CN111040955B

CN111040955B - Enhanced disease-prevention growth-promoting carbon-based microbial inoculum and preparation method and application thereof

Info

Publication number: CN111040955B
Application number: CN201911335293.8A
Authority: CN
Inventors: 王光飞; 马艳; 郭德杰; 罗佳; 宋修超
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2019-12-23
Filing date: 2019-12-23
Publication date: 2022-09-20
Anticipated expiration: 2039-12-23
Also published as: CN111040955A

Abstract

The invention discloses an enhanced disease-prevention growth-promoting carbon-based microbial inoculum as well as a preparation method and application thereof, wherein the microbial inoculum comprises the following components: biochar, yeast powder, peptone, sucrose, trichoderma longibrachiatum TB2 microbial inoculum and bacillus amyloliquefaciens BB2 microbial inoculum; the preparation method comprises the following steps: firstly, dissolving yeast powder, peptone and sucrose in water, adsorbing the yeast powder, the peptone and the sucrose on biochar, and airing or drying the biochar at low temperature to obtain a multi-component carbon-based carrier; then uniformly mixing the multi-component carbon-based carrier with a trichoderma longibrachiatum TB2 microbial inoculum and a bacillus amyloliquefaciens BB2 microbial inoculum to obtain the microbial inoculum; the multi-component carbon-based carrier provided by the application can provide nutrients and inhabitation space for the propagation of growth-promoting disease-preventing bacteria TB2 and BB2, so that the colonization of the effective bacteria of the microbial inoculum and the growth-promoting disease-preventing function of the microbial inoculum are enhanced, the microbial inoculum can improve the activity and diversity of soil microorganisms, has the growth-promoting and yield-increasing effects on crops, and can effectively prevent and control various soil-borne diseases of the crops.

Description

Enhanced disease-prevention growth-promoting carbon-based microbial inoculum and preparation method and application thereof

Technical Field

The invention relates to an agricultural planting microbial agent, in particular to an enhanced disease-prevention growth-promoting carbon-based microbial agent suitable for various cash crops, and a preparation method and application thereof.

Background

China is the first major country in agriculture, and agricultural areas and yield are the first world, but harmful microorganisms in soil are easy to obtain and propagate, the biological characters of the soil are deteriorated, the biological fertility of the soil is low, and the growth of crops is limited due to high intensification, unreasonable fertilizer application, long-term continuous cropping and the like. The disease-preventing growth-promoting microbial inoculum can improve the diversity and the biological activity of soil microorganisms, increase beneficial microorganisms, has the functions of promoting the growth of crops and preventing and controlling various diseases, and is an important means for solving the crop planting obstacle. The development of novel high-efficiency disease-preventing growth-promoting microbial inoculum has great significance for sustainable development of agriculture.

The application effect of the agricultural microbial agent for planting mainly depends on the colonization ability of effective bacteria in soil, so that the improvement of the colonization amount of the effective bacteria and the prolongation of the effective colonization time are beneficial to the exertion of the effect of the microbial agent. At present, carbon-based microbial agents exist in the market, and the carbon-based carriers, namely the microporous structures and the organic nutrient components of the biochar, are beneficial to the survival and the propagation of beneficial microbial floras, can provide living space for effective bacteria when applied to soil at the initial stage, and are beneficial to the colonization of the effective bacteria. However, the nutrition in the biochar cannot be effectively utilized by various agricultural planting effective bacteria, and the biochar has less original nutrition. The pores of the biochar are gradually occupied by original microorganisms of soil at a later stage, so that the existing colonization promoting effect is short in time, and is only 5-30 days generally. Screening agricultural disease-preventing growth-promoting bacteria capable of being enriched by biochar to develop a carbon-based microbial agent is beneficial to improving the application effect. In addition, the optimization of the carbon-based carrier to enhance the rapid colonization and occupation of the effective bacteria in the biochar so as to improve the colonization quantity and long-acting property of the effective bacteria in the soil is a technical difficulty to be solved in the field.

Disclosure of Invention

The invention provides an enhanced disease-prevention growth-promoting carbon-based microbial inoculum which takes biochar, peptone, yeast powder, sucrose, trichoderma longibrachiatum TB2 bacteria capable of being enriched by biochar and bacillus amyloliquefaciens BB2 bacteria as raw materials. Has the advantages of easy implementation, environmental protection, wide application of crops and the like, can improve the soil microbial activity and microbial flora, prevent and control various soil-borne diseases of economic crops, and has long-acting growth promoting and yield increasing effects on the crops.

In order to realize the purpose, the invention firstly provides an enhanced disease-prevention growth-promoting carbon-based microbial inoculum, which comprises the following raw materials: according to the mass percentage, the yeast powder accounts for 0.5 to 1 percent, the peptone accounts for 1 to 2 percent, the sucrose accounts for 1 to 2 percent, and the Trichoderma longibrachiatum TB2 microbial inoculum (the bacterial content is more than 5 multiplied by 10) ⁹ CFU/g) accounts for 15-35%, and Bacillus amyloliquefaciens BB2 microbial inoculum (the bacterial content is more than 6 multiplied by 10) ⁹ CFU/g) accounts for 15-35%, and biochar is used for making up the balance.

In the invention, the biochar is conventional commercial biochar, or is obtained by crushing a solid product obtained by carbonizing agricultural production organic waste at 400-600 ℃, and then sieving the crushed product with a 40-mesh sieve; the agricultural production organic waste comprises at least one of corn straw, corncob, wheat straw, rice hull and soybean straw.

Further, in the enhanced disease prevention and growth promotion carbon-based microbial inoculum disclosed by the invention, the trichoderma longibrachiatum TB2 microbial inoculum is obtained by the following method:

a) inoculating Trichoderma longibrachiatum TB2 strain into potato glucose agar culture medium, and culturing at 28 deg.C for 3 days; then transferring the Trichoderma longibrachiatum strain into a potato glucose liquid culture medium, wherein the liquid filling amount is 80m1/250m1, and then carrying out shake cultivation at 28 ℃ for 3-4 days at the rotation speed of 160rpm of a shaking table;

b) inoculating 10% (volume/mass ratio, ml/kg) of inoculum size into sterilized bran and straw solid culture medium (water content 60%, bran particle size greater than 0.5mm, adding straw to ensure air permeability, mass ratio of straw to bran of 1:9, average length of straw of 4cm, sterilizing at 126 deg.C for 60min), and culturing at 29 + -1 deg.C for 5-7 days until spore is produced;

c) naturally air drying Trichoderma longibrachiatum and bran or oven drying at 30 deg.C, pulverizing, and sieving with 100 mesh sieve to obtain Trichoderma longibrachiatum TB2 microbial inoculum with bacterial content of 5 × 10 ⁹ CFU/g。

Further, in the enhanced disease prevention and growth promotion carbon-based microbial inoculum disclosed by the invention, the bacillus amyloliquefaciens BB2 microbial inoculum is obtained by the following steps:

a) inoculating Bacillus amyloliquefaciens BB2 into a beef extract peptone agar culture medium, and culturing for 3 days at 32 ℃; then transferring the bacillus amyloliquefaciens strain into a beef extract peptone liquid culture medium, wherein the liquid loading is 80m1/250m1, and then carrying out shake cultivation at 32 ℃ for 3-5 days at the rotation speed of 160rpm of a shaker;

b) inoculating 10% (volume/mass ratio, ml/kg) of inoculum size into sterilized bran and straw solid culture medium (water content 60%, bran particle size greater than 0.5mm, adding straw to ensure air permeability, mass ratio of straw to bran of 1:9, average length of straw of 4cm, sterilizing at 126 deg.C for 60min), and culturing at 32 + -2 deg.C for 3-5 days until spore is produced;

c) naturally air drying Bacillus amyloliquefaciens and bran and straw or drying at 40 ℃, crushing and sieving with a 100-mesh sieve to obtain the Bacillus amyloliquefaciens BB2 microbial inoculum with the bacterial content of more than 6 multiplied by 10 ⁹ CFU/g。

The invention also provides a preparation method of the enhanced disease-prevention growth-promoting carbon-based microbial inoculum, which comprises the following steps:

a) fully stirring yeast powder, peptone, sucrose and water, mixing the obtained solution with biochar, fully adsorbing, and air-drying at normal temperature or drying at low temperature of 40-50 ℃ until the water content is 5-10% to obtain a multi-component carbon-based carrier;

b) uniformly mixing the multi-component carbon-based carrier with a trichoderma longibrachiatum TB2 microbial inoculum and a bacillus amyloliquefaciens BB2 microbial inoculum according to a ratio to prepare an enhanced disease prevention growth promotion carbon-based microbial inoculum;

thirdly, the invention also provides the application of the strengthened disease-preventing growth-promoting carbon-based microbial inoculum in preventing and treating soil-borne diseases of crops and promoting the growth of the crops, and the application method comprises the following steps: applying the enhanced disease-preventing growth-promoting carbon-based microbial inoculum into soil 0-7 days before transplanting or sowing crops, wherein the application amount is 15-40 kg/mu; generally speaking, the organic fertilizer can be applied alone or in combination with organic fertilizer for soil with the organic matter content higher than 20g/kg, and the effect of the organic fertilizer is better for the soil with the organic matter content lower than 20 g/kg.

Further, in the application of the microbial inoculum provided by the application, the crops are preferably hot peppers, tomatoes, green vegetables, watermelons, garlic and peach trees.

Further, in the application of the microbial inoculum provided by the application, the disease prevention comprises: preventing and treating at least one of crop epidemic disease, root rot and clubroot.

The applicant screens out disease-prevention growth-promoting strains trichoderma longibrachiatum TB2 and bacillus amyloliquefaciens BB2 which can be enriched by biochar from biochar soil and publishes the disease-prevention growth-promoting strains for the first time (see the literature, "the effects of different microbial agents on prevention and control of pepper phytophthora blight and the influence of different microbial agents on soil properties" \\ 26107; Shuicho et al, Jiangsu agricultural science, 2019, 35(4): 811-817). In subsequent researches, the applicant finds that the two strains can be propagated by colonizing pores of the biochar and utilizing biochar easily-degradable organic matters, and more importantly, the TB2 and BB2 strains can be used for promoting the growth of crops and inhibiting the growth of pathogenic bacteria in cooperation with the biochar. But the biochar has less available nutrition and has shorter propagation promoting time on TB2 and BB2 bacteria. Therefore, the method carries out pretreatment on the biochar to adsorb nutrition required by effective bacterium propagation in advance to form a reinforced carbon-based carrier, and can be favorable for the colonization of the effective bacteria in soil by combining with the microbial inoculum.

Through multiple formula improvement, pot culture tests and field tests, the applicant finds that yeast powder, peptone and sucrose can be rapidly utilized by TB2 and BB2 for propagation, and the yeast powder, the peptone and the sucrose are all dissolved in water, adsorbed by biochar in a solution form and then dried to form a multi-component carbon-based carrier, at the moment, the yeast powder, the peptone and the sucrose are stably fixed in biochar pores, so that TB2 and BB2 are conveniently applied to soil to be rapidly activated, and the yeast powder, the peptone and the sucrose in the biochar are utilized for propagation, so that the colonization efficiency of TB2 and BB2 in the soil is enhanced, and the enhanced disease prevention and growth promotion carbon-based microbial inoculum is obtained.

The application provides a strengthening type microbial inoculum can improve the microflora and the bioactivity, effectively prevent and control soil-borne diseases, lighten continuous cropping obstacles, promote the growth of crops and improve the yield of the crops, and compared with the prior art, the strengthening type microbial inoculum has the following beneficial effects:

(1) meanwhile, the fertilizer has the functions of improving soil microflora, preventing and controlling soil-borne diseases and promoting crop growth.

(2) The trichoderma longibrachiatum TB2 and bacillus amyloliquefaciens BB2 are screened in the charcoal soil, can be enriched by charcoal, and have synergistic growth promoting and disease preventing effects with the charcoal. The carbon-based carrier is matched with TB2 and BB2 to prepare a microbial inoculum, which is beneficial to the colonization of effective bacteria in soil and can enhance the functionality and increase the time effectiveness.

(3) The raw materials are reasonably compounded, and the biochar has large specific surface area and more pores and can provide living space for beneficial microorganisms; the prepared multi-component carbon-based carrier can provide nutrition and living space for rapid propagation after the TB2 bacteria and the BB2 bacteria are applied into soil, and is beneficial to stable colonization of the TB2 bacteria and the BB2 bacteria in the soil. In addition, the TB2 microbial inoculum and the BB2 microbial inoculum have high effective microbial activity, can effectively improve a soil microbial flora, improve the soil biological activity, inhibit pathogenic microorganisms and promote the growth of crops.

(4) The invention has simple field application method and is an environment-friendly agricultural planting disease-preventing growth-promoting microbial inoculum.

Drawings

FIG. 1 is a photograph of potted growth-promoting scenes of capsicum and green vegetables by each microbial inoculum;

FIG. 2 is a picture of the potted plant control effect of each bacterial agent on the blight of capsicum and the root rot of tomato;

FIG. 3 is a photograph of growth-promoting and disease-preventing scenes of green vegetables in obstacle leaf vegetable soil by using an enhanced carbon-based microbial inoculum;

FIG. 4 is a photograph of growth-promoting scenes of garlic bulbs with the enhanced carbon-based fungicide.

Detailed Description

The technology related to the present invention will be further described with reference to specific examples.

The examples relate to media and strains:

potato dextrose agar medium: peeling 200g potato, cutting into pieces, cooking for 30min, filtering with four layers of gauze to obtain filtrate, adding 20g glucose, diluting to 1L, adding 20g agar, and sterilizing at 121 deg.C for 30 min.

Potato dextrose liquid medium: peeling 200g potato, cutting into pieces, cooking for 30min, filtering with four layers of gauze to obtain filtrate, adding 20g glucose, diluting to 1L, and sterilizing at 121 deg.C for 30 min.

Beef extract peptone agar medium: 5g of beef extract, 10g of peptone, 5g of NaCl5g, 20g of agar, 1L of water and pH 7.2-7.4. Sterilizing at 121 deg.C for 30 min.

Beef extract peptone liquid medium: 5g of beef extract, 10g of peptone, 5g of NaCl, 1L of water and pH 7.2-7.4. Sterilizing at 121 deg.C for 30 min.

The preparation method of the solid culture medium comprises mixing testa Tritici and straw (length less than 4mm) at a mass ratio of 9:1, adding water to make water content in the mixture reach 60%, sterilizing at 126 deg.C for 60min, and cooling.

Trichoderma longibrachiatum (Trichoderma longibrachiatum) TB2 and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BB2 used in the examples are disclosed in the document "prevention and control effect of different microbial agents on pepper phytophthora blight and influence on soil property" leaf 26107; Shuo et al, Jiangsu agricultural science, 2019, 35(4): 811-817) ", and both strains are deposited and provided by Jiangsu province agricultural academy of sciences.

The examples relate to the starting materials:

biochar: purchased from Li Yang Shang De Sheng activated carbon factory, is rice straw and rice hull biochar;

yeast powder and peptone: purchased from Angel biology, Inc. Hubei.

EXAMPLE 1 preparation of Trichoderma longibrachiatum TB2 inoculum

a) Inoculating the Trichoderma longibrachiatum TB2 strain preserved on the inclined plane into a potato glucose agar culture medium, and culturing at 28 ℃ for 3 days to generate green conidia; and inoculating the trichoderma longibrachiatum blocks with conidia into a potato glucose liquid culture medium, wherein the liquid filling amount of the liquid culture medium is 80m1/250m1, and 3 trichoderma longibrachiatum blocks with the diameter of 8mm are inoculated into each bottle. Then culturing at 28 deg.C with shaking table rotation speed of 160rpm for 3-4 days to obtain mycelium solution.

b) Inoculating 80ml of trichoderma mycelium liquid into 1.5kg of sterilized bran and straw solid culture medium, uniformly stirring under aseptic condition, spreading the solid culture in a shallow tray (the shallow tray is soaked in formalin solution overnight for airing), wherein the thickness is 3-5cm, spreading filter cloth subjected to high-pressure sterilization treatment below the shallow tray, covering double layers of gauze subjected to high-pressure sterilization treatment above the shallow tray, and culturing at 29 +/-2 ℃ for 5-7 days until spore production is complete.

c) Naturally air drying Trichoderma longibrachiatum and bran or oven drying at 30 deg.C until water content is less than 10%, pulverizing, and sieving with 100 mesh sieve to obtain Trichoderma longibrachiatum TB2 microbial inoculum with bacterial content of more than 5 × 10 ⁹ CFU/g。

Example 2 preparation of Bacillus amyloliquefaciens BB2 microbial inoculum

a) Streaking and inoculating the bacillus amyloliquefaciens BB2 strain stored on the inclined plane into a beef extract peptone agar culture medium, and culturing for 2 days at 32 ℃; and inoculating the bacterium block with BB2 bacterial colony into beef extract peptone liquid medium, wherein the liquid loading capacity of the liquid medium is 80m1/250m1, and each bottle is inoculated with 3 bacterium blocks. Then culturing for 3-5 days at the rotating speed of 160rpm of a shaking table at 32 ℃ to obtain a bacterial liquid.

b) Inoculating 80ml of cultured bacillus liquid into 1.5kg of sterilized bran and straw solid culture medium, uniformly stirring under aseptic condition, spreading the solid culture in a shallow tray (the shallow tray is soaked in formalin solution overnight for airing) with the thickness of 3-5cm, spreading filter cloth subjected to high-pressure sterilization treatment below the shallow tray, covering double layers of gauze subjected to high-pressure sterilization treatment above the shallow tray, and culturing at 32 +/-2 ℃ for 4-6 days until spore production is complete.

c) Naturally air drying Bacillus together with bran and straw or oven drying at 40 deg.C until the water content is less than 10%, pulverizing, and sieving with 100 mesh sieve to obtain Bacillus amyloliquefaciens BB2 microbial inoculum with bacterial content of more than 6 × 10 ⁹ CFU/g。

Example 3 growth promoting effect of compounding Trichoderma longibrachiatum TB2 microbial inoculum and Bacillus amyloliquefaciens BB2 microbial inoculum on pepper and green vegetables

Taking healthy and disease-free vegetable plough layer soil of Huai' an Huang-quan, and dividing the soil into four treatments: no microbial inoculum: adding no microbial inoculum; TB2 microbial inoculum: adding 1% of trichoderma longibrachiatum TB2 microbial inoculum by mass percent prepared in the example 1; BB2 microbial inoculum: adding 1% of bacillus amyloliquefaciens BB2 microbial inoculum by mass percent prepared in the example 2; TB2+ BB2 bacterial agents: meanwhile, adding a TB2 microbial inoculum with the mass percent of 1% and a BB2 microbial inoculum with the mass percent of 1% for treatment, placing the mixture in square plastic basins, treating 2 basins each, and repeating the treatment 3 times.

The plant scene 30 days after the pepper and the green seedling vegetable are transplanted is shown in figure 1. The data in table 1 are the average of 3 replicates for each treatment.

TABLE 1 Effect of the inoculum on fresh weight of Capsici fructus and vegetables

	Capsicum (g/plant)	Green vegetables (g/plant)
			Addition treatment of a sterile agent	48.1±6.2b	59.7±7.7b
TB2 microbial inoculum	54.4±4.7ab	76.4±5.3a
			BB2 microbial inoculum	56.7±5.7ab	75.9±8.3a
TB2+ BB2 bacterial agent	60.4±7.1a	79.3±7.7a

In fig. 1, a group a is pepper and a group B is vegetable, and experimental results in fig. 1 and table 1 show that TB2 microbial inoculum and BB2 microbial inoculum which are independently applied have a good growth promotion effect on pepper and vegetable plants, and BB2 microbial inoculum and TB2 microbial inoculum are applied in a compound manner, so that the effect is the best, and the growth promotion effect on plants is remarkable. The test results in Table 1 show that the growth promoting mechanisms of TB2 and BB2 are not in conflict, and the growth promoting effects of the two bacteria can be superposed. And the colonization of TB2 and BB2 do not affect each other or can promote each other.

Example 4 prevention of Pepper and tomato by compounding Trichoderma longibrachiatum TB2 microbial inoculum and Bacillus amyloliquefaciens BB2 microbial inoculum

Taking Huaian Huang code village pepper phytophthora blight soil and Xuzhou Shuanggou tomato root rot soil, and respectively carrying out four treatments: no microbial inoculum: adding no microbial inoculum; TB 2: adding 1% of trichoderma longibrachiatum TB2 microbial inoculum prepared in the example 1 in percentage by mass; BB 2: adding 1% of bacillus amyloliquefaciens BB2 microbial inoculum prepared in the example 2 in percentage by mass; TB2+ BB 2: meanwhile, adding a TB2 microbial inoculum with the mass percent of 1% and a BB2 microbial inoculum with the mass percent of 1% for processing, and filling the mixture into a round pot. 5 pots per treatment, each treatment was repeated 3 times. The two diseased soils were planted with pepper and tomato, respectively, and 20-day potted scenes are shown in fig. 2, and the data of disease incidence in table 2 is the average value of 3 replicates of each treatment.

Incidence (%) — number of diseased plants/total plants × 100%.

Table 2 shows the influence of each microbial inoculum on the incidence rate of phytophthora blight of capsicum and the incidence rate of tomato root rot

	Incidence of Phytophthora capsici (%)	Incidence of tomato root rot (%)
			Addition treatment of a sterile agent	84.7±8.9a	62.2±7.8a
TB2 microbial inoculum	46.4±6.2b	43.9±5.2b
			BB2 microbial inoculum	37.8±3.5b	45.6±3.2b
TB2+ BB2 bacterial agent	27.4±4.9c	17.5±5.8c

In fig. 2, a group a is pepper, a group B is tomato, and experimental results in fig. 2 and table 2 show that TB2 microbial inoculum and BB2 microbial inoculum which are independently applied have better prevention and control effects on pepper phytophthora blight and tomato root rot, and BB2 microbial inoculum and TB2 microbial inoculum are applied in a compounding manner, so that the effect is best, and the prevention and control effects on soil-borne diseases are very remarkable. The disease prevention effect of the compound of TB2 and BB2 is better, which shows that the disease prevention mechanisms of TB2 and BB2 are not conflicted, and the disease prevention effects of the two bacteria can be superposed. And the colonization of TB2 and BB2 do not affect each other or can promote each other.

Example 5 colonization of effective bacteria in soil in enhanced disease-preventing growth-promoting carbon-based microbial inoculum

Mixing the microbial inoculum: 250g of Trichoderma longibrachiatum TB2 microbial inoculum of example 1 and 250g of Bacillus amyloliquefaciens BB2 of example 2 are mixed to obtain a mixed microbial inoculum for later use.

Carbon-based microbial inoculum: 500g of biochar is mixed with 250gTB2 microbial inoculum and 250gBB2 microbial inoculum to obtain a carbon-based microbial inoculum for later use.

Reinforced carbon-based microbial inoculum: dissolving 10g of peptone, 10g of sucrose and 5g of yeast powder in 50mL of water, adsorbing 475g of biochar, airing until the water content is 10%, and mixing with 250gTB2 microbial inoculum and 250gBB2 microbial inoculum to prepare the enhanced carbon-based microbial inoculum for later use.

Taking the soil of the Huaian Huang code village plough layer, and then respectively carrying out four treatments: adding no additive (blank control), adding 0.5% of mixed microbial inoculum (the mass of the microbial inoculum accounts for 0.5% of the mass of the soil), adding 1% of carbon-based microbial inoculum (the mass of the carbon-based microbial inoculum accounts for 1% of the mass of the soil), adding 1% of enhanced carbon-based microbial inoculum (the mass of the enhanced carbon-based microbial inoculum accounts for 1% of the mass of the soil), filling the mixture into a round pot, and repeating each treatment for 1 pot 3 times. The number of viable bacteria of the effective bacteria is measured respectively after 30 days and 90 days of moisture preservation, and the measuring method is a dilution coating culture method.

The results are shown in Table 3, which shows the average of 3 replicates for each treatment.

Table 3 shows the colonization of the effective bacteria in the soil by each microbial inoculum

The results in Table 3 show that the addition of biochar in the microbial inoculum can increase the colonization amount of TB2 and BB2 in soil by 24.0 percent and 39.3 percent respectively after 30 days of moisture preservation. If the microbial inoculum is combined with multi-component biochar containing peptone, yeast powder and sucrose, the colonization effect is better, and the colonization increasing rates of TB2 and BB2 are 45.1 percent and 74.2 percent respectively. After being kept wet for 90 days, the colonization numbers of TB2 and BB2 in soil can be increased by 21.4 percent and 12.5 percent respectively by combining the microbial inoculum with the biochar, and the colonization numbers of the microbial inoculum combination reinforced multi-component biochar can be increased by 239 percent and 121 percent respectively. Therefore, the colonization of effective bacteria in soil can be obviously increased by combining the microbial inoculum with the reinforced carbon-based carrier, the colonization promoting effect is not weakened along with the application time, and the colonization promoting effect is more obvious when the application time is longer.

Example 6 application in watermelon substrate cultivation

TB2 enhanced carbon-based microbial inoculum: dissolving 10g of peptone, 10g of sucrose and 5g of yeast powder in 50mL of water, adsorbing 475g of biochar, drying until the water content is 10%, and mixing with 250gTB2 microbial inoculum to prepare the enhanced carbon-based microbial inoculum for later use.

BB2 enhanced carbon-based microbial inoculum: dissolving 10g of peptone, 10g of sucrose and 5g of yeast powder in 50mL of water, adsorbing to 475g of biochar, drying until the water content is 10%, and mixing with 250gBB2 microbial inoculum to prepare the enhanced carbon-based microbial inoculum for later use.

Compounding a reinforced carbon-based microbial inoculum: dissolving 10g of peptone, 10g of sucrose and 5g of yeast powder in 50mL of water, adsorbing 475g of biochar, airing until the water content is 10%, and mixing with 250gTB2 microbial inoculum and 250gBB2 microbial inoculum to prepare the enhanced carbon-based microbial inoculum for later use.

The cultivation substrate is taken for four treatments: no addition treatment (blank control), 0.75% of TB2 enhanced carbon-based microbial inoculum (the mass of the microbial inoculum accounts for 0.75% of the mass of the matrix), 0.75% of BB2 enhanced carbon-based microbial inoculum (the mass of the microbial inoculum accounts for 0.75% of the mass of the matrix), and 1% of composite enhanced carbon-based microbial inoculum (the mass of the enhanced carbon-based microbial inoculum accounts for 1% of the mass of the matrix). Each treatment was 1 substrate tank, 10 watermelon seedlings per tank, and each treatment was repeated 3 times. The results after 15 days of planting four-leaf watermelon seedlings are shown in table 4, and the data in table 4 are the average value of 3 times of repetition of each treatment. Soil microbial activity and diversity were determined using Biolog-Eco ecological test plates.

TABLE 4 growth promoting effect of the microbial inoculum on watermelon and improvement effect on soil biological characteristics

The results in Table 4 show that, compared with the conventional control, the application of TB2 enhanced type carbon-based microbial inoculum and BB2 enhanced type carbon-based microbial inoculum can promote the growth of watermelons, the fresh weight is respectively increased by 11.0% and 14.2%, the application of the composite enhanced type carbon-based microbial inoculum can more remarkably promote the growth of watermelon plants, and the fresh weight is increased by 21.3%. Biolog carbon source utilization detection of AWCD values can be a sensitive indicator of microbial activity. The effect of the composite enhanced carbon-based microbial inoculum on improving the activity and the microbial diversity of soil microorganisms is better than that of TB2 enhanced carbon-based microbial inoculum and BB2 enhanced carbon-based microbial inoculum. Therefore, TB2 and BB2 can synergistically promote the growth of crops and improve the soil microbial traits under the strengthening carbon-based carrier.

Example 7 application in greenhouse green vegetable soil

Carbon-based microbial inoculum: 500g of biochar is mixed with 300gTB2 microbial inoculum and 200gBB2 microbial inoculum to obtain a carbon-based microbial inoculum for later use.

Reinforced carbon-based microbial inoculum: dissolving 15g of peptone, 15g of sucrose and 10g of yeast powder in 100mL of water, adsorbing 460g of biochar, airing until the water content is 10%, and mixing with 300gTB2 microbial inoculum and 200gBB2 microbial inoculum to prepare the enhanced carbon-based microbial inoculum for later use.

The greenhouse is a greenhouse for greenhouse leaf vegetables with the service life of more than 10 years in the Zhenjiakang vegetable production base of Town, Louis, Wuxi, and has serious continuous cropping obstacles and poor growth of leaf vegetables.

The experiment set up 3 treatments: conventional treatment, carbon-based microbial inoculum treatment and reinforced carbon-based microbial inoculum treatment. Each processing 3 repeated cells, the cell area is 10m ² And are randomly arranged. The conventional treatment is not added with microbial inoculum, the carbon-based microbial inoculum is treated and spread for 15 kg/mu, the reinforced carbon-based microbial inoculum is treated and spread for 15 kg/mu, the soil is uniformly spread on the soil surface, green seeds are sown after full tillage, and the actual scenes of each treatment cell after 25 days of sowing are shown in figure 3. The results of yield, morbidity and soil properties are shown in tables 5 and 6.

TABLE 5 influence of the inoculum on the yield and clubroot of green vegetables

As can be seen from FIG. 3, the green vegetables plants are stronger and thrive when the carbon-based fungicide is applied compared with the conventional control and the carbon-based fungicide.

The results in Table 5 show that compared with the conventional control, the occurrence rate of clubroot can be remarkably reduced by 51.7% by applying the carbon-based fungicide, and the yield of green vegetables is increased by 46.9%. The enhanced carbon-based microbial inoculum has better effect, the disease incidence rate is reduced by 68.3 percent, and the yield is increased by 87.7 percent.

TABLE 6 Effect of the inoculum on Trichoderma reesei Numbers, AWCD, microbial Activity and microbial diversity in Umbelliferae

The results in table 6 show that compared with the carbon-based microbial inoculum, the application of the reinforced carbon-based microbial inoculum can significantly improve the microbial Activity (AWCD) and the microbial diversity, significantly increase the colonization amount of the effective trichoderma and bacillus in the soil, and be beneficial to the prevention and growth promotion of diseases.

Example 8 application in soil for garlic vegetables

Dissolving 20g of peptone, 20g of sucrose and 10g of yeast powder in 150mL of water, adsorbing the peptone on 450g of biochar, airing the biochar until the water content is 10%, and mixing the biochar with 350gTB2 microbial inoculum and 150gBB2 microbial inoculum to prepare the enhanced carbon-based microbial inoculum.

The planting obstacle soil of Xuzhou city Fengxian Yangxian garlic is selected, and the yield of the garlic after long-term continuous cropping tends to decline year by year. 2 treatments are set: conventional treatment and reinforced carbon-based microbial inoculum treatment. Each processing 3 repeated cells, the cell area is 10m ² And are randomly arranged.

Applying 500 kg/mu Xuzhou Hebao commercial organic fertilizer and 90 kg/mu Xinyangfeng 17-17-17 compound fertilizer in conventional treatment, uniformly spreading the fertilizer on the soil surface, fully ploughing and sowing garlic;

500 kg/mu of Xuzhou Hebao organic fertilizer, 90 kg/mu of Xinyanfeng 17-17-17 compound fertilizer and 40 kg/mu of microbial inoculum are applied to the treatment of the enhanced carbon-based microbial inoculum, and the garlic is sowed after the garlic is uniformly scattered on the soil surface and fully ploughed. The results of the soil sampling analysis of the measured yield after harvest in the next year are shown in tables 7 and 8. Fig. 4 is a diagram of two treated garlic bulbs.

Table 7 shows the effect of the microbial inoculum on the yield and incidence of garlic

As can be seen from FIG. 4, the strengthened carbon-based microbial inoculum group has large garlic and higher fresh weight of a single plant.

The results in table 7 show that, compared with the conventional control, the application of the enhanced carbon-based microbial inoculum can significantly increase the garlic yield by 17.7% and reduce the garlic root rot by 51.4%.

TABLE 8 Effect of microbial Agents on Trichoderma Garriculatum population, Bacillus population, AWCD and microbial diversity

The results in Table 8 show that the application of the reinforced carbon-based microbial inoculum can obviously improve the activity and the diversity of microorganisms in soil and improve the biological fertility of the soil. The effective trichoderma and bacillus of the enhanced carbon-based microbial inoculum can stably colonize in garlic planting obstacle soil, and is beneficial to playing roles in preventing diseases and promoting growth.

Example 9 application in greenhouse peach tree soil

Dissolving 20g of peptone, 20g of sucrose and 10g of yeast powder in 150mL of water, adsorbing the peptone on 450g of biochar, airing the biochar until the water content is 10%, and mixing the biochar with 150gTB2 microbial inoculum and 350gBB2 microbial inoculum to prepare the enhanced carbon-based microbial inoculum.

The peach tree soil of Xuzhou city Feng county is selected, the soil is sandy soil and low in biological activity, and the peach age is 1 year.

Set 2 treatments: conventional treatment and reinforced carbon-based microbial inoculum treatment. Each treated 3 replicate cells, 28 peach trees per replicate cell.

Applying 1500 kg/mu of organic fertilizer for the Yingfengjiayuan and 120 kg/mu of compound fertilizer for 15-15-15-15 of Jinzhengda in a conventional treatment manner, uniformly spreading the organic fertilizer and the compound fertilizer on the surfaces of the rows of the peach trees, and then fully ploughing;

1500 kg/mu of organic fertilizer for the full-plump and good orchard, 120 kg/mu of compound fertilizer with 15-15-15 of golden land and 30 kg/mu of microbial inoculum are applied to the strengthening carbon-based microbial inoculum for treatment, and the strengthening carbon-based microbial inoculum is uniformly spread on the surfaces of the rows of the peach trees and then fully ploughed. The yield, tree height and width were measured and analyzed by soil sampling after harvest in the next year, and the results are shown in tables 9 and 10.

TABLE 9 influence of the base inocula on peach yield and breadth

The results in table 9 show that, compared with the conventional control, the application of the enhanced carbon-based fungicide can increase 9.4% of peach yield, 8.7% of peach tree width and 7.9% of peach tree height. The strengthened carbon-based microbial inoculum promotes the growth of peach trees.

TABLE 10 Effect of the inoculants on Trichoderma Takamuranus amounts, Bacillus amounts, AWCD and microbial diversity

The results in Table 10 show that the application of the enhanced carbon-based inoculant can improve the soil microbial activity (AWCD0) and the microbial diversity of peach trees and improve the soil biological fertility. The effective trichoderma and bacillus of the enhanced carbon-based microbial inoculum can stably colonize in the soil of the sand peach trees and is beneficial to playing a role in promoting growth.

The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only illustrative of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An enhanced disease-prevention growth-promoting carbon-based microbial inoculum is characterized by comprising the following raw materials, by mass, 0.5-1% of yeast powder, 1-2% of peptone, 1-2% of sucrose, 15-35% of trichoderma longibrachiatum TB2 microbial inoculum, 15-35% of bacillus amyloliquefaciens BB2 microbial inoculum and the balance of charcoal;

the strain content of the trichoderma longibrachiatum TB2 microbial inoculum is more than 5 multiplied by 10 ⁹ CFU/g；

The bacterial content of the bacillus amyloliquefaciens BB2 microbial inoculum is more than 6 multiplied by 10 ⁹ CFU/g。

2. The preparation method of the enhanced disease prevention growth promoting carbon-based microbial inoculum according to claim 1, which is characterized by comprising the following steps:

a) dissolving yeast powder, peptone and sucrose in water according to a certain proportion, mixing with biochar, adsorbing, and air drying or oven drying at 40-50 deg.C until water content is 5-10% to obtain multi-component carbon-based carrier;

b) and uniformly mixing the multi-component carbon-based carrier with a trichoderma longibrachiatum TB2 microbial inoculum and a bacillus amyloliquefaciens BB2 microbial inoculum according to a ratio to prepare the enhanced disease prevention growth promotion carbon-based microbial inoculum.

3. The method for preparing the enhanced disease prevention and growth promotion charcoal-based bacterial agent according to claim 2, wherein the enhanced disease prevention and growth promotion charcoal-based bacterial agent is prepared by

The biochar is obtained by the following method: crushing a solid product obtained by carbonizing the agricultural production organic waste at the temperature of 400-; the agricultural production organic waste comprises at least one of corn straw, corncob, wheat straw, rice hull and soybean straw.

4. The preparation method of the enhanced disease-prevention growth-promoting carbon-based microbial inoculum according to claim 2, wherein the trichoderma longibrachiatum TB2 microbial inoculum is obtained by the following method:

a) inoculating trichoderma longibrachiatum TB2 into a potato glucose agar culture medium, culturing at 28 ℃ for 3 days, transferring into a potato glucose liquid culture medium, and performing shake culture at 28 ℃ for 3-4 days to obtain a primary culture product of trichoderma longibrachiatum TB 2;

b) inoculating the primary culture of trichoderma longibrachiatum TB2 bacteria into a bran straw culture medium according to the inoculation amount of 10% by mass, and culturing for 5-7 days at 29 +/-1 ℃ to obtain a trichoderma longibrachiatum TB2 bacteria culture product;

c) and (3) air-drying or drying the culture product of the trichoderma longibrachiatum TB2 strain at 30 ℃, and then crushing and sieving by a 100-mesh sieve to obtain the trichoderma longibrachiatum TB2 strain agent.

5. The preparation method of the enhanced disease prevention and growth promotion carbon-based microbial inoculum according to claim 2, wherein the bacillus amyloliquefaciens BB2 microbial inoculum is obtained by the following method:

a) inoculating the bacillus amyloliquefaciens BB2 into a beef extract peptone agar culture medium, culturing for 3 days at 32 ℃, then transferring into a beef extract peptone liquid culture medium, and performing shake culture for 3-5 days at 32 ℃ to obtain a bacillus amyloliquefaciens BB2 primary culture product;

b) inoculating the primary culture product of the bacillus amyloliquefaciens BB2 into a wheat bran and straw culture medium according to the inoculation amount of 10% by mass, and culturing for 3-5 days at the temperature of 32 +/-2 ℃ to obtain a bacillus amyloliquefaciens BB2 culture product;

c) naturally air-drying or drying the culture product of the bacillus amyloliquefaciens BB2 at 40 ℃, and then crushing and sieving by a 100-mesh sieve to obtain the bacillus amyloliquefaciens BB2 bacterial agent.

6. The use of the enhanced disease prevention growth promoting carbon-based fungicide according to claim 1 for disease prevention growth promotion of crops;

the disease prevention comprises at least one of epidemic disease, root rot and clubroot.

7. The application of claim 6, wherein the application of the enhanced disease-preventing growth-promoting fungicide in crop disease prevention and growth promotion is to apply the enhanced disease-preventing growth-promoting fungicide into soil 0-7 days before the crops are transplanted or sowed, and the application amount is 15-40 kg/mu.

8. The use as claimed in claim 6, wherein the crop comprises at least one of pepper, tomato, green vegetables, watermelon, garlic, peach.