CN115786172B

CN115786172B - Bacillus bailii and composition and application thereof

Info

Publication number: CN115786172B
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: 2023-12-19
Anticipated expiration: 2042-09-19
Also published as: CN115786172A

Abstract

The invention discloses bacillus belicus, a composition and application thereof. The bacillus beliae is bacillus beliae YD2022BV strain and is preserved in the microorganism strain collection center of Guangdong province, and the preservation number is GDMCCNo:62736. the bacillus bailii YD2022BV strain disclosed by the invention can promote plant growth and prevent and treat plant diseases caused by bacteria such as fungi, and has very good two effects.

Description

Bacillus bailii and composition and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to bacillus beijerinckii, a composition and application thereof.

Background

Bacillus subtilis (Bacillus velezensis) is a bacterium belonging to the genus Bacillus and is highly related to Bacillus subtilis (Bacillus subtilis). During growth, it produces a series of metabolites that inhibit fungal and bacterial activity, a well known aerobic bacterium, commonly found in soil samples.

In the year 2005, spanish scientist Ruiz Garcia (International Journal of Systematic and Evolutionary Microbiology,2005, 55:191-195) isolated two strains of bacteria capable of producing bio-synthetic lipopeptide substances in salt water of malaplus belay river can produce antibacterial ability and named bacillus belay, but subsequent scientists obtained bacillus belay as subspecies of bacillus amyloliquefaciens through conventional gene 16SrRNA test.

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

Bacillus bailii is a gram-positive, motile, rod-shaped bacterium which usually forms a chain to produce spores, has an optimal growth temperature of 30-40 ℃, below 5 ℃ or above 50 ℃ and no growth, has good antibacterial and fungal infection resisting functions, and is applied to agricultural planting, livestock and aquatic products. Zhang Defeng et al, in the classification of Bacillus belicus, antagonistic function and progress of application study thereof (microbiology report Nov.20,2020,47 (11): 3634-3649), listed that Bacillus belicus has 11 naturally occurring metabolites (6 kinds of lipopeptides cyclopeptides, 3 kinds of polyketides, 1 kind of siderophores, 1 phosphoric acid oligopeptide antibiotics), can effectively inhibit fungal bacteria and nematode bodies, and counted 34 cases of biological control of Bacillus belicus in animals and plants in the middle and outer.

According to the transfer of peasant Dairy newspaper Xinhua net for 2021, 12 months and 10 days, the excellent strain CGMCC No.14384 of the first strain Bacillus bailii in China is registered in the agricultural sector in 2021, 9 months to become the first biological pesticide registered by the Bacillus bailii, thus the novel and commercial value of the biological pesticide are seen.

The bacillus belicus strain disclosed in the prior art is mainly related to inhibiting and preventing and controlling diseases of bacteria, fungi and nematode bodies, but has little research and invention for taking the growth promotion function into consideration, and has high research value in the aspect.

Disclosure of Invention

The invention aims at the problems and provides bacillus beijerinus YD2022BV, and a composition and application thereof.

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

a bacillus beleiensis strain which is bacillus beleiensis YD2022BV strain and is deposited in the Guangdong province microorganism strain collection center, wherein the deposit number is GDMCC No:62736.

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

A nutrient solution for culturing nutrient cells of bacillus clarkii 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 24-36 hours under the conditions that the temperature is 33-38 ℃ and the dissolved oxygen content is more than 90% by using the nutrient solution to obtain the vegetative cells.

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

A composition comprising the strain of bacillus beliensis YD2022BV, wherein the strain of bacillus beliensis YD2022BV is used in combination with one or more other bacteria selected from one or more of bacillus subtilis and bacillus licheniformis.

The composition is formulated as a water-soluble concentrate, dispersible concentrate, emulsion concentrate, emulsifier, suspension, microemulsion, gel, microcapsule, granule, ultra low volume liquid, wet powder, dust-able powder, or seed coating composition.

The bacillus beijerinus YD2022BV strain is applied to prevention and treatment of fungal bacterial plant diseases.

The bacillus beijerinus YD2022BV strain is applied to promoting plant growth.

Preferably, the bacillus beijerinus YD2022BV strain is useful for producing biostimulation in seeds, seedlings and/or plants, including initiating growth of said seeds, seedlings and/or plants.

Preferably, the seed, seedling and/or plant is selected from one or more of Gramineae, brassicaceae, solanaceae, cucurbitaceae, convolvulaceae, compositae, umbelliferae or Leguminosae.

Compared with the prior art, the invention has the following advantages:

the bacillus bailii YD2022BV strain disclosed by the invention can promote plant growth and prevent and treat plant diseases caused by bacteria such as fungi, and has very good two effects.

Drawings

FIG. 1 is a lineage diagram of YD2022BV strain.

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

FIG. 3 is a photomicrograph of YD2022BV vegetative cells in example 3.

Fig. 4 is a photomicrograph of the transformation stage of YD2022BV from vegetative cells to spores (YD 2022BV vegetative cell and spore mix) in example 4.

FIG. 5 is a photograph of spores of YD2022BV cultured in example 4 under a microscope.

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

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

FIG. 8 is a graph showing the effect of YD2022BV strain in example 7 on promoting growth of vegetable of Brassicaceae plant in pot culture and controlling plant diseases and insect pests.

FIG. 9 is a comparative graph of YD2022BV strain in example 7 promoting growth of Brassicaceae green vegetables.

Fig. 10 is a graph showing the effect of YD2022BV on promoting growth of cowpea of legumes in example 8.

FIG. 11 is a graph showing the comparison of the effect of YD2022BV on the promotion of growth of cucurbita moschata of Cucurbitaceae in example 9.

FIG. 12 is a graph showing the growth promoting effect of YD2022BV on the growth promotion of pepper in Solanaceae plants in example 10.

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

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

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

FIG. 16 is a graph showing the comparison of the growth promoting effect of YD2022BV on the plant Ipomoea batatas belonging to Convolvulaceae in example 12.

FIG. 17 is a graph showing the effect of YD2022BV on promoting growth of lettuce in Compositae, and root system in example 13.

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

FIG. 19 is a graph showing the comparison of the length of rice ears of YD2022BV Gramineae plant rice in example 14.

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

Detailed Description

The invention is further described below in connection with the following detailed description.

EXAMPLE 1 isolation of YD2022BV Strain

The soil sample from which the bacterial strain of the present invention was isolated was from Xinyang city of Henan province, china. 10g of a soil sample is taken and diluted with 90 ml of distilled water. After homogenization, serial dilution to 10 ^-5 . 1ml of each serial dilution was smeared onto 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 on the top, re-inoculated on nutrient agar plates and incubated for 24 hours at 37 ℃. Each candidate colony was grown in minimal medium M9 and under aerobic conditions in an orbital shaker (NEW brinswick ^TM 94E25/E25R, germany). The minimum medium M9 described above was free of iron but supplemented with 0.2% glucose.

Colony morphology: colonies showed cream or white color when bacteria grew on nutrient agar, and the shape of the colonies was regular, slightly convex and/or umbilical, with an average size diameter of 2-10 mm, see fig. 2.

Whether siderophores (siderophores) are produced: after 48 hours of incubation, the culture medium was centrifuged at 13,000Xg for 2 minutes. The CAS agar plate was punched to form a well, and then the supernatant (10. Mu.L) collected from the culture was brought into the well. After 8 hours, an orange halo around the punch indicated excretion of siderophores in the supernatant taken. The size of the discoloured halo region of each isolate was measured and recorded and the size of the discoloured halo region of the isolate was about 2-3 mm.

The isolated strain is named as YD2022BV strain, and morphological, physiological and biochemical characteristics of the YD2022BV strain are identified as gram-positive bacillus, vegetative cell thalli are arranged singly, in pairs or in chains, and are oval, mesenchyme or proximal. The colonies were grown on NA (nutrient agar) plates at 30℃for 2 days, and were rough in surface, opaque and uneven in edges. The test results are shown in Table 1, and are preliminarily determined as Bacillus belicus.

Table 1, results of morphological and physiochemical experiments of YD2022BV Strain

Note that: "+" indicates a positive reaction, and "-" indicates a negative reaction.

Example 2 identification and characterization of strains DNA identification and biological genetics

The YD2022BV strain was subjected to strain identification by the microorganism strain collection of Guangdong province at 28 th year of 2022, and was determined to be Bacillus bailii by molecular identification. The strain was deposited at the collection of microorganisms and cell cultures (abbreviated as GDMCC) of Guangdong province at month 8 of 2022, with accession number GDMCC No:62736.

according to the molecular identification result of the Guangdong province microorganism strain collection center, the 16S rRNA gene sequence obtained by sequencing the Bacillus bailii YD2022BV is shown as SEQ ID No: 1. Of these, the specific sequence of 1377bp can be clearly detected, but the specific base types at positions 427, 864 and 897 are not detected due to detection technical problems and the like, and therefore, in SEQ ID No:1 is replaced by "n" which refers to a or g or c or t/u, unknown or other bases. Nucleotide homology was compared with the registered sequence in Genebank using Blast program, and the 16S rRNA gene sequence of the bacterium had homology of 99.93% with Bacillus velezensis (Bacillus bailii) and 99.78% with Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). The gyrB gene sequence obtained by sequencing is 757bp, and the sequence is shown as SEQ ID No: 2. Nucleotide homology was compared with the registered sequence in Genebank using Blast program, and the gyrB gene sequence of the bacterium had 100% homology with Bacillus velezensis (Bacillus bailii).

Adopting MEGA software, constructing a phylogenetic tree (see figure 1) based on a 16S rRNA gene sequence by an adjacent method, repeating calculation 1000 times, displaying a numerical value of which the Bootstrap value is greater than 50% by nodes, and enabling an upper mark 'T' to represent a model strain.

EXAMPLE 3 method for culturing vegetative cells of Strain

To investigate the productivity of vegetative cells of the YD2022BV strain of the invention, it was found that the use of the culture broth of Table 2 was optimal based on multiple experiments performed under ambient conditions using different arrangements and combinations of culture ingredients and additives.

Table 2, YD2022BV Strain nutrient cell nutrient solution

Composition of the components	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. The culture medium, the yeast extract, the peptone and the sodium chloride which are shown in the table 2 are dissolved and fully mixed, the pH value is adjusted to 7.5-8, the culture solution is sealed and put into a sterilizer for sterilization at 121 ℃ for 40 minutes, and the sterilization is carried out after the culture solution is taken out, and the culture solution is put into a sterile workbench for cooling, and is inoculated after the temperature reaches 37 ℃. After the completion, the mixture is put into a constant temperature shaking table for culturing for 24 hours, the temperature is set to be 35-37 ℃, and the primary fermentation is completed.

10L of nutrient solution is prepared according to the formula shown in Table 2 for secondary fermentation. When the culture environment is close to 37 ℃, the pH value is close to 8, the culture time is close to 24 hours, the culture medium generates foam, 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 vegetative cells were in the form of a bar and translucent, and the density of the vegetative cells was 10 ⁹ ～10 ¹¹ CFU/ml liquid.

The vegetative cells can be used as described in Shi Lie 5-6. The YD2022BV nutrient cell fluid can be mixed with starch, dried in an oven at 50-60 ℃ for 24-48 hours, and ground into powder with 40-80 meshes for use.

EXAMPLE 4 cultivation method of bacterial Strain spores

To investigate the spore production capacity of the YD2022BV strain of the invention, based on multiple experiments performed under ambient conditions using different arrangements and combinations of medium components and additives, the preferred method is:

10L of the spore induction culture broth was prepared according to the formulation described in Table 3. The method comprises the steps of dividing the culture medium into 2 barrels, placing the 2 barrels into 2 20L PC distilled water plastic barrels, sealing 5L of each barrel, placing the barrels into a sterilizer, sterilizing at 121 ℃ for 60 minutes, placing the barrels into a sterile working chamber, cooling the barrels to 36-38 ℃, adding 5L of the nutrient cell culture solution obtained by the culture of the example 3 into each barrel, and fermenting and inoculating. After the completion, air filtration and return prevention air lock are carried out, magnetic stirring and air supply are started, and continuous fermentation is carried out for 72 hours in the middle of keeping the temperature of the sterile room at 35-37 ℃. Bubbles and some tiny mud particles are generated during fermentation.

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

Under the conditions of the culture medium shown in Table 3, the effect of inducing YD2022BV strain to generate spores is better, and under the condition of the best effect, the quantity of spores in the obtained liquid exceeds 10 at the alkaline pH value and the temperature of 37 DEG C ⁹ Spores/ml, concentrated spores more than 10 ¹⁴ Spores/ml. Fig. 4-5 show microscopic observations of spores after transformation of vegetative cells into spores.

Table 3, YD2022BV Strain spore induction culture solution

Composition of the components	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 promotion of biostimulation by soybean (leguminous) plants

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

And (3) carrying out surface sterilization on the soybean seeds, and storing the soybean seeds in a refrigerator at the temperature of 4 ℃ for 3-4 days to obtain the 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 YD2022BV strain were applied to seedlings of the experimental group, and the control group was applied with beads not containing YD2022BV strain.

As shown in fig. 6, the soybean seedlings on the left side are a control group, and are shorter, and yellow spots appear on the bud leaves; the soybean seedlings on the right side are used as experimental groups, and are treated by using beads containing YD2022BV strain, so that the soybean seedlings have good growth vigor and do not have any disease phenomenon. It was demonstrated that the YD2022BV strain promoted the growth of soybean seedlings, inducing a significant increase in soybean sprouts (leguminous plants).

Example 6 laboratory tests for biostimulation on rice (Gramineae).

YD2022BV Strain was grown in trypticase Soy broth (supplemented with 18 g of pure agar, oxoidThermo Scientific, lenex, 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 Buffer 0. Cell density was measured and adjusted to od600=1 (-10) ⁹ CFU/ml). After absorbing 500ml of the culture broth into 100g of tapioca starch beads, it was dried at 60℃and 0.1g (containing YD2022 BV) of dry tapioca starch beads was added to the soil and contacted with rice seedling roots. As a control, tapioca starch beads were used in which only medium without YD2022BV strain culture was absorbed.

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

As a result, as shown in FIG. 7, the left 1 rice was used as an experimental group, the left 2 rice was used as a control group to which Bacillus licheniformis (Bacillus licheniformis) strain known to promote rice growth was commercially available, and the left 3 to 5 rice was used as a control group without using the strain at all. The root and plant height of the obviously grown left 1 and left 2 rice are better than those of the left 3-5 rice, the root system and plant height of the left 1 are also higher than those of the left 2, which indicates that YD2022BV has obvious growth promoting effect on gramineous rice and is better than the existing bacillus licheniformis.

EXAMPLE 7 biological stimulation of potted and field test green vegetables (Brassicaceae)

The YD2022BV strain was incubated at 37℃for 3 days (OD 600 = 1), and a starch phospholipid broth of the YD2022BV strain was obtained for use.

The green vegetable (cabbage) seeds are firstly subjected to surface sterilization and stored for 3-4 days in the dark at 4 ℃. Sterile green vegetable seeds were sown on soil-containing foam boxes (90 mm) and in field. A foam box (90 mm) containing soil was placed in the garden. After 7 days, the experimental group was exposed to 10ml of 1/100 diluted starch phospholipid broth of YD2022BV strain, and the control group was exposed to 10ml of 1/100 diluted starch phospholipid broth without YD2022BV strain.

The result of the potting test is shown in fig. 8, and fig. 8A is an experimental group, the vegetables are bigger and the leaves are healthy; fig. 8B is a control group with smaller vegetables and yellow leaves infected with insect and fungal viruses.

The green vegetable field test, using the starch phospholipid broth produced in the same manner and at the same concentration of YD2022BV strain as described above, was used on day 7 after planting of green vegetables, grown to day 14 as shown in FIG. 9A, grown to day 21 as shown in FIG. 9B, and harvested on the last 45 days as shown in FIG. 9C (experimental group) and FIG. 9D (control group). Wherein, the left side green vegetables of fig. 9A and 9B are experimental groups and the right side green vegetables are control groups. The graph shows that the YD2022BV experiment group vegetable is larger, mature in development, healthy and bright in leaves, and has an average weight which is 1.75 times that of the control group, and no obvious insect bite phenomenon and blackening phenomenon of insect bite parts; in the control group, the vegetables are smaller, the development is slower (the vegetable coating is not drawn), the leaves are seriously bitten by insects, and the fungal infection phenomenon caused by the insect bite is lighter in average weight.

Therefore, the green vegetables treated by the YD2022BV strain not only can promote growth, but also can effectively prevent and treat diseases of fungal infection.

Example 8 biostimulation of cowpea (leguminosae)

Into 100g of corn starch500ml of YD2022BV nutrient cell culture broth (concentration 10) prepared as in example 3 was added ¹¹ CFU/ml) was dried at 60 ℃ for 24 hours and then pulverized, 1g (containing YD2022 BV) of the pulverized corn starch was added to 100ml of water mixed, and applied 3 times during the growth phase of cowpea respectively (application time is: 15 days in seedling stage, 30-35 days in vine stage and 45 days in flowering stage) to allow YD2022BV to contact with cowpea root. The control group was administered corn starch without YD2022BV. 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 group.

Example 9 biostimulation of pumpkin (Cucurbitaceae)

To 100g of tapioca starch was added 500ml of YD2022BV nutrient cell broth obtained as described in example 3, which was dried at 60℃for 24 hours and then pulverized, and 10g (containing YD2022 BV) of the pulverized tapioca starch was added to mix 2000ml of water and applied 7 days and 21 days after germination of pumpkin. The control group was administered tapioca starch without YD2022BV. Fig. 11 shows a comparison of the pumpkin planting effect for 32 days, a left 2-action control group and a right 2-action experimental group. The leaves of the experimental group were thicker, and the number of leaves was also more than 1 leaf than that of the control group on average. The YD2022BV pair is proved to promote the growth of pumpkin of Cucurbitaceae.

Example 10 biostimulation of Capsicum plants (Solanaceae)

To 100g of corn starch was added 500ml of YD2022BV nutrient cell broth prepared as described in example 3, dried at 60℃for 24 hours and then pulverized, 5g (containing YD2022 BV) of the pulverized corn starch was added 1000ml of water mixed, and the mixture was applied once every 7 days, 21 days and 30 days after germination of capsicum, by accurately irrigating the roots of plants with 5, 15 and 30 ml of water every two plants using a needle-removed syringe. The control group was administered corn starch without YD2022BV. Fig. 12A is a comparison of the effect of pepper planting just before the flowering phase for about 50 days. The capsicum plants of the YD2022BV experimental group of every two plants are obvious in height, the leaves are luxuriant, and the flowering and fruiting period is far longer than that of the control group. Fig. 12B is a comparison of the first fruit, wherein the upper 5 peppers are the control group, with smaller heads; the lower 5 peppers are the experimental group, big and average in size. The YD2022BV has obvious growth promoting effect on the growth of the peppers in the Solanaceae.

EXAMPLE 11 biostimulation of carrot (Umbelliferae)

To 100g of corn starch was added 500ml of YD2022BV nutrient cell broth prepared as described in example 3, dried at 60℃for 24 hours and then pulverized, 5g of corn starch (containing YD2022 BV) was added to 100ml of room temperature water, 20 g of carrot seeds were soaked for 20 minutes, and the control seeds were soaked in room temperature water without YD2022BV for 20 minutes, and all of the carrot seeds were drained at room temperature for 24 hours. The next day was sown on approximately 150 square meters of cultivated land at both ends, respectively, as YD2022BV seed experimental and control groups. During the test, no fertilizer or pesticide is used in the field, and the drip irrigation belt is used as a test area for more uniform drip irrigation.

The experiments were compared for 20, 35 and 70 days after sowing. Day 20 as shown in fig. 13, a is the control group and B is the experimental group. The seedling density of the control group is sparse and obviously lower; the experimental group is very thick and the plant height is obvious. Day 35 as shown in fig. 14, a is the control group and B is the experimental group. The plant height of the control group is generally 18-21 cm, the plant height of the experimental group is generally close to 29 cm, and the plant height advantage is obvious. Fruit comparison is shown in fig. 15, where 4 roots on the left are harvested in the control group and 8 roots on the right are harvested in the experimental group, which clearly dominates the plant height and root length planes, harvested within the same plot area (square). The carrots are subjected to shape comparison after being cleaned, the carrots of the experimental group are obvious in appearance standard, large in size and free from bursting; whereas the carrot shape of the control group was not standard, because harvesting was irregular shape of carrot in rainy days and internal water pressure was raised, all cracked.

Experiments prove that the seed soaking treatment of YD2022BV can effectively improve the germination rate and the development of seedlings, promote the growth of carrots of Umbelliferae and greatly improve the appearance and bursting quality of carrots.

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

To 100g of corn starch was added 500ml of YD2022BV nutrient cell broth prepared as described in example 3, which was dried at 60℃for 24 hours and then pulverized, and 5g (containing YD2022 BV) of the pulverized corn starch was added to 1000ml of water. The first irrigation was 7 days after the survival of the vine mann of the seed of sweet potato, 10 square areas (2 x5 m) of the seed of sweet potato were irrigated with the above solution, and then the same actions were repeated every 20 days but the weight of the corn starch containing YD2022BV and the water used were doubled for 3 times, namely 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, but without YD2022BV. After 110 days, the sweet potato size was aligned. As shown in fig. 16, the left five are experimental groups, the right three are control groups, the experimental groups are significantly larger than the control groups, and the average weight is more than 2.5 times of that of the control groups. Experiments prove that YD2022BV has obvious growth promoting effect on sweet potatoes in Convolvulaceae.

EXAMPLE 13 biostimulation of lettuce (Compositae)

25ml of YD2022BV vegetative cells produced as in example 3 were used to drip-irrigate directly 10 root parts of 10-day old lettuce, then drenched with water and applied repeatedly at the same dose on day 20. After 35 days of growth, the lettuce root is scooped up with a scoop, and the root is rinsed to observe the development of the root system in the period. As shown in fig. 17, the lower three are experimental groups and the upper three are control groups. The experimental group has obvious root development larger, and the plant height and stem thickness are superior to those of the control group. The YD2022BV is proved to have obvious growth promoting effect on the lettuce of the Compositae.

EXAMPLE 14 biostimulation of Rice (Gramineae)

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

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

Table 4, YD2022BV Strain promoting Rice growth in field

Example 15 prevention of cucumber yellow mosaic

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

500 g of YD2022BV strain spore powder (same as in example 14) and 50L of water were mixed, and after 30 minutes, corn starch separated from the particles by filtration was filtered by a warp cloth to prevent the nozzle from blocking, to obtain YD2022BV spore solution. For cucumber in greenhouse of 600 (15X 40) square meter, the cucumber is separated from the left and right, the right is the experimental group of YD2022BV, the left is the control group, the control group has the same condition as the experimental group, but the applied solution does not contain YD2022BV. After the germination and growing period of the cucumber (before the seedling period is finished), the YD2022BV spore solution is sprayed on the leaf surface for one time by using the pesticide application watering can in a saturation mode until the leaf surface is dripped, and the rest about 20L of YD2022BV solution is sprayed on the rhizome part of the cucumber seedling. After 30 days, the cucumbers enter the initial period, the cucumbers in the control group show obvious yellow mosaic disease as shown in fig. 20A, and the cucumbers in the experimental group show no yellow mosaic disease as shown in fig. 20B. Experiments prove that the YD2022BV and spores and metabolites thereof can effectively prevent bacterial maculopathy, probably because the iron-containing carrier 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 foregoing examples are illustrative of only a few of the many embodiments of this invention, and are thus not to be construed as limiting the scope of this invention, although they are described in detail and in detail. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.

Claims

1. Bacillus bailii strainBacillusVelezensis) Strain YD2022BV, deposited under accession number GDMCC No:62736.

2. a method of culturing the bacillus bailii strain YD2022BV vegetative cells of claim 1, wherein the method comprises the steps of: culturing the bacillus beijerinus strain YD2022BV for 24-36 hours at the temperature of 33-38 ℃ and under the condition that the dissolved oxygen content is more than 90% by using a nutrient solution to obtain vegetative cells, wherein the nutrient solution consists of 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 the balance of water, wherein the pH value is 7.5-8.

3. A method of culturing spores of bacillus beijerinckii strain YD2022BV according to claim 1, characterized in that: inoculating bacillus bailii strain YD2022BV nutrient cell culture solution into spore induction nutrient solution according to 50 v/v% of inoculation amount, and dissolving oxygen content at 30-40 DEG C>Culturing for 48-72 h under the condition of 90% to obtain spores; the spore induction nutrient solution consists of the following components in percentage by weight: 8-10 g/L of yeast extract, 40-g/L of corn steep liquor powder, 5-10 v/v of tomato juice and MgSO ₄ ·7H ₂ O 200mg/L、MnSO ₄ ·4H ₂ O20 mg/L, the balance being water, and the pH value is 7.5-8.5.

4. A composition characterized by: a vegetative cell or spore comprising bacillus belgium strain YD2022BV according to claim 1.

5. Use of bacillus beijerinus strain YD2022BV according to claim 1, for promoting the growth of plants selected from one or more of soybean, rice, green vegetable, cowpea, pumpkin, capsicum, carrot, sweet potato, lettuce, cucumber.

6. Use of bacillus beijerinus strain YD2022BV according to claim 1 for controlling cucumber yellow mosaic disease.