 80 parts of potassium fulvate, 20 parts of water and 3 parts of diammonium phosphate.