CN115786172A

CN115786172A - Bacillus belgii and composition and application thereof

Info

Publication number: CN115786172A
Application number: CN202211161189.3A
Authority: CN
Inventors: 方英培; 许耀鹏
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-09-19
Filing date: 2022-09-19
Publication date: 2023-03-14
Anticipated expiration: 2042-09-19
Also published as: CN115786172B

Abstract

The invention discloses a Bacillus belgii strain, and a composition and application thereof. The bacillus belgii is a bacillus belgii YD2022BV strain, is preserved in Guangdong province microorganism strain preservation center, and has the preservation number of GDMCCNo:62736. the Bacillus belgii YD2022BV strain can simultaneously promote the growth of plants and prevent and treat plant diseases caused by bacteria such as fungi, and both effects are very good.

Description

Bacillus belgii and composition and application thereof

Technical Field

The invention relates to the field of microorganisms, and particularly relates to a bacillus beleisi strain, and a composition and application thereof.

Background

Bacillus subtilis (Bacillus velezensis) is a bacterium belonging to the genus Bacillus and having a high affinity with Bacillus subtilis. It produces a series of metabolites that inhibit fungal and bacterial activity during growth, a well-known aerobic bacterium, commonly found in soil samples.

In 2005 Spanish scientist Ruiz Garcia (International Journal of Systematic and evolution Microbiology,2005, 55.

The Bacillus belgii is a bacterium widely distributed in the nature, has the characteristics of rapid growth and stability, has the fourth category of biohazard degree according to the national standard of the people's republic of China, the general requirements for laboratory biosafety (GB 19489-2004), and has no pollution to human beings and the environment.

The Bacillus belgii is a gram-positive, motile, rod-shaped bacterium, generally forms a chain, produces spores, has no growth at the optimal growth temperature of 30-40 ℃ and below 5 ℃ or above 50 ℃, has good antibacterial and fungal infection functions, and is applied to agricultural planting, livestock raising and aquatic products. Zhang Defeng in "Classification, antagonistic action and application research progress of Bacillus beilis" (microbiological report Nov.20,2020,47 (11): 3634-3649) lists that Bacillus beilis has 11 naturally occurring metabolites (6 lipoid cyclopeptide dipeptides, 3 polyketides, class 1 siderophores, 1 phosphooligopeptide antibiotics) and can effectively inhibit fungal bacteria and nematode bodies, and counts the outer 34 cases related to the biological control of Bacillus beilis in animals and plants.

According to the transferring of Xinhua net by farmers in the daily newspaper in 2021, 12 months and 10 days, the first Bacillus belgii excellent strain CGMCC NO.14384 in China is registered as the first biological pesticide registered by Bacillus belgii in the agricultural department in 2021 and 9 months, so that the novelty and the commercial value of the biological pesticide are seen.

The Bacillus belgii strains disclosed in the prior domestic technology are mostly about inhibiting and preventing diseases of bacteria, fungi and nematodes, but few researches and inventions considering the growth promoting function have high research value in the aspect.

Disclosure of Invention

Aiming at the problems, the invention provides a Bacillus belgii YD2022BV, a composition and application thereof.

The above object of the present invention is achieved by the following solutions:

the bacillus belgii is a bacillus belgii YD2022BV strain, is preserved in Guangdong province microorganism strain preservation center, and has the preservation number of GDMCC No:62736.

the bacillus beleisi is a vegetative cell, spore and metabolite, and comprises fresh bacteria of YD2022BV strain, frozen bacteria, dried bacteria, freeze-dried bacteria, liquid suspension of bacteria, bacteria encapsulated in spore-encapsulated form, live bacteria, dead bacteria, culture medium, and bacterial extract. The bacterial extract comprises a sterile extract, a supernatant, a bacterial lysate, and a fraction from the bacteria or a metabolite produced by the bacteria.

A nutrient solution for culturing vegetative cells of Bacillus licheniformis YD2022BV strain, which comprises the following components in percentage by weight: 1g/L of beef extract culture medium, 2g/L of yeast extract, 5g/L of peptone, 5g/L of sodium chloride and 7.5-8 of pH value; the YD2022BV strain is cultured for 24 to 36 hours by using the nutrient solution at the temperature of between 33 and 38 ℃ under the condition that the dissolved oxygen content is more than 90 percent to obtain vegetative cells.

A spore induction nutrient solution for culturing Bacillus belgii YD2022BV strain comprises the following components in percentage by weight: 8-10 g/L of yeast extract, 40g/L of corn soaking liquid powder, 5-10% (v/v) of tomato juice and MgSO (MgSO) ₄ ·7H ₂ O200 mg/L，MnSO ₄ ·4H ₂ O20 mg/L, and pH value 7.5-8.5; YD2022BV strain nutritive cell culture solution is inoculated to the spore induction nutritive solution according to the inoculation amount of 50 percent (v/v), and the dissolved oxygen content is between 30 and 40 DEG C>Culturing for 48-72 h under 90% of conditions to obtain spores.

A composition comprises the Bacillus belgii YD2022BV strain, and the Bacillus belgii YD2022BV strain can be used in combination with one or more other bacteria selected from Bacillus subtilis and Bacillus licheniformis.

The compositions are formulated as water-soluble concentrates, dispersible concentrates, emulsion concentrates, emulsifiers, suspensions, microemulsions, gels, microcapsules, granules, ultra-low volume liquids, wet powders, dustable powders, or seed coating compositions.

The Bacillus beleisi YD2022BV strain is applied to prevention and treatment of fungal bacterial plant diseases.

The application of the Bacillus belgii YD2022BV strain in promoting plant growth.

Preferably, the bacillus belgii YD2022BV strain may be used to produce biostimulation in seeds, seedlings and/or plants, including initiating growth of the seeds, seedlings and/or plants.

Preferably, the seeds, seedlings and/or plants are selected from one or more of the families gramineae, cruciferae, solanaceae, cucurbitaceae, convolvulaceae, compositae, umbelliferae or Leguminosae.

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

the Bacillus belgii YD2022BV strain can simultaneously promote the growth of plants and prevent and treat plant diseases caused by bacteria such as fungi, and both effects are very good.

Drawings

FIG. 1 is a pedigree of the YD2022BV strain.

FIG. 2 is a photograph of YD2022BV strain in a petri dish in example 1.

FIG. 3 is a photomicrograph of YD2022BV trophoblasts in example 3.

FIG. 4 is a photomicrograph of the transformation stage of YD2022BV from vegetative cells to spores (YD 2022BV vegetative cells mixed with spores) in example 4.

FIG. 5 is a microscopic photograph of spores cultured at YD2022BV in example 4.

FIG. 6 is a graph showing the effect of YD2022BV strain in promoting the growth of soybean of leguminous plants in example 5.

FIG. 7 is a graph showing the effect of YD2022BV strain in promoting the growth of rice seedlings of gramineous plants in example 6.

FIG. 8 is a comparison of the effect of YD2022BV strain in promoting the growth of crucifer vegetables and controlling pests in pots in example 7.

FIG. 9 is a comparative plot of the YD2022BV strain in example 7 for promoting the growth of Brassicaceae vegetables in the field.

FIG. 10 is a graph comparing the effect of YD2022BV on the growth promotion of cowpea of leguminous plants in example 8.

FIG. 11 is a graph showing a comparison of the effect of YD2022BV on the growth promotion of Cucurbita pepo of the Cucurbitaceae family in example 9.

FIG. 12 is a graph showing the growth promoting effect of YD2022BV on the plant height and growth promoting effect of capsicum annuum of Solanaceae in example 10.

FIG. 13 is a graph showing the effect of YD2022BV on the growth of carrot (20 days) which is an Umbelliferae plant in example 11.

FIG. 14 is a graph showing the effect of YD2022BV on the growth of carrot (35 days) which is an Umbelliferae plant in example 11.

FIG. 15 is a graph showing the comparison between the plant height and root effect of carrot harvested 70 days in example 11.

FIG. 16 is a comparison of the growth promoting effect of YD2022BV on sweet potatoes of the family Convolvulaceae in example 12.

FIG. 17 is a graph comparing the effect of YD2022BV on the growth period promoting plant height and root system of lettuce of Compositae in example 13.

FIG. 18 is a graph showing a comparison of growth promotion of rice of the Gramineae plant by YD2022BV in example 14.

FIG. 19 is a comparison of rice ear lengths of YD2022BV of a gramineous plant in example 14.

FIG. 20 is a graph showing the effect of YD2022BV on cucumber yellow leaf disease prevention and treatment d in example 15.

Detailed Description

The present invention will be further described with reference to the following embodiments.

Example 1 isolation of YD2022BV Strain

The soil sample for separating the bacterial strain is from Xinyang city of Henan province in China. A soil sample of 10g is taken and diluted with 90 ml of distilled water. After homogenization, serial dilution to 10 ^-5 . 1ml of each serial dilution was spread on nutrient agar plates and incubated at 37 ℃ for 24 hours. After the incubation period, at the highest dilution (10) ^-5 ) The most relevant colonies were found and picked, re-plated on nutrient agar plates, and incubated at 37 ℃ for 24 hours. Each candidate colony was grown in minimal medium M9 and aerobically on an orbital shaker (NEW BRUNSWICK) ^TM 94

E25/E25R, germany). The minimum medium M9 mentioned above contains no iron but is supplemented with 0.2% glucose.

Colony morphology: the colonies appeared creamy or white when the bacteria were grown on nutrient agar, the shape of the colonies was regular, slightly convex and/or umbilicus with a size average diameter of 2-10 mm, see fig. 2.

Whether siderophore (siderophore) is produced: after 48 hours of incubation, the culture broth was centrifuged at 13,000 Xg for 2 minutes to pellet the cells. The CAS agar plate was punched to form one well, and then the supernatant (10. Mu.L) collected from the culture was brought into the well. After 8 hours, an orange halo developed around the punch indicated drainage of the siderophore in the supernatant taken. The size of the halo zone of each isolate was measured and recorded, the halo zone of the isolate was approximately 2-3 mm.

The separated strain is named as YD2022BV strain, morphological, physiological and biochemical characteristics identification is carried out on the YD2022BV strain, and the strain is gram-positive bacilli, vegetative cell thalli are arranged in a single, paired or chain shape, spore ellipse, near-midlife or near-end life. When cultured on NA (nutrient agar) plates for 2 days at 30 ℃, the surface of the colonies is rough, the colonies are not transparent, and the edges are irregular. The test results are shown in Table 1 and were preliminarily determined to be Bacillus belgii.

TABLE 1 morphological and physiological and biochemical test results of YD2022BV Strain

Note: a positive reaction is indicated by "+", and a negative reaction is indicated by "-".

Example 2 identification and characterization of strains DNA identification and Biogenetics

YD2022BV strain was assigned to Guangdong province culture Collection for strain identification at 28 days 6 months 2022, and was identified as Bacillus belgii by molecular identification. The strain is preserved in Guangdong province microorganism culture collection (abbreviated as GDMCC) at 24/8/2022, and the preservation number is GDMCC No:62736.

according to the molecular identification result of Guangdong province microbial strain collection center, the 16S rRNA gene sequence obtained by sequencing Bacillus belgii YD2022BV is shown as SEQ ID No:1 is shown. Of these, a specific sequence of 1377bp could be clearly detected, but specific base types at positions 427, 864 and 897 were not detected due to problems in detection technology, etc., and thus, the nucleotide sequence shown in SEQ ID No:1 by "n" means a or g or c or t/u, unknown or other base. By comparing the nucleotide homology with the registered sequence in Genebank using Blast program, the 16S rRNA gene sequence of the strain has 99.93% homology with Bacillus velezensis (Bacillus belief) and 99.78% homology with Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). The sequence of the gyrB gene obtained by sequencing is 757bp, and the sequence is shown as SEQ ID No:2, respectively. Nucleotide homology comparisons with the registered sequences were made in Genebank using the Blast program, and the gyrB gene sequence of this strain had 100% homology with Bacillus velezensis (Bacillus belgii).

A phylogenetic tree (shown in figure 1) is constructed by adopting MEGA software based on a 16S rRNA gene sequence and an adjacency method, the nodes are repeatedly calculated for 1000 times, the Bootstrap value is more than 50% of the value, and the superscript T represents a model strain.

EXAMPLE 3 method for culturing vegetative cells of Strain

To investigate the productivity of the vegetative cells of the YD2022BV strain according to the invention, it was found that the use of the culture broth according to Table 2 works best based on multiple experiments performed under ambient conditions using different permutations and combinations of culture components and additives.

TABLE 2 nutritive cell nutrient solution of YD2022BV strain

Composition (I)	Content or value
		Beef extract culture medium	1g/L
Yeast extract	2g/L
		Peptone	5g/L
Sodium chloride (NaCl)	5g/L
		Temperature (. Degree.C.)	33～38
Dissolved oxygen content	>90％
		Incubation time (hours)	24～36
pH value	7.5～8.5

200ml of nutrient solution was prepared according to the medium formulation shown in Table 2. Completely dissolving and mixing the culture medium, the yeast extract, the peptone and the sodium chloride which are described in the table 2, adjusting the pH value to be 7.5-8, sealing the culture solution, putting the culture solution into a sterilizer at 121 ℃, sterilizing for 40 minutes, taking the culture solution out, putting the culture solution into an aseptic workbench, cooling the culture solution until the temperature reaches 37 ℃, and then inoculating the culture solution. After the fermentation is finished, putting the mixture into a constant temperature shaking table for culturing for 24 hours, setting the temperature to be 35-37 ℃, and finishing primary fermentation.

10L of nutrient solution was prepared according to the formulation shown in Table 2 for secondary fermentation. When the culture environment is close to 37 ℃, the pH value is close to 8, and the culture time is close to 24 hours, the culture medium generates foams, and the cell morphology is observed on a compound microscope (400X). For clarity, the slides may be examined under a phase contrast microscope. As shown in FIG. 3, YD2022BV trophoblasts were strip-shaped and translucent, and the density of the generated trophoblasts was calculated to be 10 ⁹ ～10 ¹¹ CFU/ml liquid.

The vegetative cells can be used as they are as described in the above-mentioned examples 5 to 6. YD2022BV nutritive cell liquid can be mixed with starch and dried in a baking oven at 50-60 ℃ for 24-48 hours and ground into powder of 40-80 meshes for use.

EXAMPLE 4 method for culturing spores of Strain

To investigate the spore productivity of the YD2022BV strains of the invention, based on multiple experiments performed under ambient conditions using different permutations and combinations of media components and additives, the preferred method was:

10L of spore induction culture solution was prepared according to the formulation described in Table 3. Dividing the mixture into 2 barrels, placing the barrels into 2 20L PC distilled water plastic barrels, sealing 5L of each barrel, placing the barrels into a sterilizer, sterilizing the barrels at 121 ℃ for 60 minutes, placing the barrels into a sterile working chamber, cooling the barrels to 36-38 ℃, adding 5L of nutrient cell culture solution obtained by culturing in the embodiment 3 into each barrel, and performing fermentation inoculation. After the completion, the mixture is connected with an air filter and an anti-return air plug, magnetic stirring and air supply are started, and the mixture is continuously fermented for 72 hours in the middle of keeping the temperature of the sterile room at 35-37 ℃. During fermentation, air bubbles and some tiny muddy particles are produced.

Spore counts can be obtained on a multiplex microscope (400X) using a Neubaur cell counting chamber. For clarity, the slides may be examined under a phase contrast microscope. The end-use spores can be harvested by high speed centrifugation, washed with buffer and re-centrifuged to obtain a concentrated volume, then freeze dried in an industrial process.

Under the culture medium conditions shown in Table 3, the effect of inducing the YD2022BV strain to produce spores is better, and when the alkaline pH value and the temperature of 37 ℃ are adopted, the optimal effect condition is realized, and the number of spores in the obtained liquid exceeds 10 ⁹ Spore/ml, and concentrated spore number more than 10 ¹⁴ Spores per ml. FIGS. 4 to 5 show the transformation process of vegetative cells into spores and the microscopic observation results of the spores after the transformation.

TABLE 3 spore induction culture solution of YD2022BV strain

Composition (A)	Content or value
		Yeast extract (g/L)	8～10
Corn steep liquor powder (g/L)	40
		Tomato juice (v/v%)	5～10
MgSO ₄ ·7H ₂ O(mg/L)	200
		MnSO ₄ ·4H ₂ O(mg/L)	20
Inoculum size (v/v%)	50％
		Temperature (. Degree.C.)	30～40
Dissolved oxygen content	>90％
		Incubation time (h)	48～72
pH value	7.5～8.5

Example 5 laboratory test for promoting biostimulation of Soybean (Leguminosae) plants

The YD2022BV strain was incubated at 37 ℃ for 3 days (OD 600= 1) using the culture solution of Table 2 to obtain a culture broth containing vegetative cells of the YD2022BV strain, 500ml of the culture broth was mixed with 100g of zeolite and kneaded to make beads, which were dried at 60 ℃ for future use.

The soybean seeds are subjected to surface sterilization and stored in a refrigerator at 4 ℃ in the dark for 3 to 4 days to obtain sterile soybean seeds. Sterile soybean seeds were sown on PET bottles (90 mm) containing soil. The PET bottles were incubated in a climatic cabinet (21 ℃ C.; 16h/d; 60-70% relative humidity). After 7 days, the above beads containing the YD2022BV strain were applied to the seedlings of the experimental group, and the beads containing no YD2022BV strain were applied to the control group.

As shown in fig. 6, the soybean seedlings on the left side were a control group, and were short, with yellow spots appearing on the bud leaves; the right soybean seedling is an experimental group, and is treated by using beads containing YD2022BV strain, so that the growth vigor is good, and the soybean seedling has no any disease phenomenon. The YD2022BV strain is shown to promote the growth of soybean seedlings and induce the obvious increase of soybean sprouts (leguminous plants).

Example 6 the laboratory tests show the biostimulating effect on rice (gramineae).

YD2022BV strain was grown in tryptone soy broth (supplemented with 18 g of pure agar, oxoid thermo Scientific, lenexa, USA) and the culture conditions of Table 2 at 37 ℃ for 3 days, the cells were plated on agar plates and incubated with 10mM MgSO ₄ ·7H ₂ 0 buffer solution. Cell density was measured and adjusted to OD600=1 (-10) ⁹ CFU/ml). After 500ml of the culture broth was absorbed into 100g of tapioca beads, which were dried at 60 ℃, 0.1g (containing YD2022 BV) of dried tapioca beads were added to the soil and contacted with the rice seedling roots. As a control, tapioca beads were used, in which only the medium without the culture of the YD2022BV strain was absorbed.

The method comprises the following steps of putting seeds in an Eppendorf tube opened in a dryer tank, and sterilizing the surfaces of the seeds of the rice for 3 hours, wherein the method comprises the following specific steps: two beakers were also placed into the desiccator tank, each beaker containing 50ml of sodium hypochlorite solution, and 1.5ml of 37% hydrochloric acid was added to each beaker. The desiccator tank was closed and the seeds were sterilized with chlorine. Eppendorf tubes containing sterile seeds were opened in the flow cabinet for 30 minutes and then placed on a wet paper filter in a petri dish. The petri dishes were sealed and wrapped in tin foil paper and stored at 4 ℃ for 3-4 days. Seeds of rice were soaked in 70% ethanol for 2 minutes, then in 1% sodium hypochlorite solution for 20 minutes, and surface sterilization was performed. After soaking, the seeds were rinsed three times in sterile water. Sterile seeds of rice were sown on PET bottles in a climate cabinet at 21 ℃; the plant level is 180umol/m ² S;16h/d;70% relative humidity.

As shown in FIG. 7, the rice of the left 1 was used as an experimental group, the rice of the left 2 was used as a control group to which a Bacillus licheniformis (Bacillus licheniformis) strain known to promote the growth of rice was purchased on the market, and the rice of the left 3 to 5 was used as a control group to which no strain was used at all. The root and plant height of the left 1 and the left 2 rice are superior to those of the left 3-5 rice, and the root system and plant height of the left 1 are also higher than those of the left 2, which shows that YD2022BV has obvious growth promoting effect on gramineous rice and is superior to the existing bacillus licheniformis.

Example 7 Pot culture test and field test of the biostimulating action of Green vegetables (Brassicaceae)

The YD2022BV strain was incubated at 37 ℃ for 3 days (OD 600= 1) to obtain a starch phospholipid culture broth of the YD2022BV strain for later use.

The seeds of the green vegetables (pakchoi) are firstly sterilized on the surface and stored in the dark at 4 ℃ for 3-4 days. Sterile green vegetable seeds were sown on foam plastic boxes (90 mm) containing soil and in field fields. A foam box (90 mm) filled with soil was placed in the garden. After 7 days, the experimental group exposed the seedlings to 10ml of 1/100 dilution of the amylophospholipid broth of YD2022BV strain, and the control group exposed the seedlings to 10ml of 1/100 dilution of the amylophospholipid broth without YD2022BV strain.

The results of the potting test are shown in fig. 8, fig. 8A is an experimental group with large vegetables and healthy leaves; FIG. 8B shows the control group, which had smaller vegetables and leaves that yellow due to insect pest and fungal virus infection.

The green vegetable field test, using a starch phospholipid culture broth produced in the same manner as above and at the same concentration of YD2022BV strain, was used on day 7 after the green vegetable was planted, grown to day 14 as shown in fig. 9A, grown to day 21 as shown in fig. 9B, and harvested for the last 45 days as shown in fig. 9C (experimental group) and fig. 9D (control group). In fig. 9A and 9B, the left side green vegetables are experimental groups, and the right side green vegetables are control groups. As can be seen from the figure, the vegetable of the YD2022BV experimental group is large, the development is mature, the leaves are healthy and bright, the average weight is 1.75 times of that of the control group, and no obvious insect bite phenomenon or blackening phenomenon of the insect bite part exists; in the control group, the vegetables are small, the development is slow (the vegetable moss is not extracted), the insect bite on the leaves is serious, and the average weight of the fungal infection phenomenon caused by the insect bite is light.

Therefore, the green vegetables treated by the YD2022BV strain can promote the growth and effectively prevent and treat diseases caused by fungal infection.

Example 8 biostimulation of cowpea (Leguminosae)

To 100g of corn starch was added 500ml of YD2022BV nutrient cell culture broth (10 concentration) prepared according to example 3 ¹¹ CFU/ml) was dried at 60 c for 24 hours and then pulverized, 1g (containing YD2022 BV) of the pulverized corn starch was added to mix 100ml of water and applied 3 times respectively at the growing period of cowpea (application time is: seedling stage 15 days, tendril stage 30-35 days and flowering stage 45 days) to allow YD2022BV to contact the cowpea roots. Control group was administered corn starch without YD2022BV. The cowpea growth results are shown in fig. 10, the lower 4 are control groups, the upper 3 are control groups, and the experimental group is obviously longer and heavier than the control groups.

Example 9 biostimulation of pumpkin (Cucurbitaceae)

To 100g of tapioca starch was added 500ml of YD2022BV nutrient cell culture solution obtained as shown in example 3, dried at 60 ℃ for 24 hours and then pulverized, and 10g (containing YD2022 BV) of the pulverized tapioca starch was added to 2000ml of mixed water and applied 7 days and 21 days after germination of pumpkin. The control group was administered tapioca starch without YD2022BV. Fig. 11 is a comparison of the pumpkin planting effect in 32 days, wherein the left 2 is a behavior control group, and the right 2 is a behavior experiment group. The leaves of the experimental group are wider and thicker, and the number of the leaves is more than 1 more than that of the control group on average. The YD2022BV pair was shown to promote the growth of cucurbitaceae pumpkins.

Example 10 biostimulation of Capsicum annuum (Solanaceae) plants

500ml of YD2022BV nutrient cell culture solution prepared by the method shown in example 3 was added to 100g of corn starch, the mixture was dried at 60 ℃ for 24 hours and then pulverized, 5g (containing YD2022 BV) of the pulverized corn starch was added to 1000ml of water and applied once every 7 days, 21 days and 30 days after the pepper germination, and the application method was to irrigate the roots of the plants with 5ml, 15 ml and 30 ml of water accurately every two plants by using a needle cylinder from which needles were removed. Control groups were administered corn starch without YD2022BV. Fig. 12A is a comparison of the effect of pepper planting just before flowering for about 50 days. Every two YD2022BV experimental groups have obvious height of pepper plants, flourishing leaves and longer flowering and fruit setting period than the control group. FIG. 12B is a graph showing a comparison of the first fruit set, wherein the top 5 peppers are control groups and the heads are smaller; the lower 5 peppers were the experimental groups, large and average in size. Proved that YD2022BV has obvious growth promoting effect on the growth of the pepper of the solanaceae.

Example 11 biostimulation of carrot (Umbelliferae)

Adding 500ml of YD2022BV nutrient cell culture solution prepared according to the method shown in example 3 to 100g of corn starch, drying the solution at 60 ℃ for 24 hours and then crushing the solution, adding 5g of corn starch (containing YD2022 BV) to 100ml of mixed room temperature water, soaking 20 g of carrot seeds for 20 minutes, soaking the control group seeds in room temperature water without YD2022BV for 20 minutes, and draining all carrot seeds at room temperature for 24 hours. The two ends of the cultivated land with 150 square meters are respectively sowed on the next day and respectively used as an YD2022BV seed experimental group and a control group. During the test period, no fertilizer or pesticide is used in the field, and drip irrigation is carried out uniformly by taking a drip irrigation tape as a test area.

The test was observed at 20 days, 35 days and 70 days after sowing. Day 20 is shown in fig. 13, where a is the control group and B is the experimental group. The seedlings in the control group are sparse and obviously short; the experimental group is very dense and the plant height is obvious. Day 35 is shown in FIG. 14, where A is the control group and B is the experimental group. The height of the control group is generally 18-21 cm, the height of the experimental group is generally close to 29 cm, and the advantages of the heights are obvious. The fruits are harvested within the same plot area (one square) on the 70 th day, the comparison of the fruits is shown in FIG. 15, 4 on the left side are harvested for a control group, 8 on the right side are harvested for an experimental group, and the experimental group is obviously superior in plant height and root length. The carrots are cleaned and then compared in shape, the carrots in the experimental group are obviously standard in appearance, large in size and free of burst; the shape of the carrots in the control group was not standardized, because the harvesting was irregular shape of the carrots in rainy days and the internal water pressure was increased, all cracked.

Experiments prove that the germination rate and the development of seedlings can be effectively improved by the soaking treatment of YD2022BV on seeds, the growth of carrot in Umbelliferae can be promoted, and the appearance and burst quality of carrot can be greatly improved.

Example 12 biostimulation of sweet Potato/sweet Potato (Convolvulaceae)

To 100g of corn starch was added 500ml of YD2022BV nutrient cell culture solution prepared as described in example 3, dried at 60 ℃ for 24 hours and then pulverized, and 5g (containing YD2022 BV) of the pulverized corn starch was added 1000ml of water mixed. The first irrigation was 7 days after vine survival of the sweet potato seeds, 10 square area of sweet potato seeds (2 x5 m) were irrigated with the above solution, and the same action was repeated every 20 days after that, but the weight of corn starch containing YD2022BV and the amount of water used were doubled, for 3 times, 7 days 5g,27 days 10g,47 days 10g, and 25g in total. The control group was also administered 25g of starch and a total of 5 liters of water simultaneously, but did not contain YD2022BV. After 110 days, the comparison is excavated to the size of the sweet potatoes. As shown in FIG. 16, the left five are experimental groups, the right three are control groups, the sweet potatoes in the experimental groups are obviously larger than the control groups, and the average weight of the sweet potatoes is more than 2.5 times that of the control groups. The experiment proves that YD2022BV has obvious growth promoting effect on sweet potatoes in Convolvulaceae.

Example 13 biostimulation of Asparagus lettuce (Compositae)

Using 25ml of YD2022BV vegetative cells generated according to example 3, 10 asparagus lettuce roots growing for 10 days were directly drip-irrigated and drenched with water, and the same amount was applied once again on day 20. After growing for 35 days, the asparagus lettuce with roots is scooped up by a shovel, and the roots are washed so as to observe the development of the roots. As shown in fig. 17, the lower three are experimental groups and the upper three are control groups. The experimental group is obviously larger in root development and is superior to the control group in plant height and stem thickness. Proved that the use of YD2022BV can lead the asparagus lettuce of the family Compositae to have obvious growth promoting effect.

Example 14 biostimulation of Rice (Gramineae)

The spore powder containing the YD2022BV strain is applied to rice together with nitrogen, phosphorus and potassium fertilizers and organic fertilizers, and sprayed for 1 time and 3 times respectively in a seedling stage, a middle stage and a flowering stage. YD2022BV spore powder takes corn starch as a carrier, accounts for 95 percent, and the rest YD2022BV strain or spores or metabolites thereof is ground into powder. The application method comprises the following steps: after 100kg of nitrogen-phosphorus-potassium fertilizer is sprayed, 1.5kg of YD2022BV spore powder is mixed with 10kg of human and animal excreta and sprayed on soil at the root of the rice. The control group was sprayed with fertilizer without YD2022BV. Control group planting areaIs 2000m ² The planting area of the experimental group is 2000m ² 。

The results are shown in table 4 and fig. 18, in which the left side of fig. 18 is the experimental group, the right side is the control group, the stem of the experimental group is significantly higher than that of the control group, fig. 19A is the experimental group (treated group), the ear length is 30cm, fig. 19B is the control group, the ear length is 21cm, and the yield of the experimental group is increased by 83.3%.

TABLE 4 growth promotion of YD2022BV strains on rice in the field

Example 15 prevention of cucumber yellow leaf disease

Cucumbers are susceptible to yellow leaf disease during the planting process, also commonly referred to as downy mildew, angular leaf spot, brown spot, etc., caused by infection with bacteria, particularly fungi. Yellow leaf diseases are also common in the planting of general melons, fruits, peppers, even bananas and the like.

500 g of YD2022BV strain spore powder (same as example 14) and 50L of water are mixed, and after 30 minutes, corn starch with particles separated by a piece of warp cloth is filtered to prevent a spray head from being blocked, so that YD2022BV spore solution is obtained. For cucumbers in a greenhouse with 600 (15X 40) square meters, the cucumbers are divided into a left experimental group and a right experimental group, wherein the right experimental group is YD2022BV, the left control group is a control group, the conditions of the control group are the same as those of the experimental group, and applied solution does not contain YD2022BV. After the cucumber germination and growth period is about 25-30 days (before the seedling stage is finished), the YD2022BV spore solution is firstly sprayed on the leaf surface once by a pesticide application spraying pot in a saturated way until the leaf surface drops, and the rest 20L YD2022BV solution is sprayed on the rhizome part of the cucumber seedling. After 30 days, the cucumbers enter the initial stage, the cucumbers in the control group have obvious yellow leaf disease symptoms as shown in figure 20A, and the cucumbers in the experimental group have no yellow leaf disease symptoms as shown in figure 20B. Experiments prove that YD2022BV and spores and metabolites thereof can effectively prevent bacterial maculopathy, probably because the iron-containing carriers of the YD2022BV metabolites can greatly absorb iron elements required by the survival of fungi, so that the propagation of the fungi is greatly inhibited.

The above-mentioned embodiments only represent a few of the embodiments of the present invention, and although the description is specific and detailed, the invention should not be construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims

1. The bacillus belgii is a bacillus belgii YD2022BV strain, is preserved in Guangdong province microorganism strain preservation center, and has the preservation number of GDMCC No:62736.

2. the Bacillus belgii of claim 1, wherein the Bacillus belgii is a vegetative cell, spore or metabolite.

3. A nutrient solution for culturing vegetative cells of the bacillus belgii YD2022BV strain of claim 2, wherein: comprises the following components in the following content: 1g/L of beef extract culture medium, 2g/L of yeast extract, 5g/L of peptone, 5g/L of sodium chloride and 7.5-8 of pH value; the YD2022BV strain is cultured for 24 to 36 hours by using the nutrient solution at the temperature of between 33 and 38 ℃ under the condition that the dissolved oxygen content is more than 90 percent to obtain vegetative cells.

4. A spore induction nutrient solution for culturing the bacillus belgii YD2022BV strain of claim 2, wherein: comprises the following components in the following content: 8-10 g/L of yeast extract, 40g/L of corn soaking liquid powder, 5-10 v/v% of tomato juice and MgSO ₄ ·7H ₂ O200mg/L，MnSO ₄ ·4H ₂ O20 mg/L, and pH value 7.5-8.5; YD2022BV strain nutritive cell culture solution is inoculated to the spore induction nutritive solution according to the inoculation amount of 50v/v%, and the dissolved oxygen content is between 30 and 40 DEG C>Culturing for 48-72 h under 90% of conditions to obtain spores.

5. A composition characterized by: comprising the Bacillus belgii YD2022BV strain of claim 1.

6. The composition of claim 5, wherein: also comprises one or more other bacteria selected from one or more of Bacillus subtilis and Bacillus licheniformis.

7. The composition according to any one of claims 5 to 6, characterized in that: the compositions are formulated as water-soluble concentrates, dispersible concentrates, emulsion concentrates, emulsifiers, suspensions, microemulsions, gels, microcapsules, granules, ultra-low volume liquids, wet powders, dustable powders, or seed coating compositions.

8. Use of the bacillus belgii YD2022BV strain of claim 1 to promote plant growth and control fungal bacterial plant diseases.

9. Use according to claim 8, characterized in that: the plant is selected from one or more of Gramineae, brassicaceae, solanaceae, cucurbitaceae, convolvulaceae, compositae, umbelliferae or Leguminosae.