CN114958673B

CN114958673B - Growth-promoting disease-preventing composite microbial agent and application thereof

Info

Publication number: CN114958673B
Application number: CN202210636479.2A
Authority: CN
Inventors: 王丽丽; 金树权; 朱诗君; 汪峰; 韩金庆
Original assignee: Ningbo Academy of Agricultural Sciences
Current assignee: Ningbo Academy of Agricultural Sciences
Priority date: 2022-06-07
Filing date: 2022-06-07
Publication date: 2023-06-02
Anticipated expiration: 2042-06-07
Also published as: CN114958673A

Abstract

The invention belongs to the technical field of microbial agents, and particularly relates to a growth-promoting disease-preventing composite microbial agent and application thereof. The growth-promoting disease-preventing composite microbial agent provided by the invention comprises a bacillus amyloliquefaciens W118 bacterial agent and a trichoderma viride LX-132 bacterial agent; the viable count of the bacillus amyloliquefaciens W118 bacterial agent in the composite microbial bacterial agent is more than or equal to 1.6X10 ¹⁰ CFU/g; the spore number of the Trichoderma viride LX-132 microbial inoculum is more than or equal to 1.0X10 ⁹ CFU/g. According to the invention, the bacillus amyloliquefaciens W118 microbial agent and the trichoderma viride LX-132 microbial agent are compounded to obtain the compound microbial agent, so that the germination rate, seedling length, root length, fresh weight of 20 plants and chlorophyll SPAD value of green vegetables can be improved, the tomato yield can be improved, and the compound microbial agent has a good control effect on tomato bacterial wilt and tomato early blight. Therefore, the composite microbial agent has good application effect.

Description

Growth-promoting disease-preventing composite microbial agent and application thereof

Technical Field

The invention belongs to the technical field of microbial agents, and particularly relates to a growth-promoting disease-preventing composite microbial agent and application thereof.

Background

Along with the adjustment of the agricultural industry structure, the vegetable cultivation area is continuously increased, the vegetable production is in large-scale, specialized and factory production, the vegetable production mode is often accompanied by high intensive planting, the facility vegetable multiple cropping index is high, the vegetable planting variety is relatively single, and the vegetable continuous cropping obstacle is caused by excessively close planting. The microbial agent is a novel fertilizer developed on the basis of microbial technology development in recent years, and is prepared by taking organic matters in the nature as carriers, adding a proper amount of beneficial microorganisms which are beneficial to improving soil structure, promoting plant growth or inhibiting plant pathogenic bacteria, and processing the microbial agent by a special process. Under the influence of biological and abiotic factors, the disease prevention effect of a single biocontrol bacterium is generally unstable under complex rhizosphere and soil conditions, the growth promotion effect is not obvious, two or more microorganisms are needed to be used together for completing the disease prevention, and an ecological environment which is favorable for hosts and unfavorable for disease development is created by means of the composite action of a plurality of microorganisms so as to better realize comprehensive control.

The compound microbial agent is generally a microbial agent which is obtained by mixing 2 or more microorganisms according to a certain proportion for propagation and culture, fully playing the interaction of flora and achieving the best application effect. The microorganisms reported to date as useful in biocontrol are of a wide variety including fungi, bacteria and actinomycetes. Trichoderma is the most widely used biocontrol fungus that can be parasitic to a variety of soil-borne disease pathogens such as Fusarium, anthrax, rhizoctonia to reduce the occurrence of disease. The bacillus can produce stress-resistant dormancy body-spore, can adapt to severe environment, has long survival time and is widely used for preparing biocontrol microbial inoculum. Regarding prevention and control of bacterial wilt, in the prior art (see 'effect of Trichoderma harzianum on tobacco bacterial wilt field control effect and biological property', zhu Hongjiang, botanicals and 20191025), only good prevention and control effect of Trichoderma harzianum on tobacco bacterial wilt is reported, and the bacterial wilt has a growth promoting effect. The effects of controlling plant diseases and promoting plant growth of the bacillus amyloliquefaciens and trichoderma viride composite microbial agents are not reported in the prior art.

Disclosure of Invention

The invention aims to provide a composite microbial agent for promoting growth and preventing diseases, which can improve the germination rate, seedling length, root length, fresh weight of 20 plants and chlorophyll SPAD value of green vegetables, can also improve the tomato yield, and has better control effect on tomato bacterial wilt and tomato early blight.

The invention provides a growth-promoting disease-preventing compound microbial agent, which comprises a bacillus amyloliquefaciens W118 bacterial agent and a trichoderma viride LX-132 bacterial agent; the viable count of the bacillus amyloliquefaciens W118 bacterial agent in the composite microbial bacterial agent is more than or equal to 1.6X10 ¹⁰ CFU/g; the spore number of the Trichoderma viride LX-132 bacterial agent LX-132 in the composite microbial agent is more than or equal to 1.0X10 ⁹ CFU/g。

Preferably, the bacillus amyloliquefaciens bacterial agent is prepared from bacillus amyloliquefaciens W118 with the preservation number of CGMCC No.12200; the trichoderma viride LX-132 microbial inoculum is prepared from trichoderma viride LX-132 with the preservation number of CGMCC No.40149.

Preferably, the mass ratio of the bacillus amyloliquefaciens W118 bacterial agent to the trichoderma viride LX-132 bacterial agent is (4:1) - (9:1).

The invention also provides a preparation method of the composite microbial agent, which comprises the following steps:

inoculating bacillus amyloliquefaciens W118 to a first fermentation culture medium for first fermentation culture to obtain a fermentation broth, and performing spray drying on the first fermentation broth to obtain a bacillus amyloliquefaciens W118 microbial inoculum;

activating Trichoderma viride LX-132, inoculating to a second fermentation medium, performing second fermentation culture to obtain a culture, and naturally air-drying the culture to obtain a Trichoderma viride LX-132 spore microbial inoculum;

and mixing the bacillus amyloliquefaciens W118 bacterial agent and the trichoderma viride LX-132 spore bacterial agent to obtain the composite microbial bacterial agent.

Preferably, the time of the first fermentation culture is 16-20 h, the rotating speed is 160-180 rpm/min, and the temperature is 30-32 ℃;

the illumination condition of the second fermentation culture is 12h illumination and 12h dark treatment until the fermentation is finished; the second fermentation culture time is 6-7 days.

The invention also provides application of the composite microbial agent prepared by the technical scheme or the preparation method in plant growth promotion and/or plant disease control.

Preferably, the plant comprises tomato or green vegetable.

Preferably, the plant disease comprises tomato bacterial wilt or tomato early blight.

Preferably, the application mode is root irrigation.

Preferably, the compound microbial agent is diluted and used, and the dilution factor of the compound microbial agent is 800-1000 times.

The invention has the beneficial effects that: the growth-promoting disease-preventing composite microbial agent provided by the invention comprises a bacillus amyloliquefaciens W118 bacterial agent and a trichoderma viride LX-132 bacterial agent; the viable count of the bacillus amyloliquefaciens W118 bacterial agent in the composite microbial bacterial agent is more than or equal to 1.6X10 ¹⁰ CFU/g; the spore number of the Trichoderma viride LX-132 microbial agent in the composite microbial agent is more than or equal to 1.0X10 ⁹ CFU/g. According to the invention, the bacillus amyloliquefaciens W118 microbial agent and the trichoderma viride LX-132 microbial agent are compounded to obtain the compound microbial agent, so that the germination rate, seedling length, root length, fresh weight of 20 plants and chlorophyll SPAD value of green vegetables can be improved, the tomato yield can be improved, and the compound microbial agent has a good control effect on tomato bacterial wilt. The results of the examples show that: the method has the advantage that the growth promoting effect on green vegetables is best when the mass ratio of the bacillus amyloliquefaciens W118 microbial inoculum to the trichoderma viride LX-132 microbial inoculum is (4:1) - (9:1). In tomato planting, the composite microbial agent provided by the invention is compared with a single microbial agent, namely bacillus amyloliquefaciens W118 microbial agent and trichoderma viride LX-132 microbial inoculum is best in plant height and chlorophyll SPAD value, and the acre yield reaches 6234.4kg, the acre yield of tomatoes with single application of bacillus amyloliquefaciens W118 microbial inoculum reaches 5687.1kg, and the acre yield of tomatoes with single application of trichoderma viride LX-132 microbial inoculum reaches 5214.1kg. The composite microbial agent provided by the invention has a good control effect on tomato bacterial wilt, the maximum control effect reaches 65.7% after the composite microbial agent is applied, the control effect of the bacillus amyloliquefaciens W118 bacterial agent on tomato bacterial wilt reaches 45.5% and the control effect of the trichoderma viride LX-132 bacterial agent on tomato bacterial wilt reaches 28.5%. The composite microbial agent has good control effect on early blight of tomatoes, the maximum control effect reaches 67.7% after the composite microbial agent is applied, and the control effect on early blight of tomatoes is better than that of a single microbial agent, and the control effects of bacillus amyloliquefaciens W118 microbial agent and trichoderma viride LX-132 microbial agent on early blight of tomatoes respectively reach 43.2% and 35.4%. Therefore, the composite microbial agent has good application prospect in growth promotion and disease prevention.

Description of biological preservation

Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), W118, which is preserved in China general microbiological culture collection center (CGMCC) for 3-10 of 2016, with a preservation number of CGMCC No.12200 and a preservation unit address: beijing Chaoyang area North Chenxi Lu No.1, 3.

Trichoderma viride (Trichoderma viride) LX-132 is preserved in China general microbiological culture collection center (CGMCC) for 3 months and 21 days in 2022, and the preservation number is CGMCC No.40149, and the preservation unit address is: beijing Chaoyang area North Chenxi Lu No.1, 3.

Bacillus 15 deposited in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) with a deposit number of CGMCC No.19003 and a deposit address: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a Bacillus amyloliquefaciens W118 bacterial agent prepared in example 1;

FIG. 2 is a Trichoderma viride LX-132 microbial inoculum prepared in example 1;

FIG. 3 is a bacillus 15 inoculant prepared in comparative example 1;

FIG. 4 shows the results of the measurement of the growth promoting effect of green vegetables by the composite microbial agents of example 3 and comparative examples 2 to 8, wherein W118+LX-132 is the result of the measurement of example 3; w118+15 is the measurement result of comparative example 2, and w118+15+lx-132 is the measurement result of comparative example 3; 15+LX-132 is the measurement result of comparative example 4; clear water is the measurement result of comparative example 5; w118 is the measurement result of comparative example 6; 15 is the measurement result of comparative example 7; LX-132 is the measurement result of comparative example 8;

FIG. 5 shows the growth promoting effect of the different compound microorganism dilution factors on green vegetables according to example 5;

FIG. 6 shows the effect of the soil mixing, watering and spraying of example 6.

Detailed Description

The invention provides a growth-promoting disease-preventing compound microbial agent which comprises a bacillus amyloliquefaciens W118 bacterial agent and a trichoderma viride LX-132 bacterial agent; the viable count of the bacillus amyloliquefaciens W118 bacterial agent in the composite microbial bacterial agent is more than or equal to 1.6X10 ¹⁰ CFU/g, preferably ≡1.67X10 ¹⁰ CFU/g; the spore number of the Trichoderma viride LX-132 microbial agent in the composite microbial agent is more than or equal to 1.0X10 ⁹ CFU/g, preferably ≡1.67X10 ⁹ CFU/g。

In the invention, the bacillus amyloliquefaciens W118 bacterial agent is preferably prepared from bacillus amyloliquefaciens W118 with the preservation number of CGMCC No.12200; the trichoderma viride LX-132 microbial inoculum is preferably prepared from trichoderma viride LX-132 with the preservation number of CGMCC No.40149. According to the invention, the bacillus amyloliquefaciens and trichoderma viride are compounded and applied to further improve the germination rate, seedling length, root length, fresh weight of 20 plants and chlorophyll SPAD value of green vegetables, and improve the field control effect of tomato bacterial wilt and tomato early blight.

In the present invention, the mass ratio of the bacillus amyloliquefaciens W118 bacterial agent to the trichoderma viride LX-132 bacterial agent is (4:1) to (9:1), more preferably (5:1) to (8:1), and even more preferably (5:1).

The invention provides a preparation method of a composite microbial agent, which comprises the following steps: inoculating bacillus amyloliquefaciens W118 to a first fermentation culture medium for first fermentation culture to obtain a fermentation broth, and performing spray drying on the first fermentation broth to obtain a bacillus amyloliquefaciens W118 microbial inoculum; activating Trichoderma viride LX-132, inoculating to a second fermentation medium, performing second fermentation culture to obtain a culture, and naturally air-drying the culture to obtain a Trichoderma viride LX-132 spore microbial inoculum; and mixing the bacillus amyloliquefaciens W118 bacterial agent and the trichoderma viride LX-132 spore bacterial agent to obtain the composite microbial bacterial agent.

In the invention, the bacillus amyloliquefaciens W118 is preferably used for preparing bacillus amyloliquefaciens W118 seed liquid before inoculation, the culture medium adopted in the preparation of the bacillus amyloliquefaciens W118 seed liquid is preferably a modified NA liquid culture medium, and the composition of the modified NA liquid culture medium is preferably 10g of glucose, 5g of peptone and 3g of beef extract in every 1000mL of sterile distilled water; the culture conditions for preparing the bacillus amyloliquefaciens W118 seed liquid are preferably 30 ℃ for 24 hours at the rotating speed of 170rpm/min, and the bacillus amyloliquefaciens W118 seed liquid is obtained after the culture is completed. In the invention, the strain concentration of the bacillus amyloliquefaciens W118 seed liquid is preferably more than or equal to 4.0x10 ⁹ CFU/mL。

After the bacillus amyloliquefaciens W118 seed liquid is obtained, the bacillus amyloliquefaciens W118 seed liquid is preferably inoculated into a first fermentation culture medium for first fermentation culture. In the present invention, the seed liquid of Bacillus amyloliquefaciens W118 is inoculated in an amount of preferably 5 to 6% by volume of the first fermentation medium. In the invention, the first fermentation medium takes distilled water as a solvent, and preferably comprises the following components in mass concentration: bean cake powder 15g/L, corn flour 30g/L, yeast extract 33.3g/L,Glucose 2g/L, calcium carbonate 2g/L, dipotassium hydrogen phosphate 0.2g/L, disodium hydrogen phosphate 2g/L, manganese sulfate 0.2g/L and magnesium sulfate 0.2g/L. In the present invention, the pH of the fermentation medium is preferably 7.0 to 7.2, more preferably 7.1 to 7.2, and still more preferably 7.2. The fermentation medium is preferably subjected to high-temperature sterilization treatment, the temperature of the high-temperature sterilization is preferably 121-123 ℃, and the time of the high-temperature sterilization is preferably 0.5h. In the present invention, the time of the first fermentation culture is preferably 16 to 20 hours, more preferably 17 to 19 hours, and still more preferably 17 hours. The first fermentation temperature is preferably 30 to 32 ℃, more preferably 31 to 32 ℃, and still more preferably 32 ℃. The first fermentation speed is preferably 160 to 180rpm/min, more preferably 170 to 180rpm/min, and still more preferably 180rpm/min. In the fermentation process, sampling is started after the fermentation is performed for 12 hours, sampling is performed every 2 hours, colony plate counting and microscopic examination are performed, and fermentation is stopped after 2 times of viable bacteria and spore proportion have no obvious change, so that bacillus amyloliquefaciens W118 fermentation liquor is obtained. The bacillus amyloliquefaciens W118 bacterial agent is obtained after the bacillus amyloliquefaciens W118 fermentation liquid is preferably spray-dried and dried, and the effective viable bacteria content of the bacillus amyloliquefaciens W118 bacterial agent is preferably more than or equal to 2.0x10 ¹⁰ CFU/g。

According to the invention, after the activation culture of the trichoderma viride LX-132, inoculating the trichoderma viride LX-132 to a fermentation culture medium for fermentation culture to obtain a culture, and naturally air-drying the culture to obtain the trichoderma viride LX-132 spore microbial inoculum.

In the present invention, the culture medium for activating Trichoderma viride LX-132 is preferably PDA culture medium, the temperature of the activation culture of Trichoderma viride LX-132 is 28 ℃, and the time of the activation culture of Trichoderma viride LX-132 is 72 hours. After the activation culture, the invention preferably cuts the activated Trichoderma viride LX-132 strain into a lawn and inoculates the lawn to a wheat grain mixture culture medium; the means used for cutting the activated Trichoderma viride LX-132 strain into lawn is preferably a punch. In the present invention, the effect of inoculating Trichoderma viride LX-132 strain to the kernel medium prior to fermentation culture is to allow Trichoderma viride LX-132 to produce more conidia.

In the present invention, the wheat grain mixture medium is preferably packed in an edible fungus bag, and the edible fungus bag is preferably 20×36cm in size. In the invention, the inoculation quantity is that each edible fungus bag is preferably a piece of fungus moss; the edible fungi bag mouth is preferably fastened by a cotton plug after inoculation. In the present invention, the inoculation is preferably followed by a constant temperature culture.

In the invention, the wheat grain mixture culture medium preferably comprises the following components in percentage by mass: 80% of wheat grains, 10% of wheat bran, 9% of rice hulls and 1% of calcium carbonate. In the invention, the preparation method of the wheat grain mixture culture medium preferably comprises the following steps: the wheat grains, the wheat bran and the rice hulls are boiled for 30 minutes after being absorbed with water, so as to obtain a boiling liquid; filtering the obtained cooking liquid to obtain a mixture of wheat grains, wheat bran and rice hulls, spreading out and naturally airing to obtain a cooked mixture, filling the cooked mixture into an edible fungus bag, wherein the mass of the cooked mixture filled into the edible fungus bag is preferably 400g, and preferably adding 1% by mass of calcium carbonate into the edible fungus bag after filling the cooked mixture for high-temperature sterilization; the high-temperature sterilization condition is preferably 121 ℃,30min and cooling for standby.

In the present invention, the temperature of the constant temperature culture is preferably 25 ℃; the constant temperature culture time is preferably 4d; after the constant temperature culture, white mycelium is fully distributed on the surface of the mixture of the wheat grain mixture culture medium in the edible fungus bag, and the wheat grain mixture culture medium is slightly turned over after green spores appear at part of the bag openings. The effect of the gentle tumbling is to allow oxygen to enter, producing the greatest amount of spores. After the overturning, the bag opening of the edible fungus bag is opened, and then the wheat grain mixture culture medium is transferred into a tray for tray fermentation, so that a culture is obtained. The tray is preferably covered with a preservative film, and the number of layers of the preservative film is preferably 1; water drops accumulated on the preservative film are shaken off every 1-2 days; the illumination condition of the tray culture is preferably 12h illumination and 12h dark treatment until the fermentation is finished; the time of the tray fermentation culture is preferably 6-7 d; the tray culture is used for inducing trichoderma spores to generate. The invention adopts light and dark culture treatment of 12h illumination and 12h darkness, which is beneficial to inducing the trichoderma viride LX-132 to generate more spores.

In the invention, the time for naturally drying the culture is preferably 24-48 h, the culture is screened after naturally drying to obtain the trichoderma viride LX-132 microbial inoculum, and the screening is preferably a 200-mesh metal screen.

In the invention, after the bacillus amyloliquefaciens W118 bacterial agent and the trichoderma viride LX-132 bacterial agent are obtained, the bacillus amyloliquefaciens W118 bacterial agent and the trichoderma viride LX-132 bacterial agent are mixed to obtain the composite microbial bacterial agent.

In the present invention, the plant preferably comprises tomato or green vegetable.

In the present invention, the plant disease preferably includes tomato bacterial wilt or tomato early blight.

In the present invention, the mode of application is preferably root irrigation.

In the present invention, the complex microbial agent is preferably diluted and used, and the dilution factor of the complex microbial agent is preferably 800 to 1000 times, more preferably 800 to 900 times, and even more preferably 800 times.

In the invention, the composite microbial agent is applied to a field or a seedling tray.

When the compound microbial agent is applied to a field, the application amount of the diluent of the compound microbial agent is preferably 200mL per plant, and the application time is preferably one or more of the seedling stage, the flowering and fruiting stage, and more preferably three of the seedling stage, the flowering and fruiting stage. In the specific implementation process of the invention, when the compound microbial agent is applied to a field, the dilution multiple of the compound microbial agent is 800 times, the application amount of the diluent of the compound microbial agent is 200mL of each plant, and the application time is three periods of seedling stage, flowering and fruit setting stage and fruiting stage.

When the compound microbial agent is applied to the seedling raising tray, the application amount of the diluent of the compound microbial agent is preferably 1-2L per time calculated by the 32-hole seedling raising tray, the application times are preferably 2 times, the application times are 1 time on the same day as sowing, and the interval time between two adjacent times is preferably 5-7 d, and more preferably 6d. In the specific implementation process of the invention, when the compound microbial agent is applied to a seedling raising tray, 2L of the 800-time diluent of the compound microbial agent is applied each time by using a 32-hole seedling raising tray, the application time is 2 times on the day of sowing, and the interval time of 2 times is 7d.

For further explanation of the present invention, the growth-promoting and disease-preventing composite microbial agent and the application thereof provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

EXAMPLE 1 preparation of Bacillus amyloliquefaciens W118 bacterial agent

(1) Culture medium

Improved NA liquid medium: 10g of glucose, 5g of peptone, 0.5g of yeast powder, 3g of beef extract and 1L of sterile water.

First fermentation medium: 450g of bean cake powder, 900g of corn meal, 100g of yeast extract, 60g of glucose, 60g of calcium carbonate, 6g of dipotassium hydrogen phosphate, 60g of disodium hydrogen phosphate, 6g of manganese sulfate and 6g of magnesium sulfate, and adding distilled water to 30L.

(2) Preparation method of microbial inoculum

Selecting single colony of Bacillus amyloliquefaciens W118, inoculating into modified NA liquid culture medium, and culturing at 30deg.C for 24 hr at 170rpm/min to obtain seed solution of Bacillus amyloliquefaciens W118 with strain concentration of 1.0X10 ⁹ CFU/ml. Inoculating seed liquid of bacillus amyloliquefaciens W118 with the inoculum size of 5% by volume ratio into a fermentation tank, fermenting by using a first fermentation culture medium, wherein the fermentation temperature is 32 ℃, the fermentation speed is 180rpm/min, the fermentation pH is 7.0, sampling is started after fermentation is carried out for 12 hours, sampling is carried out every 2 hours, colony plate counting and microscopic examination are carried out, and when the fermentation is carried out for 17 hours, the fermentation is stopped without obvious change of the viable count and the spore ratio for 2 times. After fermentation, the viable count of the fermentation liquor of the bacillus amyloliquefaciens W118 reaches 4.0x10 ⁹ CFU/ml, the spore amount accounts for 85 percent, and then spray drying is carried out to obtain the bacillus amyloliquefaciens W118 microbial inoculum (effective activity)The bacterial content is 2.0X10 ¹⁰ CFU/g). The microbial inoculum of the bacillus amyloliquefaciens W118 finally prepared is shown in figure 1.

EXAMPLE 2 preparation of Trichoderma viride LX-132 microbial inoculum

(1) Culture medium

PDA medium: 200g of potato, 20g of glucose, 20g of agar and 1000mL of distilled water.

A second fermentation medium, wherein the second fermentation medium is a wheat grain mixture medium: 80% of wheat grains, 10% of wheat bran, 9% of rice hulls and 1% of calcium carbonate (mass ratio).

The preparation method of the wheat grain mixture culture medium comprises the following steps:

(1) Selecting wheat, wheat bran and rice husk without plant diseases and insect pests, and placing the wheat, the wheat bran and the rice husk in a clear water container until the wheat grain mixture is saturated with water;

(2) Taking the mixture in the step (1), and putting the mixture into boiling water to be boiled for 30 minutes;

(3) Filtering out excessive water from the boiled mixture, spreading out, naturally airing the water on the surface, bagging, filling about 400g of the boiled mixture into each edible fungus bag, and adding 1% by mass of calcium carbonate. The cotton plug is tied by the rubber band to seal the edible fungi bag, and the opening of the bag is wrapped by newspaper.

(4) Sterilizing the bagged cooked wheat grain mixture culture medium in a high-pressure steam sterilizing pot at 121deg.C for 30min, and cooling.

(2) Preparation method of microbial inoculum

After activation of Trichoderma viride LX-132 on PDA plates, the cut fungus coating was inoculated into a wheat grain mixture medium in an edible fungus bag using a puncher, and the bag mouth was then fastened with a cotton plug. The strain bags are slightly turned over and then are placed at a constant temperature of 25 ℃ for culture. After 3-4 days of culture, white mycelium begins to be fully distributed on the surface of the wheat grain mixture, and green spores appear at part of the bag openings.

Turning over the wheat mixture culture medium, opening a bag opening, transferring into a tray for tray fermentation, covering a layer of preservative film on the tray, performing 12h illumination and 12h darkness treatment, and inducing trichoderma spores to generate. Water drops accumulated on the preservative film were shaken off every 2 days, and the agglomerated cultures were loosened with a wood plate. Inoculation ofAfter 7 days, spores can be piled up and fully covered on the surface of wheat grain mixture, at this time, the fresh-keeping film is removed, the culture substrate is naturally air-dried for 48h, and the culture substrate is sieved by using 200-mesh metal sieve so as to obtain a large quantity of trichoderma viride LX-132 microbial inoculum (effective viable bacteria content is 1.0X10) ¹⁰ CFU/g). The finally prepared Trichoderma viride LX-132 microbial inoculum is shown in figure 2.

Example 3 Effect of Complex microbial inoculants on growth promoting effects of greengroceries

(1) Test materials: the test crop is green vegetables, and the variety is heat resistant 605; the composite microbial inoculants are bacillus amyloliquefaciens W118 inoculant and trichoderma viride LX-132 inoculant composite, the dilution factor is 800 times (mass ratio), and the test is carried out on the building 11 of Ningbo national institute of agricultural science institute at 9 and 20 days 2021.

(2) The mass ratio of the bacillus amyloliquefaciens W118 microbial agent prepared in the example 1 to the trichoderma viride LX-132 microbial agent prepared in the example 2 is 1:1.

the test method comprises the following steps:

(a) Germination rate test: 1.25g of the compound microbial agent is diluted to 1L by 800 times with clean water, poured into a plastic box filled with 100 green vegetable seeds, stirred until each seed can be contacted with the microbial agent diluent, poured out of the compound liquid after 5 hours, wrapped by 8 layers of gauze, placed into a 25 ℃ incubator for germination acceleration, and counted the germination rate after 24 hours;

(b) Growth test: the untreated green vegetable seeds are sown in 32-hole seedling trays, the root irrigation is carried out 1 time on the sowing day, 800 times of clear water is applied to dilute the compound microbial inoculum, 2L of the compound microbial inoculum is irrigated on each seedling tray, then 1 time is applied every 7 days, the seedling is put into a 25 ℃ incubator, the seedling length of the green vegetable seedlings is measured by a ruler after 10 days, the SPAD value of leaves is measured by a chlorophyll meter (KonicaMinolta), the whole seedling is pulled out, the root length of the green vegetable seedlings is measured by a ruler, and the fresh weight of 20 whole seedlings is measured by a balance.

Example 4-1 Effect of different Mass ratio Complex microbial inoculants on growth promoting effects of greengroceries

The effect of the different mass ratios of the compound microbial agents on the growth promoting effect of green vegetables was further determined on the basis of the results of example 3.

(1) Test materials: the test crop is green vegetables, and the variety is heat resistant 605; the composite microbial agents are bacillus amyloliquefaciens W118 bacterial agent and trichoderma viride LX-132 bacterial agent, and the test is carried out on the building 11 of the national institute of agricultural science of Ningbo city 10 month 8 days 2021.

(2) The mass ratio of the bacillus amyloliquefaciens microbial agent to the trichoderma viride microbial agent prepared in the example 1 to the trichoderma viride microbial agent prepared in the example 2 is 5:1.

(3) The test method was the same as in example 3.

Example 4-2

The process is carried out in the manner of example 4-1, except that the mass ratio of the bacillus amyloliquefaciens microbial agent to the trichoderma viride microbial agent in the composite microbial agent is 0:1.

examples 4 to 3

The method is carried out in the manner of example 4-1, wherein the mass ratio of the bacillus amyloliquefaciens microbial agent to the trichoderma viride microbial agent in the composite microbial agent is 2:1.

examples 4 to 4

The method is carried out in the manner of example 4-1, wherein the mass ratio of the bacillus amyloliquefaciens microbial agent to the trichoderma viride microbial agent in the composite microbial agent is 8:1.

examples 4 to 5

The method is carried out in the manner of example 4-1, wherein the mass ratio of the bacillus amyloliquefaciens microbial agent to the trichoderma viride microbial agent in the composite microbial agent is 1:0.

examples 4 to 6

The procedure of example 4-1 was followed except that only clean water was used without adding the complex microbial agent, and the amount of the clean water applied was the same as the amount of the complex microbial agent applied.

Example 5 Effect of different Mass ratios of Complex microbial inoculants on growth promoting effects of greengroceries

The mass ratio of the bacillus amyloliquefaciens W118 microbial agent to the trichoderma viride LX-132 microbial agent in the composite microbial agent is 3: 1. 4: 1. 5:1. 6: 1. 7: 1. 8:1. 9: 1. 10:1 total 8 treatments, and determining the influence of the compound microbial agents with different proportions on the growth promotion effect of the green vegetables. The rest of the test procedure is the same as in example 3.

Example 6 determination of optimal dilution of Compound microbial agent

Test materials: the test crop is green vegetables, and the variety is heat resistant 605; the composite microbial agent is bacillus amyloliquefaciens W118 bacterial agent and trichoderma viride LX-132 bacterial agent with the mass ratio of 5: the 1 complex was tested on day 28, 10, 2021, at Ningbo city, national institute of agricultural science, laboratory building 11.

Five treatments of 5 dilution factors (100-fold, 300-fold, 500-fold, 800-fold, 1000-fold) of the composite microbial inoculum were set, while a control group to which only clean water was applied was set. And (5) determining the optimal dilution multiple of the compound microbial agent on the growth promotion effect of the green vegetables. The rest of the test procedure is the same as in example 3.

Example 7 determination of mode of application of Compound microbial inoculant

Test materials: the test crop is green vegetables, and the variety is heat resistant 605; the composite microbial agent is bacillus amyloliquefaciens W118 bacterial agent and trichoderma viride LX-132 bacterial agent in a mass ratio of 5:1, and the test is carried out on 11 th day of 2021 in 11 th day of Ningbo city agricultural science institute laboratory building 11.

Test treatment: the test sets 3 different application modes of the compound microbial agent, namely spraying treatment, soil mixing treatment and root irrigation treatment.

The test method comprises the following steps: (1) spray treatment: 2.5g of the compound microbial agent is taken and diluted by 800 times with clean water. Selecting green vegetable seeds with complete appearance, sowing the green vegetable seeds in seedling trays, spraying and diluting the composite microbial agent for 1 time on the day of sowing, spraying 1L of the composite microbial agent on each seedling tray, and then applying 1 time every 7 days, wherein the total spraying time is 2 times;

(2) And (3) mixing soil: 2.5g of composite microbial agent is mixed in 1250g of matrix according to the mass ratio of 2 percent, the mixed matrix is provided with a seedling tray, and the seedling tray is internally sown with complete green vegetable seeds;

(3) Root irrigation treatment: 2.5g of the compound microbial agent is taken and diluted by 800 times with clean water. Selecting green vegetable seeds with complete appearance, sowing 2.5g (800 times of diluent) of the 1 st root-irrigation compound microbial agent in a seedling raising tray, 1L of root irrigation per seedling raising tray, and then applying 1 time every 7 days for 2 times;

the three treated microbial inoculum are 2.5g, the microbial inoculum numbers are consistent, and the microbial inoculum is placed into an illumination incubator until the test is finished after sowing, the incubator is kept at the temperature of 25 ℃ and the humidity of 70% RH, and the illumination intensity is 24000Lux. After 10 days, the seedling length of the green vegetable seedlings was measured with a ruler, the SPAD value of the leaves was measured with a chlorophyll meter (Konica Minolta), the whole seedlings were pulled out, the root length of the green vegetable seedlings was measured with a ruler, and the fresh weight of the 20 whole seedlings was measured with a balance.

Example 8 field application Effect of the composite microbial agent for controlling tomato bacterial wilt and promoting tomato growth

The test crop is tomato and the variety is peach star. The field test was performed at the Ningbo city agricultural science institute, the Xun base.

The test set up 4 treatments in total, respectively:

treatment 1: the composite microbial agent is prepared from bacillus amyloliquefaciens W118 bacterial agent prepared in example 1 and trichoderma viride LX-132 bacterial agent prepared in example 2 in a mass ratio of 5:1, the effective viable count content of the bacillus amyloliquefaciens W118 in the composite microbial agent is 1.67 multiplied by 10 ¹⁰ CFU/g, effective viable count content of Trichoderma viride LX-132 in composite microbial agent is 1.67×10 ⁹ CFU/g。

Treatment 2: bacillus amyloliquefaciens W118 microbial inoculum with effective viable bacteria content of 2.0X10 ¹⁰ CFU/g。

Treatment 3: trichoderma viride LX-132 microbial inoculum with effective viable bacteria content of 1.0X10 ¹⁰ CFU/g。

Treatment 4: and (5) clean water.

The dilution times of the treated microbial inoculum are all 800 times.

Application of Entec compound fertilizer 700 kg/hm to base fertilizer during tomato planting ^-2 And 7000kg hm ^-2 The common organic fertilizer of (2) is respectively applied with a microbial inoculum or clear water after being diluted 800 times according to treatment in the seedling stage, the flowering and fruit setting stage and the fruiting stage of the tomatoes, 200mL of each plant is applied by root irrigation, the black mulching film is uniformly covered 3 days before transplanting, the root setting water is fully irrigated on the same day of transplanting, and then the test requirements are not needed any morePerforming watering operation; the greenhouse ventilation opening is opened in the morning and closed in the evening every day, the air temperature is lower than 10 ℃, the heating device is opened, picking is started for 4 months and 20 days, each treatment is repeated for 3 times, and each cell is 30m ² And (3) arranging random granules, planting 2 rows in each cultivation furrow, and planting double plants.

The plant height and the SPAD value of mature leaves of the tomato plants are investigated in the fruit swelling period (3 months and 20 days), the plant height is measured by a tape, and the chlorophyll measurement is measured by a KonicaMinolta chlorophyll meter; the yield of tomato fruits is counted in the harvest period, and the occurrence of tomato bacterial wilt is counted after the occurrence of tomato plants for 2 times (5 months 15 and 5 months 29 days).

Investigation of tomato bacterial wilt disease: according to the leaf wilting degree of the plants, the disease stage is divided into 5 stages: grade 0, normal plants; stage 1, plant leaf wilting less than or equal to 25%; grade 2, plant leaf wilting of 26 percent or less than 50 percent; grade 3, plant leaf wilting of 51% or more and 75% or less; stage 4, plant leaves wilting or dying at a rate of 76%.

Example 9 field application Effect of composite microbial inoculant for controlling early blight of tomato

Investigation of tomato early blight condition: the disease classification criteria were as follows: level 0: no disease spots; stage 1: the area of the disease spots is less than or equal to 25 percent; 2 stages: the area of the disease spots is more than or equal to 26% and less than or equal to 50%;3 stages: the area of the disease spots is more than or equal to 51% and less than or equal to 75%;4 stages: the area of the disease spots is more than or equal to 76 percent. Counting the occurrence of early blight of tomato after the occurrence of tomato plants for 2 times, wherein the counting time is 5 months, 20 days and 6 months, 4 days. The rest of the procedure is as in example 8.

Comparative example 1 preparation of Bacillus 15 preparation

(1) Culture medium

The formula of the improved NA liquid culture medium comprises the following components: 10g of glucose, 5g of peptone, 0.5g of yeast powder, 3g of beef extract and 1L of sterile water.

The formula of the fermentation medium comprises: 450g of bean cake powder, 300g of corn meal, 300g of soybean meal, 100g of yeast extract, 200g of glucose, 60g of calcium carbonate, 6g of dipotassium hydrogen phosphate, 60g of disodium hydrogen phosphate, 6g of manganese sulfate and 6g of magnesium sulfate, and adding distilled water to 30L.

(2) Preparation method

The strain is bacillus 15 and is preserved in China general microbiological culture collection center (CGMCC) in the 11 th month 25 of 2019, the preservation number is CGMCC No.19003, and the preservation unit address is: beijing Chaoyang area North Chenxi Lu No.1, 3.

Selecting single colony of bacillus 15, inoculating into improved NA liquid culture medium, and obtaining bacillus 15 seed liquid with seed liquid strain concentration of 1.0X10.30 deg.C at 170rpm/min for 24 hr ⁹ CFU/ml。

Regulating pH of fermentation medium to 7.2, transferring seed liquid of bacillus 15 into the fermentation tank at an inoculum size of 5% by volume, fermenting for 12 hr, sampling, counting colony plate and microscopic examination, and stopping fermentation with no obvious change in viable count and spore ratio for 2 times until 18 hr, wherein viable count of bacillus 15 reaches 3.2X10 ⁹ CFU/ml, the quantity of spores accounts for 85 percent, and the fermentation liquor of the bacillus 15 is dried by spray drying to obtain the bacillus 15 microbial inoculum. The final preparation of the bacillus 15 microbial inoculum is shown in figure 3.

Comparative example 2

The bacillus amyloliquefaciens W118 microbial agent prepared in the example 1 and the bacillus 15 microbial agent prepared in the comparative example 1 are used for obtaining a composite microbial agent, and the mass ratio of the bacillus amyloliquefaciens W118 microbial agent to the bacillus 15 microbial agent is 1:1 and the rest of the conditions are the same as in example 3.

Comparative example 3

The bacillus amyloliquefaciens W118 microbial agent prepared in example 1, the trichoderma viride LX-132 microbial agent prepared in example 2 and the bacillus 15 microbial agent prepared in comparative example 1 are applied to obtain a composite microbial agent, wherein the mass ratio of the bacillus amyloliquefaciens W118 microbial agent to the trichoderma viride LX-132 microbial agent to the bacillus 15 microbial agent is 1:1:1 and the rest of the conditions are the same as in example 3.

Comparative example 4

The Trichoderma viride LX-132 microbial agent prepared in the example 2 and the bacillus 15 microbial agent prepared in the comparative example 1 are applied to obtain a composite microbial agent, wherein the mass ratio of the bacillus 15 microbial agent to the Trichoderma viride LX-132 microbial agent in the composite microbial agent is 1:1 and the rest of the conditions are the same as in example 3.

Comparative example 5

The composite microbial agent was not added, and the composite microbial agent was replaced with clear water, and the other conditions were the same as in example 3.

Comparative example 6

Only the Bacillus amyloliquefaciens W118 bacterial agent prepared in example 1 was added, and the other conditions were the same as in example 3.

Comparative example 7

Only the Bacillus 15 strain prepared in comparative example 1 was added, and the other conditions were the same as in example 3.

Comparative example 8

Only the Trichoderma viride LX-132 microbial inoculum prepared in example 2 was added, and the other conditions were the same as in example 3.

Application example 1

The results of the measurement of the growth promotion effect of green vegetables by the composite microbial agents of example 3 and comparative examples 2 to 8 are shown in Table 1 and FIG. 4.

(3) The test results are shown in Table 1.

TABLE 1 Effect of different microbial inoculants on growth promoting effects of greengroceries

As is clear from Table 1, the Bacillus amyloliquefaciens W118 bacterial agent and the Trichoderma viride LX-132 bacterial agent of example 3 had the best growth promoting effect.

Application example 2

The test results of example 4 were measured by the method of example 3 and are shown in Table 2.

TABLE 2 Effect of different mass ratios of Compound microbial inoculants on growth promoting effects of greengroceries

As is clear from the results shown in Table 2, when the mass ratio of the Bacillus amyloliquefaciens W118 bacterial agent to the Trichoderma viride LX-132 bacterial agent is in the range of (5:1) to (8:1), the growth promoting effect of the composite microbial agent on green vegetables is excellent.

Application example 3

The test results of example 5 were determined using the test method of example 3 and are shown in table 3:

TABLE 3 influence of Compound microbial inoculants in different ratios on growth promoting effect of green vegetables

According to Table 3, the composite microbial agent has better growth promoting effect on green vegetables when the mass ratio of the bacillus amyloliquefaciens W118 bacterial agent to the trichoderma viride LX-132 bacterial agent in the composite microbial agent is in the range of (4:1) - (9:1), wherein the composite microbial agent has the best growth promoting effect on green vegetables when the mass ratio of the bacillus amyloliquefaciens W118 bacterial agent to the trichoderma viride LX-132 bacterial agent in the composite microbial agent is 5:1. The mass ratio of the bacillus amyloliquefaciens W118 microbial agent to the trichoderma viride LX-132 microbial agent in the composite microbial agent is 5:1, the effective viable bacteria content of the bacillus amyloliquefaciens W118 bacterial agent in the composite bacterial agent is 1.67 multiplied by 10 ¹⁰ The effective viable bacteria content of the Trichoderma viride LX-132 microbial inoculum in the CFU/g composite microbial inoculum is 1.67 multiplied by 10 ⁹ CFU/g。

Application example 4

The test results of example 6 were measured by the test method of example 3, and the results are shown in Table 4 and FIG. 5.

TABLE 4 growth promoting effect of different Compound microorganism dilution factors on green vegetables

From the results shown in Table 4 and FIG. 5, the compound microbial agent has effects on the germination rate, stem length, root length, fresh weight and chlorophyll SPAD of the green vegetable seeds at different dilution factors, and each index shows best when the dilution factor of the compound microbial agent is 800 times, and then the dilution factor of the compound microbial agent is 1000 times. When the dilution multiple of the compound microbial agent is 500 times, the indexes of the compound microbial agent are not different from those of the clear water contrast. Therefore, the optimal dilution multiple of the compound microbial agent in application is 800-1000 times, and the compound microbial agent is best for promoting the growth of green vegetables.

Application example 5

The seedling length of the green vegetable seedling, SPAD value of the green vegetable leaf, root length of the green vegetable seedling, and fresh weight of 20 whole seedlings of example 7 were measured, and the results are shown in table 5. The effects of the soil mixing, irrigation and spraying of example 7 are shown in FIG. 6.

TABLE 5 growth promoting effect of different application modes on green vegetables

As can be seen from table 5 and fig. 6, different application modes have effects on the germination rate, root length, fresh weight and chlorophyll SPAD of the green vegetable seeds, wherein the composite microbial agent has the best performance in terms of the germination rate, root length, fresh weight and chlorophyll SPAD of the green vegetable when the composite microbial agent is applied by root irrigation, and therefore, the best application mode is root irrigation.

Application example 6

The plant height, chlorophyll SPAD value and acre yield/kg of the example 8 are counted, meanwhile, the condition of the tomato bacterial wilt is investigated, and the condition index and the prevention and treatment effect of the tomato bacterial wilt are calculated;

disease index = Σ (number of disease stages x number of plants of the disease stage)/(number of maximum disease stages x total number of plants) ×100;

control effect (%) = (control disease index-treatment disease index)/control disease index x 100. The results are shown in tables 6 and 7.

TABLE 6 Effect of different microbial inoculants on tomato plant growth promotion

Treatment of	Plant height/cm	Chlorophyll SPAD value	Mu yield/kg
				Process 1	77.2	51.1	6234.4
Process 2	73.2	46.8	5687.1
				Process 3	72.1	47.1	5214.1
Process 4	70.4	46.1	5023.6

As can be seen from Table 6, the compound microbial inoculum is better than the single microbial inoculum in plant height, chlorophyll SPAD value and acre yield, the acre yield reaches 6234.4kg, and the acre yield reaches 5687.1kg.

TABLE 7 control effects of different microbial inoculants on tomato bacterial wilt

According to Table 7, the compound microbial agent has better control effect on tomato bacterial wilt, the maximum control effect reaches 65.7% after the compound microbial agent is applied, and the control effect on tomato bacterial wilt is better than that of a single microbial agent, and the control effects on tomato bacterial wilt by the bacillus amyloliquefaciens W118 microbial agent and the trichoderma viride LX-132 microbial agent reach 45.5% and 28.5% respectively.

Application example 7

Investigation on the condition of the early blight of the tomato is carried out in the embodiment 9, and the disease index and the control effect of the early blight of the tomato are calculated;

disease index (%) =Σ (disease number×plant number of the disease number)/(maximum disease number×total plant number) ×100;

control effect (%) = (control disease index-treatment disease index)/control disease index x 100. The results are shown in Table 8.

Table 8 control effects of different microbial inoculants on early blight of tomato

According to Table 8, the composite microbial agent has good control effect on early blight of tomatoes, the maximum control effect reaches 67.7% after the composite microbial agent is applied, and the control effect on early blight of tomatoes is better than that of a single microbial agent, and the control effects on early blight of tomatoes by the bacillus amyloliquefaciens W118 microbial agent and the trichoderma viride LX-132 microbial agent reach 43.2% and 35.4% respectively.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. The composite microbial agent for promoting growth and preventing diseases is characterized by comprising a bacillus amyloliquefaciens W118 bacterial agent and a trichoderma viride LX-132 bacterial agent; the viable count of the bacillus amyloliquefaciens W118 bacterial agent in the composite microbial bacterial agent is more than or equal to 1.6X10 ¹⁰ CFU/g; the spore number of the Trichoderma viride LX-132 microbial agent in the composite microbial agent is more than or equal to 1.0X10 ⁹ CFU/g；

The preservation number of the bacillus amyloliquefaciens W118 is CGMCC No.12200; the preservation number of the trichoderma viride LX-132 is CGMCC No.40149.

2. The composite microbial agent of claim 1, wherein the mass ratio of the bacillus amyloliquefaciens W118 bacterial agent to the trichoderma viride LX-132 bacterial agent is (4:1) - (9:1).

3. The preparation method of the composite microbial agent as claimed in claim 1 or 2, which is characterized by comprising the following steps:

4. The method according to claim 3, wherein the first fermentation culture is performed for 16-20 hours at a rotation speed of 160-180 rpm and at a temperature of 30-32 ℃;

the illumination condition of the second fermentation culture is 12h illumination 12h dark treatment until the fermentation is finished; the second fermentation culture time is 6-7 days.

5. The application of the compound microbial agent of claim 1 or 2 or the compound microbial agent prepared by the preparation method of claim 3 or 4 in plant growth promotion and/or plant disease control, wherein the plant disease comprises tomato bacterial wilt or tomato early blight.

6. The use according to claim 5, wherein the application is root irrigation.

7. The use according to claim 5, wherein the compound microbial agent is diluted and used, and the dilution factor of the compound microbial agent is 800-1000 times.