CN112029693A

CN112029693A - Bacillus amyloliquefaciens with growth promoting effect and capable of preventing and treating sesame stem blight and fusarium wilt and application thereof

Info

Publication number: CN112029693A
Application number: CN202011056275.9A
Authority: CN
Inventors: 赵辉; 刘新涛; 倪云霞; 刘红彦; 何碧珀
Original assignee: Institute of Plant Protection of Henan Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Henan Academy of Agricultural Sciences
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2020-12-04
Anticipated expiration: 2040-09-30
Also published as: CN112029693B

Abstract

The invention relates to a Bacillus amyloliquefaciens strain with growth promoting effect and capable of preventing and treating sesame stalk blight and fusarium wilt, wherein the Bacillus amyloliquefaciens strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Bam62, is preserved in China center for type culture collection, and has the preservation address: wuhan university in Wuhan, China, the preservation number: CCTCC NO: m2019303, date of deposit: year 2019, month 4, and day 28. The bacillus amyloliquefaciens Bam62 is derived from farmland soil, is safe and environment-friendly, is easy to culture, does not generate drug resistance after being used, does not pollute the environment, has no residue, is environment-friendly and pollution-free, is harmless to people and livestock, and meets the current requirements on environment protection and safety of pesticides.

Description

Bacillus amyloliquefaciens with growth promoting effect and capable of preventing and treating sesame stem blight and fusarium wilt and application thereof

Technical Field

The invention relates to bacillus amyloliquefaciens with a growth promoting effect and capable of preventing and treating sesame stalk blight and fusarium wilt and application thereof, and belongs to the technical field of microorganisms.

Background

Sesame is an important high-quality oil crop in China and an important export commodity, and has great economic value. Because the growing period of sesame is generally between 5 months and 10 months, the whole growing period is in a season of high temperature and raininess, and the period is just the period of plant pathogenic bacteria infection and disease activity, sesame diseases and insect pests are very common, which becomes an important factor harming sesame production, especially the occurrence of soil-borne diseases such as sesame stem blight and blight is serious, and the sesame seed growth is one of the main factors causing low and unstable sesame yield.

Sesame stem blight and wilt often cause the whole plant of sesame in the field to die in pieces, can cause 20-30% of yield reduction, can cause the serious annual yield reduction to be more than 80%, even absolutely, has destructiveness, and has serious influence on the stability and development of industries such as planting, processing, trade and the like of sesame in China. The stem blight and the fusarium wilt have the characteristics of serious harm, gradual aggravation, hidden and difficult diagnosis of early symptoms, complex infection and the like. In the aspect of prevention, the sesame seed oil has the characteristics of high prevention difficulty, poor prevention effect, high prevention cost and the like, so that the prevention and treatment of the stem blight and the fusarium wilt have an important effect on the yield stabilization and yield increase of the sesame.

In addition, in the agricultural production of China, chemical pesticides are used for a long time in a large amount, so that the pollution of the chemical pesticides in China is increasingly serious, certain environmental pollution is caused, the physical health and food safety of people are greatly influenced, and the problem to be solved in the agricultural production at present is solved urgently.

With the application of biological control technology in agriculture, a plurality of beneficial microorganism functional bacteria are also widely applied to the fields of pesticides and fertilizers, and an effective method and a solution way are provided for realizing environment-friendly, pollution-free and efficient agricultural production. At present, fewer biocontrol bacteria are used for simultaneously preventing and controlling the stem blight and the fusarium wilt of the sesame, and fewer biocontrol strains with growth promoting effects are used, so that the development and utilization of the biocontrol bacteria with broad-spectrum prevention effect and growth promoting effects have important theoretical and practical significance for preventing and controlling soil-borne diseases.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the bacillus amyloliquefaciens which has the growth promoting effect and can prevent and treat sesame stem blight and fusarium wilt and the application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a Bacillus amyloliquefaciens strain with growth promoting effect and capable of preventing and treating sesame stalk blight and fusarium wilt is a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Bam62, and is preserved in China center for type culture collection with the preservation address: wuhan university in Wuhan, China, the preservation number: CCTCC NO: m2019303, date of deposit: year 2019, month 4, and day 28.

The bacillus amyloliquefaciens Bam62 is a gram-positive bacterium, and a bacterial colony cultured on an LB culture medium for 48 hours at the temperature of 28 ℃ is milky white, opaque, irregular in edge, and has a bulge, a surface fold and a roughness.

Bacillus amyloliquefaciens Bam62 catalase reaction, anaerobic growth test, gelatin hydrolysis, starch hydrolysis, V-P reaction, M.R reaction, nitrate reduction, urease reaction, D-glucose utilization, D-mannitol utilization and maltose utilization are all positive; tyrosine hydrolysis, phenylalanine deaminase reaction, indole production and citrate utilization are all negative; the growth is not carried out in 7% NaCl, the growth temperature is 25-30 ℃, and the growth pH is 7.

The preparation method of the fermentation liquor of the bacillus amyloliquefaciens Bam62 comprises the following steps:

(1) strain activation and culture: inoculating bacillus amyloliquefaciens Bam62 to an NYDA culture medium, and performing activated culture at 28 ℃ for 24 hours; inoculating the activated bacillus amyloliquefaciens Bam62 into a BPY culture medium, and carrying out shake culture for 24h at the temperature of 28 ℃, the initial pH value of 7.0 and the speed of 180 rpm/min;

(2) preparing a seed solution: inoculating activated and cultured Bacillus amyloliquefaciens Bam62 into a seed tank, and culturing at 28 deg.C, initial pH7.0, speed of 180rpm/min and dissolved oxygen of more than 40% for 24h to prepare seed solution;

(3) and (3) sterilization: sterilizing the pipeline, the air filter, the fermentation tank and the culture medium;

(4) fermentation: after sterilization, inoculating the seed solution into a fermentation medium of a fermentation tank, wherein the inoculation amount of the seed solution is 3% of the volume of the fermentation medium, and fermenting for 48h under the conditions of temperature of 28-32 ℃, initial pH of 7.0, speed of 180rpm/min and dissolved oxygen of more than 70% to obtain the spore with the spore content of 10⁹～10¹⁰CFU/mL fermentation broth.

The fermentation medium is composed of 10g of soluble starch, 3g of ammonium sulfate, 5g of glucose, 0.7g of magnesium sulfate, 0.5g of monopotassium phosphate, 1g of calcium carbonate, 1g of defoaming agent and the balance of distilled water per 1000mL, and is sterilized at 121 ℃ for 30 min.

The preparation method of the bacillus amyloliquefaciens Bam62 wettable powder comprises the following steps:

(1) separation and purification: preparing high-content bacterial pulp by using diatomite, wherein the mass ratio of the diatomite to the fermentation liquor is 1:10, adding the diatomite into the fermentation liquor for three times, adding 1/3 of diatomite each time, and centrifuging for 6 hours at the speed of 4000rpm/min to obtain the bacterial pulp with the spore content of 10¹⁰～10¹¹CFU/g of Bacillus amyloliquefaciens bacterial pulp;

(2) and (3) mixing auxiliary agents: comprises the following components in percentage by mass: 67.5% of bacillus amyloliquefaciens bacterial pulp, 3.5% of a dispersing agent Morwet D-425 naphthalene sulfonate polycondensate, 2% of a wetting agent Morwet EFW and 27% of a mixture of diatomite and kaolin with the mass ratio of 1: 1;

(3) and (3) freeze drying: filling the uniformly mixed auxiliary agent into a tray of a vacuum freeze dryer, and carrying out vacuum freeze drying for 22h to obtain freeze-dried powder;

(4) mixing and crushing: filling the freeze-dried powder into a three-dimensional mixer, and stirring, shearing and uniformly mixing to obtain the product with the spore content of 10⁹～10¹⁰CFU/g bacillus amyloliquefaciens wettable powder.

The bacillus amyloliquefaciens is applied to the aspect of promoting the growth of crops.

The bacillus amyloliquefaciens is applied to the prevention and treatment of sesame stalk blight and fusarium wilt.

The bacillus amyloliquefaciens is applied to promoting the growth of crops and preventing and treating sesame stem blight and fusarium wilt at the same time.

The invention has the beneficial effects that:

through a pot experiment of oppositional screening, disease prevention and growth promotion, the bacillus amyloliquefaciens strain Bam62 which can simultaneously prevent and treat sesame stem blight and has growth promotion effects on sesame, wheat and corn is obtained.

The bacillus amyloliquefaciens Bam62 is derived from farmland soil, is safe and environment-friendly, is easy to culture, does not generate drug resistance after being used, does not pollute the environment, has no residue, is environment-friendly and pollution-free, is harmless to people and livestock, and meets the current environment-friendly and safety requirements of people on pesticides.

The prevention effect of the strain Bam62 on sesame stem blight and fusarium wilt is close to that of a chemical carbendazim, and the occurrence of stem blight and fusarium wilt can be reduced.

Drawings

FIG. 1 shows the results of the antagonism of the strain Bam62 against Septoria phaseoloides, Fusarium oxysporum and Fusarium graminearum;

FIG. 2 shows the colony morphology of strain Bam 62;

FIG. 3 shows the growth promoting effect of strain Bam62 on sesame, wheat and corn for 5 days;

FIG. 4 shows the growth promoting effect of strain Bam62 on sesame, wheat and corn for 20 days.

Detailed Description

The following examples further illustrate the embodiments of the present invention in detail.

The experimental materials used in the invention:

NYDA culture medium: 7g of beef extract, 7g of peptone, 10g of glucose and 20g of agar, adding distilled water to a constant volume of 1L, mixing uniformly, subpackaging in triangular bottles, and sterilizing at 121 ℃ for 30 min.

LB culture medium: 10g of peptone, 5g of yeast extract, 10g of sodium chloride and 20g of agar, adding distilled water to a constant volume of 1L, mixing uniformly, subpackaging in triangular bottles, and sterilizing at 121 ℃ for 30 min.

BPY medium: 5g of beef extract, 10g of peptone, 10g of glucose, 5g of yeast extract and 5g of sodium chloride, adding distilled water to a constant volume of 1L, mixing uniformly, subpackaging in triangular bottles, and sterilizing at 121 ℃ for 30 min.

PDA culture medium: 200g of potatoes (cut into small blocks with the side length of about 1cm, put into 800mL of distilled water to be boiled for 15min, filtered by double-layer gauze and then retained in filtrate), 20g of glucose and 20g of agar, adding distilled water to fix the volume to 1000mL, and sterilizing at 121 ℃ for 30 min.

The pathogenic bacteria, other raw materials and reagents used by the invention are all purchased from the market.

Example 1 isolation and screening of the Strain Bam62

The strain Bam62 is used for separating soil, collecting soil from the Conyza sativa Linn sesame field in Yuanyang county of New county, Henan province, weighing 1g of soil, adding 10mL of sterile water, fully oscillating to obtain soil suspension, shaking the soil suspension uniformly, and diluting by 10% to obtain 10% of soil suspension²、10³Or 10⁴Taking 0.1mL of diluent, coating the diluent on an NYDA culture medium, carrying out inverted culture in a constant-temperature incubator at 28 ℃ until bacterial colonies grow out, picking bacterial colonies with different sizes, colors and forms within 72 hours, and carrying out streak purification culture on the surface of a KB solid culture medium; then inoculating the purified strain into a BPY culture medium, carrying out shake culture for 24h at the temperature of 28 ℃, the initial pH value of 7.0 and the speed of 180rpm/min to obtain a fermentation liquid of the strain, carrying out bacteriostatic activity determination, and screening biocontrol bacteria.

Oxford cup method: inoculating target pathogenic bacteria (i.e., fusarium phaseoloides, fusarium oxysporum and fusarium graminearum) on a PDA culture medium, culturing the fusarium oxysporum and the fusarium graminearum at 25 ℃ and 28 ℃ for 5-10 days, and activating for later use. The method comprises the steps of beating pathogenic bacteria colonies into circular mycelium blocks with the diameter of 5mm by using a sterilized puncher, respectively inoculating (placing) target pathogenic bacteria blocks and an Oxford cup on a PDA (personal digital assistant) flat plate, then placing fermentation liquor of strains in the Oxford cup, enabling the distance between the pathogenic bacteria blocks and antagonistic strains to be 50mm, and simultaneously setting independently inoculated pathogenic bacteria blocks as a control. After the treatment is repeated for 4 times, the result of the confrontation culture is checked after 5 to 7 days according to the growth rate of the target bacteria control, and the result of the bacteriostasis effect of the strain Bam62 is shown in figure 1.

A toxic medium containing method: inoculating target pathogenic bacteria (i.e., fusarium phaseoloides, fusarium oxysporum and fusarium graminearum) on a PDA culture medium, culturing the fusarium oxysporum and the fusarium graminearum at 25 ℃ and 28 ℃ for 5-10 days, and activating for later use. Then mixing the fermentation liquor of the strain filtered by the microporous filter membrane of the bacterial filter (for 2 times) into the cooled PDA culture medium, and shaking up; 1mL of strain fermentation broth filtrate was mixed per 100mL of PDA medium. Then, a sterilized puncher is used for punching pathogenic bacteria colonies into circular mycelium blocks with the diameter of 5mm, the target pathogenic bacteria blocks are connected to the center of a PDA (personal digital assistant) flat plate, and meanwhile, the pathogenic bacteria blocks which are independently inoculated are set as a control. Each treatment was repeated 4 times, and the results of the confrontation culture were examined after 5 to 7 days based on the growth rate of the target bacteria control, and are shown in FIG. 1.

Results of screening biocontrol bacteria by an Oxford cup method and a toxic medium-containing method show that the bacterial strain Bam62 has the highest bacteriostatic rate on the sphaerulina phaseoloides and the fusarium oxysporum, and the bacteriostatic results of the bacterial strain Bam62 are shown in table 1.

TABLE 1 results of bacteriostasis of antagonistic strain Bam62

Example 2 identification of the Strain Bam62

(1) Strain morphology and physiological and biochemical:

the bacterial strain Bam62 is a gram-positive bacterium, and the bacterial colony cultured on an LB culture medium at 28 ℃ for 48 hours is milky white, opaque, irregular in edge, bulged, wrinkled and rough in surface (as shown in figure 2).

The strain Bam62 catalase reaction, the anaerobic growth test, the gelatin hydrolysis, the starch hydrolysis, the V-P reaction, the M.R reaction, the nitrate reduction, the urease reaction, the D-glucose utilization, the D-mannitol utilization and the maltose utilization are all positive; tyrosine hydrolysis, phenylalanine deaminase reaction, indole production and citrate utilization are all negative; the growth is not carried out in 7% NaCl, the growth temperature is 25-30 ℃, and the growth pH is 7.

(2)16S rDNA sequence analysis:

the strain Bam62 is sequenced, the 16S rDNA gene sequencing result is shown in a sequence table SEQ ID NO.1, and the identification result is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).

Example 3 preparation of fermentation broth of Bacillus amyloliquefaciens Bam62

(3) and (3) sterilization: sterilizing the pipeline, the air filter, the fermentation tank and the culture medium according to the production process standard;

(4) fermentation: after sterilization, inoculating the seed solution into a fermentation medium of a fermentation tank, wherein the inoculation amount of the seed solution is 3% of the volume of the fermentation medium, and fermenting for 48h under the conditions that the temperature is 28-32 ℃, the initial pH value is 7.0, the speed is 180rpm/min, and the dissolved oxygen is more than 70%; sampling every 12h for 1 time, measuring pH value, observing the existence of mixed bacteria, and finally obtaining the concentration (spore content) of 10⁹～10¹⁰CFU/mL fermentation broth.

Wherein the fermentation medium, per 1000mL, is composed of 10g of soluble starch, 3g of ammonium sulfate, 5g of glucose, 0.7g of magnesium sulfate, 0.5g of monopotassium phosphate, 1g of calcium carbonate, 1g of antifoaming agent and the balance of distilled water, and is sterilized at 121 ℃ for 30 min.

Example 4 preparation method of wettable powder of Bacillus amyloliquefaciens Bam62

The preparation process of wettable powder includes high speed centrifugation of fermented liquid to eliminate water, stirring thallus with dispersant, wetting agent and carrier, vacuum drying, mixing and crushing.

(1) Separation and purification: preparing high-content bacteria slurry with diatomaceous earth (adsorbent) at a mass ratio of 1:10, adding diatomaceous earth into the fermentation broth three times, each time adding 1/3 diatomaceous earth, centrifuging at 4000rpm/min for 6 hr by disc centrifuge to obtain spore with spore content of 10¹⁰～10¹¹CFU/g of Bacillus amyloliquefaciens mash.

(2) And (3) mixing auxiliary agents: comprises the following components in percentage by mass: the spore content is 10¹⁰～10¹¹67.5 percent of CFU/g of bacillus amyloliquefaciens bacterial pulp, 3.5 percent of dispersant Morwet D-425 naphthalene sulfonate polycondensate, 2 percent of wetting agent Morwet EFW and 27 percent of mixture (carrier) of diatomite and kaolin;

wherein the wetting agent Morwet EFW is a mixture of alkyl naphthalene sulfonate and an anionic wetting agent; the mass ratio of the diatomite to the kaolin is 1: 1.

(3) And (3) freeze drying: and (3) filling the uniformly mixed auxiliary agent into a tray of a vacuum freeze dryer, wherein the thickness of the tray is 1cm, and carrying out vacuum freeze drying for 22h to obtain freeze-dried powder.

(4) Mixing and crushing: putting the freeze-dried powder into a three-dimensional mixer, stirring, shearing and uniformly mixing to obtain the product with the spore content of 10⁹～ 10¹⁰CFU/g bacillus amyloliquefaciens wettable powder.

Example 5 disease prevention test of Bacillus amyloliquefaciens Bam62

Planting sesame in flowerpots filled with sterile soil, planting 1 plant in each flowerpot, and selecting sesame with consistent growth vigor to perform a disease prevention test of biocontrol bacteria Bam62 when the leaf period is 6-8. The test treatment uses Phaseolus vulgaris (pathogenic bacteria of sesame stalk blight) and Fusarium oxysporum (pathogenic bacteria of sesame wilt) as target pathogens, and the inoculation concentration (spore content) of the target pathogens is 1.2 × 10⁷CFU/mL, the inoculum size of each strain was 1 mL.

The viable bacteria concentration in the fermentation liquor of the bacillus amyloliquefaciens Bam62Degree (spore content) of 2.37X 10¹⁰CFU/mL, when using, diluting the fermentation liquid with sterile water 100 times to make the viable bacteria concentration of the dilution liquid 2.37 × 10⁸CFU/mL; the viable bacteria concentration of the bacillus amyloliquefaciens Bam62 wettable powder is 2.62 multiplied by 10¹⁰CFU/mL, when using, diluting the fermentation liquid with sterile water 100 times to make the viable bacteria concentration of the dilution liquid 2.62X 10⁸CFU/mL; the control drug was 10¹⁰CFU/g Bacillus subtilis wettable powder (commercially available) and 50% carbendazim wettable powder (commercially available), 10%¹⁰The bacillus subtilis wettable powder is diluted by 100 times by using sterile water, and the 50 percent carbendazim wettable powder is used according to 100mg/kg of effective components; clear water treatment was used as control.

Each liquid medicine is used for treating 10 plants, the liquid medicine is sprayed around roots of the plants in the seedling stage, the amount of the liquid medicine treated by each plant is 10mL, the liquid medicine is sprayed for 1 time every 7 days and is sprayed for 3 times totally, the disease condition is investigated after the inoculation of pathogenic bacteria for 20 days, and the disease index and the prevention and treatment effect are calculated. The test is divided into the steps of immediately inoculating pathogenic bacteria after treating sesame seedlings by using a medicament and inoculating the pathogenic bacteria after treating the sesame seedlings for 7 days by using the medicament.

The results of pot culture experiments for preventing sesame stem blight showed that (as shown in Table 2), sesame seedlings were respectively treated with Bam62 fermentation broth, Bam62 wettable powder, and 10¹⁰The bacterial pathogens are inoculated immediately after the CFU/g bacillus subtilis wettable powder and the 50% carbendazim wettable powder are subjected to root irrigation treatment, the control effects on the sesame stem blight are 56.22%, 66.65%, 36.42% and 55.25% respectively, the control effect of the Bam62 wettable powder is slightly higher than that of the carbendazim treatment, and the control effect of the Bam62 fermentation liquor treatment and the control effect of the carbendazim treatment are not obviously different. And the medicament treatment is carried out on the sesame seedlings for 7 days, then the pathogenic bacteria treatment is carried out, the control effects of the medicament treatment on sesame stem blight are respectively 74.24%, 75.60%, 50.51% and 47.44%, and the control effects of the Bam62 fermentation liquor and Bam62 wettable powder are obviously higher than that of 50% carbendazim wettable powder treatment. After the sesame seedlings are irrigated with the Bam62 fermentation liquor for 7 days, the pathogenic bacteria are inoculated, the biocontrol effect is obviously improved, which is related to the colonization time of the bacillus amyloliquefaciens, and the application potential of the bacillus amyloliquefaciens Bam62 in the aspect of preventing and treating sesame stalk blight is better.

TABLE 2 prevention and control of sesame stalk blight by the strain Bam62

Note: the lower case in the table indicates that the difference reaches a significant level of 0.05, as shown in the table below.

The results of pot culture experiments for preventing sesame wilt showed that (as shown in Table 3), sesame seedlings were separately treated with Bam62 fermentation broth, Bam62 wettable powder and 10¹⁰After the CFU/g bacillus subtilis wettable powder and the 50% carbendazim wettable powder are irrigated to roots, pathogenic bacteria are immediately inoculated, the control effects on sesame wilt are respectively 41.83%, 39.44%, 36.03% and 48.65%, and the control effects of the Bam62 fermentation liquor and the Bam62 wettable powder are lower than those of the carbendazim treatment, but the control effects are 10% of the Bam62 fermentation liquor and the Bam62 wettable powder¹⁰The difference of the CFU/g bacillus subtilis wettable powder treatment control effect is not obvious. After the sesame seedlings are treated by the agents for 7 days, the sesame seedlings are inoculated with pathogenic bacteria for treatment, the control effects of the treatment of the agents on sesame wilt are respectively 54.45%, 56.67%, 55.56% and 51.11%, and the control effects of Bam62 fermentation liquor are respectively 10% and 54.45%¹⁰The control effect difference of CFU/g bacillus subtilis wettable powder and 50% carbendazim wettable powder treatment is not obvious, the control effect of Bam62 fermentation liquor and Bam62 wettable powder is obviously higher than that of carbendazim treatment, and the biocontrol bacterium Bam62 has better application potential in the aspect of preventing and treating sesame wilt.

TABLE 3 preventive and therapeutic effects of the strain Bam62 on sesame wilt

Example 5 potted plant growth promotion test

Treating sesame, wheat and corn seeds with different concentrations of Bam62 fermentation liquor with a stock solution concentration of 3.22 × 10¹⁰CFU/mL, diluting the fermentation broth respectively 50 times (50X), 100 times (100X), 500 times (500X) and 1000 times (1000X) in the test treatment, planting the treated plant seeds in a flowerpot by taking sterile water treatment as a control, and performing the growth promotion test of the growth of plant seedlingsEach treatment was repeated 3 times, and after 5 days, emergence rate, root length and plant height were observed, and after 20 days, plant height, root length, fresh weight and dry weight were measured. See fig. 3-4.

The Bam62 fermentation liquor has a promoting effect on the growth of sesame (as shown in Table 4), and the diluted Bam62 fermentation liquor has no influence on the emergence of sesame seedlings; the strain height and root growth of sesame can be remarkably promoted within 5 days by diluting 50 x-100 x with Bam62 fermentation liquor, and the strain height and root growth of sesame can be remarkably promoted within 20 days by diluting 50 x-500 x with Bam62 fermentation liquor; the fresh weight and the dry weight of the plant can be obviously increased by diluting 50 x-500 x of Bam62 fermentation liquor.

TABLE 4 influence of Bam62 fermentation broth on sesame growth

Treatment of

5d rate of emergence/%)

5d plant height/cm

5d root length/cm

20d plant height/cm

20d root length/cm

Fresh weight/g of 20 days

20d dry weight/g

Dilution

50X

100a

2.36a

6.81a

16.43a

6.63a

9.92a

0.32a

Dilution 100X

100a

2.32a

6.51ab

15.16b

6.16a

8.35ab

0.29ab

Dilution

500X

99.25a

2.04b

5.8bc

13.62c

5.26b

6.73b

0.25bc

Dilution

1000X

98a

1.95b

5.5c

11.21d

4.44c

5.99cd

0.22cd

Control

98.25a

1.9b

5.27c

11.01d

4.63c

4.93d

0.18d

The Bam62 fermentation liquor has a promoting effect on the growth of wheat (as shown in Table 5), and the Bam62 fermentation liquor can promote the emergence of wheat seedlings; the strain height and root length growth of wheat can be remarkably promoted in 5 days by diluting the fermentation liquor Bam62 by 500 x-1000 x, and the strain height growth of wheat can be remarkably promoted in 20 days by diluting the fermentation liquor Bam62 by 50 x-1000 x; the fresh weight and the dry weight of the plants can be obviously increased by diluting 100 x-500 x.

TABLE 5 influence of Bam62 fermentation broth on wheat growth

Treatment of

5d rate of emergence/%)

5d plant height/cm

5d root length/cm

20d plant height/cm

20d root length/cm

Fresh weight/g of 20 days

20d dry weight/g

Dilution

50X

100a

6.39c

3.53c

33.86c

11.32a

5.8b

0.39c

Dilution

100X

97.5ab

7.5b

3.82c

37.21ab

11.44a

6.79a

0.48ab

Dilution

500X

100a

10.77a

6.7a

38.16a

12.35a

6.89a

0.51a

Dilution 1000X

97.5ab

10.39a

6.34a

35.43bc

12.12a

6.2b

0.43bc

Control

92.5b

7.62b

4.65b

29.51d

11.4a

6.2b

0.42c

The Bam62 fermentation liquor has a promoting effect on the growth of the corn (as shown in Table 6), and the diluted Bam62 fermentation liquor has no influence on the emergence of the corn; the Bam62 fermentation liquor can remarkably promote the height of a corn plant, and the corn root growth can be remarkably promoted by diluting 100 x-1000 x; the Bam62 fermentation liquor can obviously increase the fresh weight and dry weight of plants.

TABLE 6 influence of Bam62 fermentation broth on corn growth

Treatment of

5d rate of emergence/%)

5d plant height/cm

5d root length/cm

20d plant height/cm

20d root length/cm

Fresh weight/g of 20 days

20d dry weight/g

Dilution

50X

99.75a

6.23b

7.66c

51.03a

21.1bc

39.07b

2.78b

Dilution

100X

99.75a

6.27b

9.62b

51.91a

21.6ab

39.73ab

2.84a

Dilution 500X

98.25a

6.23b

11.66a

52.24a

22.33ab

40.52a

2.88a

Dilution 1000X

98.25a

7.67a

13.04a

52.35a

22.6a

40.26a

2.87a

Control

98.25a

4.77c

7.38c

48.26b

20.19c

37.41c

2.63c

50 x-1000 x diluted Bam62 fermentation liquor has certain growth promotion effect on the rate of emergence and growth of plants, 100 x-500 x diluted Bam62 fermentation liquor has obvious promotion effect on the emergence of most plants, the height of seedlings, the root length, the fresh weight and the dry weight, and therefore 100 x-500 x diluted Bam62 fermentation liquor is recommended to be used in the using process.

The above description is only a preferred embodiment of the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> institute of plant protection of academy of agricultural sciences of Henan province

<120> bacillus amyloliquefaciens with growth promoting effect and capable of preventing and treating sesame stalk blight and fusarium wilt and application thereof

<130> identification of Strain

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1348

<212> DNA

<213> Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)

<400> 1

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ctcgcggttt cgctgccctt tgttctgtcc attgtagcac gtgtgtagcc caggtcataa 180

ggggcatgat gatttgacgt catccccacc ttcctccggt ttgtcaccgg cagtcacctt 240

agagtgccca actgaatgct ggcaactaag atcaagggtt gcgctcgttg cgggacttaa 300

cccaacatct cacgacacga gctgacgaca accatgcacc acctgtcact ctgcccccga 360

aggggacgtc ctatctctag gattgtcaga ggatgtcaag acctggtaag gttcttcgcg 420

ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg cccccgtcaa ttcctttgag 480

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taatcctgtt cgctccccac gctttcgctc ctcagcgtca gttacagacc agagagtcgc 660

cttcgccact ggtgttcctc cacatctcta cgcatttcac cgctacacgt ggaattccac 720

tctcctcttc tgcactcaag ttccccagtt tccaatgacc ctccccggtt gagccggggg 780

ctttcacatc agacttaaga aaccgcctgc gagcccttta cgcccaataa ttccggacaa 840

cgcttgccac ctacgtatta ccgcggctgc tggcacgtag ttagccgtgg ctttctggtt 900

aggtaccgtc aaggtgccgc cctatttgaa cggcacttgt tcttccctaa caacagagct 960

ttacgatccg aaaaccttca tcactcacgc ggcgttgctc cgtcagactt tcgtccattg 1020

cggaagattc cctactgctg cctcccgtag gagtctgggc cgtgtctcag tcccagtgtg 1080

gccgatcacc ctctcaggtc ggctacgcat cgttgccttg gtgagccgtt acctcaccaa 1140

ctagctaatg cgccgcgggt ccatctgtaa gtggtagccg aagccacctt ttatgtctga 1200

accatgcggt tcaaacaacc atccggtatt agccccggtt tcccggagtt atcccagtct 1260

tacaggcagg ttacccacgt gttactcacc cgtccgccgc taacatcagg gagcaagctc 1320

ccatctgtcc gctcgactgc atgatagc 1348

Claims

1. The Bacillus amyloliquefaciens strain has the growth promoting effect and can prevent and control sesame stalk blight and fusarium wilt, and is characterized in that the Bacillus amyloliquefaciens strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Bam62, is preserved in China center for type culture collection, and has the preservation address: wuhan university in Wuhan, China, the preservation number: CCTCC NO: m2019303, date of deposit: year 2019, month 4, and day 28.

2. The bacillus amyloliquefaciens of claim 1, wherein the bacillus amyloliquefaciens Bam62 is a gram positive bacterium and a colony cultured on LB medium at 28 ℃ for 48 hours is milky white, opaque, irregular in edge, raised, wrinkled and rough in surface.

3. The bacillus amyloliquefaciens of claim 1, wherein the bacillus amyloliquefaciens Bam62 catalase reaction, anaerobic growth test, gelatin hydrolysis, starch hydrolysis, V-P reaction, M.R reaction, nitrate reduction, urease reaction, D-glucose utilization, D-mannitol utilization, maltose utilization are all positive; tyrosine hydrolysis, phenylalanine deaminase reaction, indole production and citrate utilization are all negative; the growth is not carried out in 7% NaCl, the growth temperature is 25-30 ℃, and the growth pH is 7.

4. A method of preparing a fermentation broth of bacillus amyloliquefaciens Bam62 according to claim 1, comprising the steps of:

5. The process according to claim 4, wherein the fermentation medium is composed of 10g of soluble starch, 3g of ammonium sulfate, 5g of glucose, 0.7g of magnesium sulfate, 0.5g of potassium dihydrogen phosphate, 1g of calcium carbonate, 1g of antifoaming agent and the balance of distilled water per 1000mL, and sterilized at 121 ℃ for 30 min.

6. A method for preparing bacillus amyloliquefaciens Bam62 wettable powder according to claim 1, comprising the following steps:

7. Use of the bacillus amyloliquefaciens of claim 1 to promote growth of a crop.

8. The use of the bacillus amyloliquefaciens of claim 1 for controlling sesame stalk blight and blight.

9. Use of the bacillus amyloliquefaciens of claim 1 to promote crop growth and control sesame stalk blight and blight simultaneously.